TY - JOUR
A1 - Chowdhury, Mita Mullick
A1 - Dosche, Carsten
A1 - Loehmannsröben, Hans-Gerd
A1 - Leimkühler, Silke
T1 - Dual role of the molybdenum cofactor biosynthesis protein MOCS3 in tRNA thiolation and molybdenum cofactor biosynthesis in humans
JF - The journal of biological chemistry
N2 - We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are beta-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.
Y1 - 2012
U6 - https://doi.org/10.1074/jbc.M112.351429
SN - 0021-9258
VL - 287
IS - 21
SP - 17297
EP - 17307
PB - American Society for Biochemistry and Molecular Biology
CY - Bethesda
ER -
TY - JOUR
A1 - Coelho, Catarina
A1 - Foti, Alessandro
A1 - Hartmann, Tobias
A1 - Santos-Silva, Teresa
A1 - Leimkühler, Silke
A1 - Romao, Maria Joao
T1 - Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase
JF - Nature chemical biology
N2 - Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-angstrom resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-angstrom resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.
Y1 - 2015
U6 - https://doi.org/10.1038/NCHEMBIO.1895
SN - 1552-4450
SN - 1552-4469
VL - 11
IS - 10
SP - 779
EP - +
PB - Nature Publ. Group
CY - New York
ER -
TY - JOUR
A1 - Coelho, Catarina
A1 - Mahro, Martin
A1 - Trincao, Jose
A1 - Carvalho, Alexandra T. P.
A1 - Ramos, Maria Joao
A1 - Terao, Mineko
A1 - Garattini, Enrico
A1 - Leimkühler, Silke
A1 - Romao, Maria Joao
T1 - The first mammalian aldehyde oxidase crystal structure insights into substrate specificity
JF - The journal of biological chemistry
N2 - Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 angstrom. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.
Y1 - 2012
U6 - https://doi.org/10.1074/jbc.M112.390419
SN - 0021-9258
VL - 287
IS - 48
SP - 40690
EP - 40702
PB - American Society for Biochemistry and Molecular Biology
CY - Bethesda
ER -
TY - JOUR
A1 - Contin, Andrea
A1 - Frasca, Stefano
A1 - Vivekananthan, Jeevanthi
A1 - Leimkühler, Silke
A1 - Wollenberger, Ursula
A1 - Plumere, Nicolas
A1 - Schuhmann, Wolfgang
T1 - A pH Responsive Redox Hydrogel for Electrochemical Detection of Redox Silent Biocatalytic Processes. Control of Hydrogel Solvation
JF - Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis
N2 - The control of bioelectrocatalytic processes by external stimuli for the indirect detection of non-redox active species was achieved using an esterase and a redox enzyme both integrated within a redox hydrogel. The poly( vinyl) imidazole Os(bpy)(2)Cl hydrogel displays pH-responsive properties. The esterase catalysed reaction leads to a local pH decrease causing protonation of imidazole moieties thus increasing hydrogel solvation and mobility of the tethered Os-complexes. This is the key step to enable improved electron transfer between an aldehyde oxidoreductase and the polymer-bound Os-complexes. The off-on switch is further integrated in a biofuel cell system for self-powered signal generation.
KW - pH responsive hydrogel
KW - External stimuli
KW - Biofuel cell
KW - Self-powered biosensor
KW - Solvation
Y1 - 2015
U6 - https://doi.org/10.1002/elan.201400621
SN - 1040-0397
SN - 1521-4109
VL - 27
IS - 4
SP - 938
EP - 944
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Correia, Marcia A. S.
A1 - Otrelo-Cardoso, Ana Rita
A1 - Schwuchow, Viola
A1 - Clauss, Kajsa G. V. Sigfridsson
A1 - Haumann, Michael
A1 - Romao, Maria Joao
A1 - Leimkühler, Silke
A1 - Santos-Silva, Teresa
T1 - The Escherichia coli Periplasmic Aldehyde Oxidoreductase Is an Exceptional Member of the Xanthine Oxidase Family of Molybdoenzymes
JF - ACS chemical biology
N2 - The xanthine oxidase (XO) family comprises molybdenum-dependent enzymes that usually form homodimers (or dimers of heterodimers/trimers) organized in three domains that harbor two [2Fe-2S] clusters, one FAD, and a Mo cofactor. In this work, we crystallized an unusual member of the family, the periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli. This is the first example of an E. coli protein containing a molybdopterin-cytosine-dinucleotide cofactor and is the only heterotrimer of the XO family so far structurally characterized. The crystal structure revealed the presence of an unexpected [4Fe-4S] cluster, anchored to an additional 40 residues subdomain. According to phylogenetic analysis, proteins containing this cluster are widely spread in many bacteria phyla, putatively through repeated gene transfer events. The active site of PaoABC is highly exposed to the surface with no aromatic residues and an arginine (PaoC-R440) making a direct interaction with PaoC-E692, which acts as a base catalyst. In order to understand the importance of R440, kinetic assays were carried out, and the crystal structure of the PaoC-R440H variant was also determined.
Y1 - 2016
U6 - https://doi.org/10.1021/acschembio.6b00572
SN - 1554-8929
SN - 1554-8937
VL - 11
SP - 2923
EP - 2935
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Dahl, Jan-Ulrik
A1 - Radon, Christin
A1 - Bühning, Martin
A1 - Nimtz, Manfred
A1 - Leichert, Lars I.
A1 - Denis, Yann
A1 - Jourlin-Castelli, Cecile
A1 - Iobbi-Nivol, Chantal
A1 - Mejean, Vincent
A1 - Leimkühler, Silke
T1 - The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia
coli That Also Affects Molybdenum Cofactor Biosynthesis
JF - JOURNAL OF BIOLOGICAL CHEMISTRY
N2 - The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a Delta tusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the Delta tusA mutant. Characterization of the Delta tusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes.
Y1 - 2013
U6 - https://doi.org/10.1074/jbc.M112.431569
SN - 0021-9258
VL - 288
IS - 8
SP - 5426
EP - 5442
PB - AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
CY - BETHESDA
ER -
TY - JOUR
A1 - Dahl, Jan-Ulrik
A1 - Urban, Alexander
A1 - Bolte, Andrea
A1 - Sriyabhaya, Promjit
A1 - Donahue, Janet L.
A1 - Nimtz, Manfred
A1 - Larson, Timothy J.
A1 - Leimkühler, Silke
T1 - The identification of a novel protein involved in Molybdenum Cofactor Biosynthesis in Escherichia coli
JF - The journal of biological chemistry
N2 - Background: In Moco biosynthesis, sulfur is transferred from L-cysteine to MPT synthase, catalyzing the conversion of cPMP to MPT.
Results: The rhodanese-like protein YnjE is a novel protein involved in Moco biosynthesis.
Conclusion: YnjE enhances the rate of conversion of cPMP to MPT and interacts with MoeB and IscS. S
ignificance: To understand the mechanism of sulfur transfer and the role of rhodaneses in the cell.
Y1 - 2011
U6 - https://doi.org/10.1074/jbc.M111.282368
SN - 0021-9258
VL - 286
IS - 41
SP - 35801
EP - 35812
PB - American Society for Biochemistry and Molecular Biology
CY - Bethesda
ER -
TY - JOUR
A1 - De Sousa Mota, Cristiano
A1 - Diniz, Ana
A1 - Coelho, Catarina
A1 - Santos-Silva, Teresa
A1 - Esmaeeli Moghaddam Tabalvandani, Mariam
A1 - Leimkühler, Silke
A1 - Cabrita, Eurico J.
A1 - Marcelo, Filipa
A1 - Romão, Maria João
T1 - Interrogating the inhibition mechanisms of human aldehyde oxidase by X-ray crystallography and NMR spectroscopy
BT - the raloxifene case
JF - Journal of medicinal chemistry / American Chemical Society
N2 - Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.
Y1 - 2021
U6 - https://doi.org/10.1021/acs.jmedchem.1c01125
SN - 0022-2623
SN - 1520-4804
VL - 64
IS - 17
SP - 13025
EP - 13037
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Dey, Pradip
A1 - Adamovski, Miriam
A1 - Friebe, Simon
A1 - Badalyan, Artavazd
A1 - Mutihac, Radu-Cristian
A1 - Paulus, Florian
A1 - Leimkühler, Silke
A1 - Wollenberger, Ursula
A1 - Haag, Rainer
T1 - Dendritic polyglycerol-poly(ethylene glycol)-based polymer networks for biosensing application
JF - ACS applied materials & interfaces
N2 - This work describes the formation of a new dendritic polyglycerol-poly(ethylene glycol)-based 3D polymer network as a matrix for immobilization of the redox enzyme periplasmatic aldehyde oxidoreductase to create an electrochemical biosensor. The novel network is built directly on the gold surface, where it simultaneously stabilizes the enzyme for up to 4 days. The prepared biosensors can be used for amperometric detection of benzaldehyde in the range of 0.8-400 mu M.
KW - biosensors
KW - hydrogel
KW - amperometry
KW - dendritic
Y1 - 2014
U6 - https://doi.org/10.1021/am502018x
SN - 1944-8244
VL - 6
IS - 12
SP - 8937
EP - 8941
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Dietzel, Uwe
A1 - Kuper, Jochen
A1 - Doebbler, Jennifer A.
A1 - Schulte, Antje
A1 - Truglio, James J.
A1 - Leimkühler, Silke
A1 - Kisker, Caroline
T1 - Mechanism of substrate and inhibitor binding of Rhodobacter capsulatus xanthine dehydrogenase
N2 - Rhodobacter capsulatus xanthine dehydrogenase (XDH) is an (alpha beta)(2) heterotetrameric cytoplasmic enzyme that resembles eukaryotic xanthine oxidoreductases in respect to both amino acid sequence and structural fold. To obtain a detailed understanding of the mechanism of substrate and inhibitor binding at the active site, we solved crystal structures of R. capsulatus XDH in the presence of its substrates hypoxanthine, xanthine, and the inhibitor pterin-6- aldehyde using either the inactive desulfo form of the enzyme or an active site mutant (E(B)232Q) to prevent substrate turnover. The hypoxanthine-and xanthine-bound structures reveal the orientation of both substrates at the active site and show the importance of residue GluB-232 for substrate positioning. The oxygen atom at the C-6 position of both substrates is oriented toward Arg(B)-310 in the active site. Thus the substrates bind in an orientation opposite to the one seen in the structure of the reduced enzyme with the inhibitor oxypurinol. The tightness of the substrates in the active site suggests that the intermediate products must exit the binding pocket to allow first the attack of the C-2, followed by oxidation of the C-8 atom to form the final product uric acid. Structural studies of pterin-6-aldehyde, a potent inhibitor of R. capsulatus XDH, contribute further to the understanding of the relative positioning of inhibitors and substrates in the binding pocket. Steady state kinetics reveal a competitive inhibition pattern with a K-i of 103.57 +/- 18.96 nM for pterin-6-aldehyde.
Y1 - 2009
UR - http://www.jbc.org/
U6 - https://doi.org/10.1074/jbc.M808114200
SN - 0021-9258
ER -
TY - JOUR
A1 - Dong, Chao
A1 - Yang, Jing
A1 - Reschke, Stefan
A1 - Leimkühler, Silke
A1 - Kirk, Martin L.
T1 - Vibrational Probes of Molybdenum Cofactor-Protein Interactions in Xanthine Dehydrogenase
JF - Inorganic chemistry
N2 - The pyranopterin dithiolene (PDT) ligand is an integral component of the molybdenum cofactor (Moco) found in all molybdoenzymes with the sole exception of nitrogenase. However, the roles of the PDT in catalysis are still unknown. The PDT is believed to be bound to the proteins by an extensive hydrogen bonding network, and it has been suggested that these interactions may function to fine-tune Moco for electron- and atom-transfer reactivity in catalysis. Here, we use resonance Raman (rR) spectroscopy to probe Moco-protein interactions using heavy-atom congeners of lumazine, molecules that bind tightly to both wild-type xanthine dehydrogenase (wt-XDH) and its Q102G and Q197A variants following enzymatic hydroxylation to the corresponding violapterin product molecules. The resulting enzyme-product complexes possess intense near-IR absorption, allowing high-quality rR spectra to be collected on wt-XDH and the Q102G and Q197A variants. Small negative frequency shifts relative to wt-XDH are observed for the low-frequency Moco vibrations. These results are interpreted in the context of weak hydrogen-bonding and/or electrostatic interactions between Q102 and the -NH2 terminus of the PDT, and between Q197 and the terminal oxo of the Mo equivalent to O group. The Q102A, Q102G, Q197A, and Q197E variants do not appreciably affect the kinetic parameters k(red) and k(red)/K-D, indicating that a primary role for these glutamine residues is to stabilize and coordinate Moco in the active site of XO family enzymes but to not directly affect the catalytic throughput. Raman frequency shifts between wt-XDH and its Q102G variant suggest that the changes in the electron density at the Mo ion that accompany Mo oxidation during electron-transfer regeneration of the catalytically competent active site are manifest in distortions at the distant PDT amino terminus. This implies a primary role for the PDT as a conduit for facilitating enzymatic electron-transfer reactivity in xanthine oxidase family enzymes.
Y1 - 2017
U6 - https://doi.org/10.1021/acs.inorgchem.7b00028
SN - 0020-1669
SN - 1520-510X
VL - 56
SP - 6830
EP - 6837
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Duffus, Benjamin R.
A1 - Schrapers, Peer
A1 - Schuth, Nils
A1 - Mebs, Stefan
A1 - Dau, Holger
A1 - Leimkühler, Silke
A1 - Haumann, Michael
T1 - Anion binding and oxidative modification at the molybdenum cofactor of formate dehydrogenase from Rhodobacter capsulatus studied by X-ray absorption spectroscopy
JF - Inorganic chemistry
N2 - Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O-2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo=S) and oxo (Mo=O) bonds, were observed in the presence of azide (N-3(-)) or cyanate (OCN-). Azide provided best protection against O-2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.
Y1 - 2020
U6 - https://doi.org/10.1021/acs.inorgchem.9b01613
SN - 0020-1669
SN - 1520-510X
VL - 59
IS - 1
SP - 214
EP - 225
PB - American Chemical Society
CY - Washington, DC
ER -
TY - JOUR
A1 - Forlani, Fabio
A1 - Cereda, Angelo
A1 - Freuer, Andrea
A1 - Nimtz, Manfred
A1 - Leimkühler, Silke
A1 - Pagani, Silvia
T1 - The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form
N2 - After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified L-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved
Y1 - 2005
SN - 0014-5793
ER -
TY - JOUR
A1 - Foti, Alessandro
A1 - Dorendorf, Frank
A1 - Leimkühler, Silke
T1 - A single nucleotide polymorphism causes enhanced radical oxygen species production by human aldehyde oxidase
JF - PLoS one
N2 - Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. The physiological function of mammalian AOX isoenzymes is still unclear, however, human AOX (hAOX1) is an emerging enzyme in phase-I drug metabolism. Indeed, the number of xenobiotics acting as hAOX1 substrates is increasing. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified within the hAOX1 gene. SNPs are a major source of inter-individual variability in the human population, and SNP-based amino acid exchanges in hAOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. In this report we selected ten novel SNPs resulting in amino acid exchanges in proximity to the FAD site of hAOX1 and characterized the purified enzymes after heterologous expression in Escherichia coli. The hAOX1 variants were characterized carefully by quantitative differences in their ability to produce superoxide radical. ROS represent prominent key molecules in physiological and pathological conditions in the cell. Our data reveal significant alterations in superoxide anion production among the variants. In particular the SNP-based amino acid exchange L438V in proximity to the isoalloxanzine ring of the FAD cofactor resulted in increased rate of superoxide radical production of 75%. Considering the high toxicity of the superoxide in the cell, the hAOX1-L438V SNP variant is an eventual candidate for critical or pathological roles of this natural variant within the human population.
Y1 - 2017
U6 - https://doi.org/10.1371/journal.pone.0182061
SN - 1932-6203
VL - 12
SP - 18338
EP - 18347
PB - PLoS
CY - San Fransisco
ER -
TY - JOUR
A1 - Foti, Alessandro
A1 - Hartmann, Tobias
A1 - Coelho, Catarina
A1 - Santos-Silva, Teresa
A1 - Romao, Maria Joao
A1 - Leimkühler, Silke
T1 - Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms
JF - Drug metabolism and disposition : the biological fate of chemicals
N2 - Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme’s role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies.
Y1 - 2016
U6 - https://doi.org/10.1124/dmd.115.068395
SN - 0090-9556
SN - 1521-009X
VL - 44
SP - 1277
EP - 1285
PB - American Society for Pharmacology and Experimental Therapeutics
CY - Bethesda
ER -
TY - JOUR
A1 - Fraesdorf, Benjamin
A1 - Radon, Christin
A1 - Leimkühler, Silke
T1 - Characterization and interaction studies of two isoforms of the dual localized 3-mercaptopyruvate sulfurtransferase TUM1 from humans
JF - The journal of biological chemistry
N2 - Background: Localization and identification of interaction partners of two splice variants of the human 3-mercaptopyruvate sulfurtransferase TUM1. Results: We show that TUM1 interacts with proteins involved in Moco and FeS cluster biosynthesis. Conclusion: Human TUM1 is a dual localized protein in the cytosol and mitochondria with distinct roles in sulfur transfer and interaction partners. Significance: The study contributes to the sulfur transfer pathway for the biosynthesis of sulfur-containing biofactors.
The human tRNA thiouridine modification protein (TUM1), also designated as 3-mercaptopyruvate sulfurtransferase (MPST), has been implicated in a wide range of physiological processes in the cell. The roles range from an involvement in thiolation of cytosolic tRNAs to the generation of H2S as signaling molecule both in mitochondria and the cytosol. TUM1 is a member of the sulfurtransferase family and catalyzes the conversion of 3-mercaptopyruvate to pyruvate and protein-bound persulfide. Here, we purified and characterized two novel TUM1 splice variants, designated as TUM1-Iso1 and TUM1-Iso2. The purified proteins showed similar kinetic behavior and comparable pH and temperature dependence. Cellular localization studies, however, showed a different localization pattern between the isoforms. TUM1-Iso1 is exclusively localized in the cytosol, whereas TUM1-Iso2 showed a dual localization both in the cytosol and mitochondria. Interaction studies were performed with the isoforms both in vitro using the purified proteins and in vivo by fluorescence analysis in human cells, using the split-EGFP system. The studies showed that TUM1 interacts with the l-cysteine desulfurase NFS1 and the rhodanese-like protein MOCS3, suggesting a dual function of TUM1 both in sulfur transfer for the biosynthesis of the molybdenum cofactor, and for the thiolation of tRNA. Our studies point to distinct roles of each TUM1 isoform in the sulfur transfer processes in the cell, with different compartmentalization of the two splice variants of TUM1.
KW - Fluorescence
KW - Mitochondria
KW - Molybdenum
KW - Sulfur
KW - Transfer RNA (tRNA)
Y1 - 2014
U6 - https://doi.org/10.1074/jbc.M114.605733
SN - 0021-9258
SN - 1083-351X
VL - 289
IS - 50
SP - 34543
EP - 34556
PB - American Society for Biochemistry and Molecular Biology
CY - Bethesda
ER -
TY - JOUR
A1 - Frasca, Stefano
A1 - Rojas, Oscar
A1 - Salewski, Johannes
A1 - Neumann, Bettina
A1 - Stiba, Konstanze
A1 - Weidinger, Inez M.
A1 - Tiersch, Brigitte
A1 - Leimkühler, Silke
A1 - Koetz, Joachim
A1 - Wollenberger, Ursula
T1 - Human sulfite oxidase electrochemistry on gold nanoparticles modified electrode
JF - Bioelectrochemistry : an international journal devoted to electrochemical aspects of biology and biological aspects of electrochemistry ; official journal of the Bioelectrochemical Society
N2 - The present study reports a facile approach for sulfite biosensing, based on enhanced direct electron transfer of a human sulfite oxidase (hSO) immobilized on a gold nanoparticles modified electrode. The spherical core shell AuNPs were prepared via a new method by reduction of HAuCl4 with branched poly(ethyleneimine) in an ionic liquids resulting particles with a diameter less than 10 nm. These nanoparticles were covalently attached to a mercaptoundecanoic acid modified Au-electrode where then hSO was adsorbed and an enhanced interfacial electron transfer and electrocatalysis was achieved. UV/Vis and resonance Raman spectroscopy, in combination with direct protein voltammetry, are employed for the characterization of the system and reveal no perturbation of the structural integrity of the redox protein. The proposed biosensor exhibited a quick steady-state current response, within 2 s, a linear detection range between 0.5 and 5.4 mu M with a high sensitivity (1.85 nA mu M-1). The investigated system provides remarkable advantages in the possibility to work at low applied potential and at very high ionic strength. Therefore these properties could make the proposed system useful in the development of bioelectronic devices and its application in real samples.
KW - Direct electron transfer
KW - Gold nanoparticle
KW - Human sulfite oxidase
KW - Ionic liquid
KW - Sulfite biosensor
Y1 - 2012
U6 - https://doi.org/10.1016/j.bioelechem.2011.11.012
SN - 1567-5394
VL - 87
SP - 33
EP - 41
PB - Elsevier
CY - Lausanne
ER -
TY - JOUR
A1 - Friemel, Martin
A1 - Marelja, Zvonimir
A1 - Li, Kuanyu
A1 - Leimkühler, Silke
T1 - The N-Terminus of Iron-Sulfur Cluster Assembly Factor ISD11 Is Crucial for Subcellular Targeting and Interaction with L-Cysteine Desulfurase NFS1
JF - Biochemistry
N2 - Assembly of iron sulfur (FeS) clusters is an important process in living cells. The initial sulfur mobilization step for FeS cluster biosynthesis is catalyzed by L-cysteine desulfurase NFS1, a reaction that is localized in mitochondria in humans. In humans, the function of NFS1 depends on the ISD11 protein, which is required to stabilize its structure. The NFS1/ISD11 complex further interacts with scaffold protein ISCU and regulator protein frataxin, thereby forming a quaternary complex for FeS cluster formation. It has been suggested that the role of ISD11 is not restricted to its role in stabilizing the structure of NFS1, because studies of single-amino acid variants of ISD11 additionally demonstrated its importance for the correct assembly of the quaternary complex. In this study, we are focusing on the N-terminal region of ISD11 to determine the role of N-terminal amino acids in the formation of the complex with NFS1 and to reveal the mitochondria) targeting sequence for subcellular localization. Our in vitro studies with the purified proteins and in vivo studies in a cellular system show that the first 10 N-terminal amino acids of ISD11 are indispensable for the activity of NFS1 and especially the conserved "LYR" motif is essential for the role of ISD11 in forming a stable and active complex with NFS1.
Y1 - 2017
U6 - https://doi.org/10.1021/acs.biochem.6b01239
SN - 0006-2960
VL - 56
SP - 1797
EP - 1808
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Fujihara, Kenji M.
A1 - Zhang, Bonnie Z.
A1 - Jackson, Thomas D.
A1 - Ogunkola, Moses
A1 - Nijagal, Brunda
A1 - Milne, Julia V.
A1 - Sallman, David A.
A1 - Ang, Ching-Seng
A1 - Nikolic, Iva
A1 - Kearney, Conor J.
A1 - Hogg, Simon J.
A1 - Cabalag, Carlos S.
A1 - Sutton, Vivien R.
A1 - Watt, Sally
A1 - Fujihara, Asuka T.
A1 - Trapani, Joseph A.
A1 - Simpson, Kaylene J.
A1 - Stojanovski, Diana
A1 - Leimkühler, Silke
A1 - Haupt, Sue
A1 - Phillips, Wayne A.
A1 - Clemons, Nicholas J.
T1 - Eprenetapopt triggers ferroptosis, inhibits NFS1 cysteine desulfurase, and synergizes with serine and glycine dietary restriction
JF - Science Advances
N2 - The mechanism of action of eprenetapopt (APR-246, PRIMA-1MET) as an anticancer agent remains unresolved, al-though the clinical development of eprenetapopt focuses on its reported mechanism of action as a mutant-p53 reactivator. Using unbiased approaches, this study demonstrates that eprenetapopt depletes cellular antioxidant glutathione levels by increasing its turnover, triggering a nonapoptotic, iron-dependent form of cell death known as ferroptosis. Deficiency in genes responsible for supplying cancer cells with the substrates for de novo glutathione synthesis (SLC7A11, SHMT2, and MTHFD1L), as well as the enzymes required to synthesize glutathione (GCLC and GCLM), augments the activity of eprenetapopt. Eprenetapopt also inhibits iron-sulfur cluster biogenesis by limit-ing the cysteine desulfurase activity of NFS1, which potentiates ferroptosis and may restrict cellular proliferation. The combination of eprenetapopt with dietary serine and glycine restriction synergizes to inhibit esophageal xenograft tumor growth. These findings reframe the canonical view of eprenetapopt from a mutant-p53 reactivator to a ferroptosis inducer.
Y1 - 2022
U6 - https://doi.org/10.1126/sciadv.abm9427
SN - 2375-2548
VL - 8
IS - 37
PB - American Assoc. for the Advancement of Science
CY - Washington
ER -
TY - JOUR
A1 - Garrido, Claudia
A1 - Leimkühler, Silke
T1 - The inactivation of human aldehyde oxidase 1 by hydrogen peroxide and superoxide
JF - Drug metabolism and disposition / American Society for Pharmacology and Experimental Therapeutics
N2 - Mammalian aldehyde oxidases (AOX) are molybdo-flavoenzymes of pharmacological and pathophysiologic relevance that are involved in phase I drug metabolism and, as a product of their enzymatic activity, are also involved in the generation of reactive oxygen species. So far, the physiologic role of aldehyde oxidase 1 in the human body remains unknown. The human enzyme hAOX1 is characterized by a broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into their corresponding carboxylic acids, and hydroxylating various heteroaromatic rings. The enzyme uses oxygen as terminal electron acceptor to produce hydrogen peroxide and superoxide during turnover. Since hAOX1 and, in particular, some natural variants produce not only H2O2 but also high amounts of superoxide, we investigated the effect of both ROS molecules on the enzymatic activity of hAOX1 in more detail. We compared hAOX1 to the high-O-2(.-)-producing natural variant L438V for their time-dependent inactivation with H2O2/O-2(.-) during substrate turnover. We show that the inactivation of the hAOX1 wild-type enzyme is mainly based on the production of hydrogen peroxide, whereas for the variant L438V, both hydrogen peroxide and superoxide contribute to the time-dependent inactivation of the enzyme during turnover. Further, the level of inactivation was revealed to be substrate-dependent: using substrates with higher turnover numbers resulted in a faster inactivation of the enzymes. Analysis of the inactivation site of the enzyme identified a loss of the terminal sulfido ligand at the molybdenum active site by the produced ROS during turnover.
Y1 - 2021
U6 - https://doi.org/10.1124/dmd.121.000549
SN - 1521-009X
SN - 0090-9556
VL - 49
IS - 9
SP - 729
EP - 735
PB - American Society for Pharmacology and Experimental Therapeutics
CY - Bethesda
ER -
TY - JOUR
A1 - Gisin, Jonathan
A1 - Mueller, Alexandra
A1 - Pfaender, Yvonne
A1 - Leimkühler, Silke
A1 - Narberhaus, Franz
A1 - Masepohl, Bernd
T1 - A Rhodobacter capsulatus member of a universal permease family imports molybdate and other oxyanions
N2 - Molybdenum (Mo) is an important trace element that is toxic at high concentrations. To resolve the mechanisms underlying Mo toxicity, Rhodobacter capsulatus mutants tolerant to high Mo concentrations were isolated by random transposon Tn5 mutagenesis. The insertion sites of six independent isolates mapped within the same gene predicted to code for a permease of unknown function located in the cytoplasmic membrane. During growth under Mo-replete conditions, the wild-type strain accumulated considerably more Mo than the permease mutant. For mutants defective for the permease, the high-affinity molybdate importer ModABC, or both transporters, in vivo Mo-dependent nitrogenase (Mo-nitrogenase) activities at different Mo concentrations suggested that ModABC and the permease import molybdate in nanomolar and micromolar ranges, respectively. Like the permease mutants, a mutant defective for ATP sulfurylase tolerated high Mo concentrations, suggesting that ATP sulfurylase is the main target of Mo inhibition in R. capsulatus. Sulfate-dependent growth of a double mutant defective for the permease and the high-affinity sulfate importer CysTWA was reduced compared to those of the single mutants, implying that the permease plays an important role in sulfate uptake. In addition, permease mutants tolerated higher tungstate and vanadate concentrations than the wild type, suggesting that the permease acts as a general oxyanion importer. We propose to call this permease PerO (for oxyanion permease). It is the first reported bacterial molybdate transporter outside the ABC transporter family.
Y1 - 2010
UR - http://jb.asm.org/
U6 - https://doi.org/10.1128/Jb.00742-10
SN - 0021-9193
ER -
TY - JOUR
A1 - Haenzelmann, Petra
A1 - Dahl, Jan U.
A1 - Kuper, Jochen
A1 - Urban, Alexander
A1 - Mueller-Theissen, Ursula
A1 - Leimkühler, Silke
A1 - Schindelin, Hermann
T1 - Crystal structure of YnjE from Escherichia coli, a sulfurtransferase with three rhodanese domains
N2 - Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple-domain rhodanese-like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well- characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N-terminal inactive rhodanese-like domain. Phylogenetic analysis reveals that YnjE triple-domain homologs can be found in a variety of other gamma-proteobacteria, in addition, some single-, tandem-, four and even six-domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3- mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C-terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.
Y1 - 2009
UR - http://www.proteinscience.org/
U6 - https://doi.org/10.1002/pro.260
SN - 0961-8368
ER -
TY - JOUR
A1 - Hahn, Aaron
A1 - Engelhard, Christopher
A1 - Reschke, Stefan
A1 - Teutloff, Christian
A1 - Bittl, Robert
A1 - Leimkühler, Silke
A1 - Risse, Thomas
T1 - Structural Insights into the Incorporation of the Mo Cofactor into Sulfite Oxidase from Site-Directed Spin Labeling
JF - Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition
N2 - Mononuclear molybdoenzymes catalyze a broad range of redox reactions and are highly conserved in all kingdoms of life. This study addresses the question of how the Mo cofactor (Moco) is incorporated into the apo form of human sulfite oxidase (hSO) by using site-directed spin labeling to determine intramolecular distances in the nanometer range. Comparative measurements of the holo and apo forms of hSO enabled the localization of the corresponding structural changes, which are localized to a short loop (residues 263-273) of the Moco-containing domain. A flap-like movement of the loop provides access to the Moco binding-pocket in the apo form of the protein and explains the earlier studies on the in vitro reconstitution of apo-hSO with Moco. Remarkably, the loop motif can be found in a variety of structurally similar molybdoenzymes among various organisms, thus suggesting a common mechanism of Moco incorporation.
KW - biocatalysis
KW - cofactors
KW - enzymes
KW - EPR spectroscopy
KW - protein structures
Y1 - 2015
U6 - https://doi.org/10.1002/anie.201504772
SN - 1433-7851
SN - 1521-3773
VL - 54
IS - 40
SP - 11865
EP - 11869
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Hahn, Aaron
A1 - Reschke, Stefan
A1 - Leimkühler, Silke
A1 - Risse, Thomas
T1 - Ketoxime coupling of p-Acetylphenylalanine at neutral pH for site-directed spin labeling of human sulfite oxidase
JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry
N2 - Site-directed spin labeling of the unnatural amino acid p-acetylphenylalanine (p-AcPhe) using oxime based coupling chemistry is successfully applied to investigate human sulfite oxidase (hSO), a protein containing an essential cysteine residue, which impedes the use of thiol based coupling chemistry. The protein was found to be sensitive toward typical reaction conditions of oxime coupling, namely, acidic reaction conditions and elevated temperatures. Thus, coupling at neutral pH and room temperature is mandatory. Three catalysts described in the literature to accelerate the reaction rate have been tested. Best spin labeling efficiencies were observed for p-methoxyaniline, while the other catalysts described in the literature to have even better performance for oxime coupling at neutral pH were substantially less active or led to precipitation of the protein. A clear correlation of spin labeling efficiency with the local environment of the residue is found, shedding some light on the importance of the sterically demanding reaction complex between p-AcPhe, the aniline catalyst, and the spin label for the reaction rate. The analysis of the line shape has shown that its interpretation in terms of local environment is more challenging as compared to the well-established spin labels based on cysteine chemistry. To this end the results presented here indicate that the larger steric demand of the spin labeled p-AcPhe can induce structural effects instead of reporting on them.
Y1 - 2014
U6 - https://doi.org/10.1021/jp503471j
SN - 1520-6106
VL - 118
IS - 25
SP - 7077
EP - 7084
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Hahnewald, Rita
A1 - Leimkühler, Silke
A1 - Vilaseca, Antonia
A1 - Acquaviva-Bourdain, Cecile
A1 - Lenz, Ulrike
A1 - Reiss, Jochen
T1 - A novel MOCS2 mutation reveals coordinated expression of the small and large subunit of molybdopterin synthase
JF - Molecular genetics and metabolism
N2 - The small and large subunits of molybdopterin (MPT) synthase (MOCS2A and MOCS2B), are both encoded by the MOCS2 gene in overlapping and shifted open reading frames (ORFs), which is a highly unusual structure for eukaryotes. Theoretical analysis of genomic sequences suggested that the expression of these overlapping ORFs is facilitated by the use of alternate first exons leading to alternative transcripts. Here, we confirm the existence of these overlapping transcripts experimentally. Further, we identified a deletion in a molybdenum cofactor deficient patient, which removes the start codon for the small subunit (MOCS2A). We observed undisturbed production of both transcripts, while Western blot analysis demonstrated that MOCS2B, the large subunit, is unstable in the absence of MOCS2A. This reveals new insights into the expression of this evolutionary ancient anabolic system.
KW - molybdenum cofactor deficiency
KW - MOCS2
KW - overlapping reading frames
Y1 - 2006
U6 - https://doi.org/10.1016/j.ymgme.2006.04.008
SN - 1096-7192
VL - 89
IS - 3
SP - 210
EP - 213
PB - Elsevier
CY - San Diego
ER -
TY - JOUR
A1 - Hall, James
A1 - Reschke, Stefan
A1 - Cao, Hongnan
A1 - Leimkühler, Silke
A1 - Hille, Russ
T1 - The reductive half-reaction of xanthine dehydrogenase from rhodobacter capsulatus the role of GLU(232) in catalysis
JF - The journal of biological chemistry
N2 - Background: Kinetic characterization of wild-type xanthine dehydrogenase and variants. Results: Comparison of the pH dependence of both k(red) and k(red)/K-d, as well as k(cat) and k(cat)/K-m. Conclusion: Ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of xanthine. Significance: Examining the contributions of Glu(232) to catalysis is essential for understanding the mechanism of xanthine dehydrogenase.
The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that k(red), the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both k(red) and k(red)/K-d from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pK(a) of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.
KW - Enzyme Kinetics
KW - Glutamate
KW - Glutamine
KW - Isotope Effect
KW - Ultraviolet-visible Spectroscopy (UV-visible Spectroscopy)
KW - Xanthine
KW - Xanthine Dehydrogenase
KW - Xanthine Oxidase
KW - pH Dependence
Y1 - 2014
U6 - https://doi.org/10.1074/jbc.M114.603456
SN - 0021-9258
SN - 1083-351X
VL - 289
IS - 46
SP - 32121
EP - 32130
PB - American Society for Biochemistry and Molecular Biology
CY - Bethesda
ER -
TY - JOUR
A1 - Han, Xiao Xia
A1 - Li, Junbo
A1 - Öner, Ibrahim Halil
A1 - Zhao, Bing
A1 - Leimkühler, Silke
A1 - Hildebrandt, Peter
A1 - Weidinger, Inez M.
T1 - Nickel electrodes as a cheap and versatile platform for studying structure and function of immobilized redox proteins
JF - Analytica chimica acta : an international journal devoted to all branches of analytical chemistry
N2 - Practical use of many bioelectronic and bioanalytical devices is limited by the need of expensive materials and time consuming fabrication. Here we demonstrate the use of nickel electrodes as a simple and cheap solid support material for bioelectronic applications. The naturally nanostructured electrodes showed a surprisingly high electromagnetic surface enhancement upon light illumination such that immobilization and electron transfer reactions of the model redox proteins cytochrome b(5) (Cyt b(5)) and cytochrome c (Cyt c) could be followed via surface enhanced resonance Raman spectroscopy. It could be shown that the nickel surface, when used as received, promotes a very efficient binding of the proteins upon preservation of their native structure. The immobilized redox proteins could efficiently exchange electrons with the electrode and could even act as an electron relay between the electrode and solubilized myoglobin. Our results open up new possibility for nickel electrodes as an exceptional good support for bioelectronic devices and biosensors on the one hand and for surface enhanced spectroscopic investigations on the other hand. (C) 2016 Elsevier B.V. All rights reserved.
KW - Ni electrodes
KW - Redox proteins
KW - Surface enhanced Raman spectroscopy
KW - Electron relay
KW - Biocompatibility
KW - Electron transfer
Y1 - 2016
U6 - https://doi.org/10.1016/j.aca.2016.08.053
SN - 0003-2670
SN - 1873-4324
VL - 941
SP - 35
EP - 40
PB - Elsevier
CY - Amsterdam
ER -
TY - JOUR
A1 - Hartmann, Tobias
A1 - Leimkühler, Silke
T1 - The oxygen-tolerant and NAD+-dependent formate dehydrogenase from Rhodobacter capsulatus is able to catalyze the reduction of CO2 to formate
JF - The FEBS journal
N2 - The formate dehydrogenase from Rhodobactercapsulatus (RcFDH) is an oxygen-tolerant protein with an ()(2) subunit composition that is localized in the cytoplasm. It belongs to the group of metal and NAD(+)-dependent FDHs with the coordination of a molybdenum cofactor, four [Fe4S4] clusters and one [Fe2S2] cluster associated with the -subunit, one [Fe4S4] cluster and one FMN bound to the -subunit, and one [Fe2S2] cluster bound to the -subunit. RcFDH was heterologously expressed in Escherichiacoli and characterized. Cofactor analysis showed that the bis-molybdopterin guanine dinucleotide cofactor is bound to the FdsA subunit containing a cysteine ligand at the active site. A turnover rate of 2189min(-1) with formate as substrate was determined. The back reaction for the reduction of CO2 was catalyzed with a k(cat) of 89min(-1). The preference for formate oxidation shows an energy barrier for CO2 reduction of the enzyme. Furthermore, the FMN-containing and [Fe4S4]-containing -subunit together with the [Fe2S2]-containing -subunit forms a diaphorase unit with activities for both NAD(+) reduction and NADH oxidation. In addition to the structural genes fdsG, fdsB, and fdsA, the fds operon in R.capsulatus contains the fdsC and fdsD genes. Expression studies showed that RcFDH is only active when both FdsC and FdsD are present. Both proteins are proposed to be involved in bis-molybdopterin guanine dinucleotide modification and insertion into RcFDH.
KW - FeS cluster
KW - FMN
KW - formate dehydrogenase
KW - molybdenum cofactor (Moco)-binding chaperone
KW - molybdoenzyme
Y1 - 2013
U6 - https://doi.org/10.1111/febs.12528
SN - 1742-464X
SN - 1742-4658
VL - 280
IS - 23
SP - 6083
EP - 6096
PB - Wiley-Blackwell
CY - Hoboken
ER -
TY - JOUR
A1 - Hartmann, Tobias
A1 - Schrapers, Peer
A1 - Utesch, Tillmann
A1 - Nimtz, Manfred
A1 - Rippers, Yvonne
A1 - Dau, Holger
A1 - Mroginski, Maria Andrea
A1 - Haumann, Michael
A1 - Leimkühler, Silke
T1 - The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions
JF - Biochemistry
N2 - Formate dehydrogenases (FDHs) are capable of performing the reversible oxidation of formate and are enzymes of great interest for fuel cell applications and for the production of reduced carbon compounds as energy sources from CO2. Metal containing FDHs in general contain a highly conserved active site, comprising a molybdenum (or tungsten) center coordinated by two molybdopterin guanine dinucleotide molecules, a sulfido and a (seleno-)cysteine ligand, in addition to a histidine and arginine residue in the second coordination sphere. So far, the role of these amino acids in catalysis has not been studied in detail, because of the lack of suitable expression systems and the lability or oxygen sensitivity of the enzymes. Here, the roles of these active site residues is revealed using the Mo-containing FDH from Rhodobacter capsulatus. Our results show that the cysteine ligand at the Mo ion is displaced by the formate substrate during the reaction, the arginine has a direct role in substrate binding and stabilization, and the histidine elevates the pK(a) of the active site cysteine. We further found that in addition to reversible formate oxidation, the enzyme is further capable of reducing nitrate to nitrite. We propose a mechanistic scheme that combines both functionalities and provides important insights into the distinct mechanisms of C-H bond cleavage and oxygen atom transfer catalyzed by formate dehydrogenase.
Y1 - 2016
U6 - https://doi.org/10.1021/acs.biochem.6b00002
SN - 0006-2960
VL - 55
SP - 2381
EP - 2389
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Hartmann, Tobias
A1 - Terao, Mineko
A1 - Garattini, Enrico
A1 - Teutloff, Christian
A1 - Alfaro, Joshua F.
A1 - Jones, Jeffrey P.
A1 - Leimkühler, Silke
T1 - The impact of single nucleotide polymorphisms on human aldehyde oxidase
JF - Drug metabolism and disposition : the biological fate of chemicals
N2 - Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N-1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95% and a yield of 50 mu g/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.
Y1 - 2012
U6 - https://doi.org/10.1124/dmd.111.043828
SN - 0090-9556
VL - 40
IS - 5
SP - 856
EP - 864
PB - American Society for Pharmacology and Experimental Therapeutics
CY - Bethesda
ER -
TY - JOUR
A1 - Hasnat, Muhammad Abrar
A1 - Zupok, Arkadiusz
A1 - Olas-Apelt, Justyna Jadwiga
A1 - Müller-Röber, Bernd
A1 - Leimkühler, Silke
T1 - A-type carrier proteins are involved in [4Fe-4S] cluster insertion into the radical S-adenosylmethionine protein MoaA for the synthesis of active molybdoenzymes
JF - Journal of bacteriology
N2 - Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical Sadenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression.
IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.
KW - iron-sulfur clusters
KW - Moco biosynthesis
KW - MoaA
KW - A-type carrier protein
KW - FNR
KW - nitrate reductase
KW - molybdenum cofactor
Y1 - 2021
U6 - https://doi.org/10.1128/JB.00086-21
SN - 1098-5530
VL - 203
IS - 12
PB - American Society for Microbiology
CY - Washington
ER -
TY - JOUR
A1 - Herter, Susanne
A1 - McKenna, Shane M.
A1 - Frazer, Andrew R.
A1 - Leimkühler, Silke
A1 - Carnell, Andrew J.
A1 - Turner, Nicholas J.
T1 - Galactose Oxidase Variants for the Oxidation of Amino Alcohols in Enzyme Cascade Synthesis
JF - ChemCatChem : heterogeneous & homogeneous & bio- & nano-catalysis ; a journal of ChemPubSoc Europe
N2 - The use of selected engineered galactose oxidase (GOase) variants for the oxidation of amino alcohols to aldehydes under mild conditions in aqueous systems is reported. GOase variant F-2 catalyses the regioselective oxidation of N-carbobenzyloxy (Cbz)-protected 3-amino-1,2-propanediol to the corresponding -hydroxyaldehyde which was then used in an aldolase reaction. Another variant, M3-5, was found to exhibit activity towards free and N-Cbz-protected aliphatic and aromatic amino alcohols allowing the synthesis of lactams such as 3,4-dihydronaphthalen-1(2H)-one, 2-pyrrolidone and valerolactam in one-pot tandem reactions with xanthine dehydrogenase (XDH) or aldehyde oxidase (PaoABC).
KW - aldehyde oxidase
KW - amino alcohols
KW - cascade reactions
KW - enzyme catalysis
KW - lactams
Y1 - 2015
U6 - https://doi.org/10.1002/cctc.201500218
SN - 1867-3880
SN - 1867-3899
VL - 7
IS - 15
SP - 2313
EP - 2317
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Kalimuthu, Palraj
A1 - Leimkühler, Silke
A1 - Bernhardt, Paul V.
T1 - Xanthine dehydrogenase electrocatalysis autocatalysis and novel activity
JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry
N2 - The enzyme xanthine dehydrogenase (XDH) from the purple photosynthetic bacterium Rhodobacter capsulatus catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid as part of purine metabolism. The native electron acceptor is NAD(+) but herein we show that uric acid in its 2-electron oxidized form is able to act as an artificial electron acceptor from XDH in an electrochemically driven catalytic system. Hypoxanthine oxidation is also observed with the novel production of uric acid in a series of two consecutive 2-electron oxidation reactions via xanthine. XDH exhibits native activity in terms of its pH optimum and inhibition by allopurinol.
Y1 - 2011
U6 - https://doi.org/10.1021/jp111809f
SN - 1520-6106
VL - 115
IS - 11
SP - 2655
EP - 2662
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Kalimuthu, Palraj
A1 - Leimkühler, Silke
A1 - Bernhardt, Paul V.
T1 - Catalytic electrochemistry of xanthine dehydrogenase
JF - The journal of physical chemistry : B, Condensed matter, materials, surfaces, interfaces & biophysical chemistry
N2 - We report the mediated electrocatalytic voltammetry of the molybdoenzyme xanthine dehydrogenase (XDH) from Rhodobacter capsulatus at a thiol-modified Au electrode. The 2-electron acceptor N-methylphenazinium methanesulfonate (phenazine methosulfate, PMS) is an effective artificial electron transfer partner for XDH instead of its native electron acceptor NAD(+). XDH catalyzes the oxidative hydroxylation of hypoxanthine to xanthine and xanthine to uric acid. Cyclic voltammetry was used to generate the active (oxidized) form of the mediator. Simulation of the catalytic voltammetry across a broad range of substrate and PMS concentrations at different sweep rates was achieved with the program DigiSim to yield a set of consistent rate and equilibrium constants that describe the catalytic system. This provides the first example of the mediated electrochemistry of a xanthine dehydrogenase (or oxidase) that is uncomplicated by interference from product oxidation. A remarkable two-step, sequential oxidation of hypoxanthine to uric acid via xanthine by XDH is observed.
Y1 - 2012
U6 - https://doi.org/10.1021/jp307374z
SN - 1520-6106
VL - 116
IS - 38
SP - 11600
EP - 11607
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Kaufmann, Hans Paul
A1 - Duffus, Benjamin R.
A1 - Mitrova, Biljana
A1 - Iobbi-Nivol, Chantal
A1 - Teutloff, Christian
A1 - Nimtz, Manfred
A1 - Jaensch, Lothar
A1 - Wollenberger, Ulla
A1 - Leimkühler, Silke
T1 - Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamie N-Oxide Reductase
JF - Biochemistry
N2 - The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.
Y1 - 2018
U6 - https://doi.org/10.1021/acs.biochem.7b01108
SN - 0006-2960
VL - 57
IS - 7
SP - 1130
EP - 1143
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Kaufmann, Paul
A1 - Duffus, Benjamin R.
A1 - Teutloff, Christian
A1 - Leimkühler, Silke
T1 - Functional Studies on Oligotropha carboxidovorans Molybdenum-Copper CO Dehydrogenase Produced in Escherichia coli
JF - Biochemistry
N2 - The Mo/Cu-dependent CO dehydrogenase (CODH) from Oligotropha carboxidovorans is an enzyme that is able to catalyze both the oxidation of CO to CO2 and the oxidation of H-2 to protons and electrons. Despite the close to atomic resolution structure (1.1 angstrom), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of O. carboxidovorans CODH in Escherichia coli. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H-2 oxidation of Mo/Cu-dependent CODHs in the future.
Y1 - 2018
U6 - https://doi.org/10.1021/acs.biochem.8b00128
SN - 0006-2960
VL - 57
IS - 19
SP - 2889
EP - 2901
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Kuecuekgoeze, Goekhan
A1 - Terao, Mineko
A1 - Garattini, Enrico
A1 - Leimkühler, Silke
T1 - Direct Comparison of the Enzymatic Characteristics and Superoxide Production of the Four Aldehyde Oxidase Enzymes Present in Mouse
JF - Drug metabolism and disposition : the biological fate of chemicals
N2 - Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4, and mAOX2, which are the products of distinct genes, are present in the mouse. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes has never been performed. In this report, the four catalytically active mAOX enzymes were purified after heterologous expression in Escherichia coli. The kinetic parameters of the four mouse AOX enzymes were determined and compared with the use of six predicted substrates of physiologic and toxicological interest, i.e., retinaldehyde, N1-methylnicotinamide, pyridoxal, vanillin, 4-(dimethylamino) cinnamaldehyde (p-DMAC), and salicylaldehyde. While retinaldehyde, vanillin, p-DMAC, and salycilaldehyde are efficient substrates for the four mouse AOX enzymes, N1-methylnicotinamide is not a substrate of mAOX1 or mAOX4, and pyridoxal is notmetabolized by any of the purified enzymes. Overall, mAOX1, mAOX2, mAOX3, and mAOX4 are characterized by significantly different KM and kcat values for the active substrates. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40% in relation to moles of substrate converted, and mAOX1, the homolog to the human enzyme, produces a rate of approximately 30% of superoxide radicals with the same substrate.
Y1 - 2017
U6 - https://doi.org/10.1124/dmd.117.075937
SN - 0090-9556
SN - 1521-009X
VL - 45
SP - 947
EP - 955
PB - American Society for Pharmacology and Experimental Therapeutics
CY - Bethesda
ER -
TY - JOUR
A1 - Küçükgöze, Gökhan
A1 - Leimkühler, Silke
T1 - Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities
JF - PLOS ONE
N2 - Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. In mouse, the isoforms mAOX1, mAOX3, mAOX4 and mAOX2 are present. We previously established a codon-optimized heterologous expression systems for the mAOX1-4 isoforms in Escherichia coli that gives yield to sufficient amounts of active protein for kinetic characterizations and sets the basis in this study for site-directed mutagenesis and structure-function studies. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes on a larger number of substrates has never been performed. Here, thirty different structurally related aromatic, aliphatic and N-heterocyclic compounds were used as substrates, and the kinetic parameters of all four mAOX enzymes were directly compared. The results show that especially mAOX4 displays a higher substrate selectivity, while no major differences between mAOX1, mAOX2 and mAOX3 were identified. Generally, mAOX1 was the enzyme with the highest catalytic turnover for most substrates. To understand the factors that contribute to the substrate specificity of mAOX4, site-directed mutagenesis was applied to substitute amino acids in the substrate-binding funnel by the ones present in mAOX1, mAOX3, and mAOX2. An increase in activity was obtained by the amino acid exchange M1088V in the active site identified to be specific for mAOX4, to the amino acid identified in mAOX3.
Y1 - 2018
U6 - https://doi.org/10.1371/journal.pone.0191819
SN - 1932-6203
VL - 13
IS - 1
PB - Public Library of Science
CY - San Fransisco
ER -
TY - JOUR
A1 - Laun, Konstantin
A1 - Duffus, Benjamin R.
A1 - Wahlefeld, Stefan
A1 - Katz, Sagie
A1 - Belger, Dennis Heinz
A1 - Hildebrandt, Peter
A1 - Mroginski, Maria Andrea
A1 - Leimkühler, Silke
A1 - Zebger, Ingo
T1 - Infrared spectroscopy flucidates the inhibitor binding sites in a metal-dependent formate dehydrogenase
JF - Chemistry - a European journal
N2 - Biological carbon dioxide (CO2) reduction is an important step by which organisms form valuable energy-richer molecules required for further metabolic processes. The Mo-dependent formate dehydrogenase (FDH) from Rhodobacter capsulatus catalyzes reversible formate oxidation to CO2 at a bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor. To elucidate potential substrate binding sites relevant for the mechanism, we studied herein the interaction with the inhibitory molecules azide and cyanate, which are isoelectronic to CO2 and charged as formate. We employed infrared (IR) spectroscopy in combination with density functional theory (DFT) and inhibition kinetics. One distinct inhibitory molecule was found to bind to either a non-competitive or a competitive binding site in the secondary coordination sphere of the active site. Site-directed mutagenesis of key amino acid residues in the vicinity of the bis-MGD cofactor revealed changes in both non-competitive and competitive binding, whereby the inhibitor is in case of the latter interaction presumably bound between the cofactor and the adjacent Arg587.
KW - CO2 reduction
KW - DFT
KW - formate oxidation
KW - inhibition kinetics
KW - IR
KW - spectroscopy
KW - molybdoenzyme
Y1 - 2022
U6 - https://doi.org/10.1002/chem.202201091
SN - 0947-6539
SN - 1521-3765
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Leimkühler, Silke
T1 - Transition metals in catalysis
BT - the functional relationship of Fe-S clusters and molybdenum or tungsten cofactor-containing enzyme systems
JF - Inorganics : open access journal
Y1 - 2021
U6 - https://doi.org/10.3390/inorganics9010006
SN - 2304-6740
VL - 9
IS - 1
PB - MDPI
CY - Basel
ER -
TY - JOUR
A1 - Leimkühler, Silke
A1 - Charcosset, M.
A1 - Latour, P.
A1 - Dorche, C.
A1 - Kleppe, S.
A1 - Scaglia, F.
A1 - Szymczak, I.
A1 - Schupp, P.
A1 - Hahnewald, Rita
A1 - Reiss, J.
T1 - Ten novel mutations in the molybdenum cofactor genes MOCS1 and MOCS2 and in vitro characterization of a MOCS2 mutation that abolishes the binding ability of molybdopterin synthase
N2 - Molybdenum cofactor deficiency (MIM#252150) is a severe autosomal- recessive disorder with a devastating outcome. The cofactor is the product of a complex biosynthetic pathway involving four different genes (MOCS1, MOCS2, MOCS3 and GEPH). This disorder is caused almost exclusively by mutations in the MOCS1 or MOCS2 genes. Mutations affecting this biosynthetic pathway result in a lethal phenotype manifested by progressive neurological damage via the inactivation of the molybdenum cofactor-dependent enzyme, sulphite oxidase. Here we describe a total of ten novel disease-causing mutations in the MOCS1 and MOCS2 genes. Nine out of these ten mutations were classified as pathogenic in nature, since they create a stop codon, affect constitutive splice site positions, or change strictly conserved motifs. The tenth mutation abolishes the stop codon of the MOCS2B gene, thus elongating the corresponding protein. The mutation was expressed in vitro and was found to abolish the binding affinities of the large subunit of molybdopterin synthase (MOCS2B) for both precursor Z and the small subunit of molybdopterin synthase (MOCS2A)
Y1 - 2005
SN - 0340-6717
ER -
TY - JOUR
A1 - Leimkühler, Silke
A1 - Lemaire, Olivier N.
A1 - Iobbi-Nivol, Chantal
T1 - Bacterial Molybdoenzymes
BT - Chaperones, Assembly and Insertion
JF - Molybdenum and tungsten enzymes : biochemistry
N2 - The biogenesis of molybdoenzymes is a cytoplasmic event requiring both the folded apoenzymes and the matured molybdenum cofactor. The structure and the complexity of the molybdenum cofactor varies in each molybdoenzyme family and consequently different accessory proteins are required for the maturation of the respective enzymes. Thus, for enzymes of both the DMSO reductase and xanthine oxidase families, specific chaperones exist which are dedicated to increase the stability and the folding of specific members of each family. In this review, we describe the role of these chaperones for molybdoenzyme maturation. We present a model which describes step by step the mechanism of the maturation of representative molybdoenzymes from each family.
Y1 - 2016
SN - 978-1-78262-391-5
SN - 978-1-78262-089-1
U6 - https://doi.org/10.1039/9781782623915-00117
VL - 5
SP - 117
EP - 142
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Leimkühler, Silke
A1 - Mendel, Ralf-Rainer
T1 - Molybdenum Cofactor Biosynthesis
JF - Molybdenum and tungsten enzymes: biochemistry
N2 - The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes with the exception of nitrogenase, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into three steps in eukaryotes, and four steps in bacteria and archaea: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5′GTP, (ii) in the second step the two sulfur molecules are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into molybdopterin to form Moco and (iv) additional modification of Moco occurs in bacteria and archaea with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review will focus on the biosynthesis of Moco in bacteria, humans and plants.
Y1 - 2016
SN - 978-1-78262-391-5
SN - 978-1-78262-089-1
SN - 978-1-78262-881-1
U6 - https://doi.org/10.1039/9781782623915
VL - 5
SP - 100
EP - 116
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Lemaire, Olivier N.
A1 - Honore, Flora A.
A1 - Tempel, Sebastien
A1 - Fortier, Emma M.
A1 - Leimkühler, Silke
A1 - Mejean, Vincent
A1 - Iobbi-Nivol, Chantal
T1 - Shewanella decolorationis LDS1 Chromate Resistance
JF - Applied and environmental microbiology
N2 - The genus Shewanella is well known for its genetic diversity, its outstanding respiratory capacity, and its high potential for bioremediation. Here, a novel strain isolated from sediments of the Indian Ocean was characterized. A 16S rRNA analysis indicated that it belongs to the species Shewanella decolorationis. It was named Shewanella decolorationis LDS1. This strain presented an unusual ability to grow efficiently at temperatures from 24 degrees C to 40 degrees C without apparent modifications of its metabolism, as shown by testing respiratory activities or carbon assimilation, and in a wide range of salt concentrations. Moreover, S. decolorationis LDS1 tolerates high chromate concentrations. Indeed, it was able to grow in the presence of 4 mM chromate at 28 degrees C and 3 mM chromate at 40 degrees C. Interestingly, whatever the temperature, when the culture reached the stationary phase, the strain reduced the chromate present in the growth medium. In addition, S. decolorationis LDS1 degrades different toxic dyes, including anthraquinone, triarylmethane, and azo dyes. Thus, compared to Shewanella oneidensis, this strain presented better capacity to cope with various abiotic stresses, particularly at high temperatures. The analysis of genome sequence preliminary data indicated that, in contrast to S. oneidensis and S. decolorationis S12, S. decolorationis LDS1 possesses the phosphorothioate modification machinery that has been described as participating in survival against various abiotic stresses by protecting DNA. We demonstrate that its heterologous production in S. oneidensis allows it to resist higher concentrations of chromate. IMPORTANCE Shewanella species have long been described as interesting microorganisms in regard to their ability to reduce many organic and inorganic compounds, including metals. However, members of the Shewanella genus are often depicted as cold-water microorganisms, although their optimal growth temperature usually ranges from 25 to 28 degrees C under laboratory growth conditions. Shewanella decolorationis LDS1 is highly attractive, since its metabolism allows it to develop efficiently at temperatures from 24 to 40 degrees C, conserving its ability to respire alternative substrates and to reduce toxic compounds such as chromate or toxic dyes. Our results clearly indicate that this novel strain has the potential to be a powerful tool for bioremediation and unveil one of the mechanisms involved in its chromate resistance.
KW - Shewanella
KW - bioremediation
KW - chromium
KW - decolorization
KW - dndBCDE
KW - dyes
KW - temperature
Y1 - 2019
U6 - https://doi.org/10.1128/AEM.00777-19
SN - 0099-2240
SN - 1098-5336
VL - 85
IS - 18
PB - American Society for Microbiology
CY - Washington
ER -
TY - JOUR
A1 - Lim, Sze Chern
A1 - Friemel, Martin
A1 - Marum, Justine E.
A1 - Tucker, Elena J.
A1 - Bruno, Damien L.
A1 - Riley, Lisa G.
A1 - Christodoulou, John
A1 - Kirk, Edwin P.
A1 - Boneh, Avihu
A1 - DeGennaro, Christine M.
A1 - Springer, Michael
A1 - Mootha, Vamsi K.
A1 - Rouault, Tracey A.
A1 - Leimkühler, Silke
A1 - Thorburn, David R.
A1 - Compton, Alison G.
T1 - Mutations in LYRM4, encoding ironsulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes
JF - Human molecular genetics
N2 - Ironsulfur clusters (ISCs) are important prosthetic groups that define the functions of many proteins. Proteins with ISCs (called ironsulfur or FeS proteins) are present in mitochondria, the cytosol, the endoplasmic reticulum and the nucleus. They participate in various biological pathways including oxidative phosphorylation (OXPHOS), the citric acid cycle, iron homeostasis, heme biosynthesis and DNA repair. Here, we report a homozygous mutation in LYRM4 in two patients with combined OXPHOS deficiency. LYRM4 encodes the ISD11 protein, which forms a complex with, and stabilizes, the sulfur donor NFS1. The homozygous mutation (c.203GT, p.R68L) was identified via massively parallel sequencing of 1000 mitochondrial genes (MitoExome sequencing) in a patient with deficiency of complexes I, II and III in muscle and liver. These three complexes contain ISCs. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died while a neonate. Complex IV was also deficient in her skeletal muscle. Several other FeS proteins were also affected in both patients, including the aconitases and ferrochelatase. Mutant ISD11 only partially complemented for an ISD11 deletion in yeast. Our in vitro studies showed that the l-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. Our findings are consistent with a defect in the early step of ISC assembly affecting a broad variety of FeS proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.
Y1 - 2013
U6 - https://doi.org/10.1093/hmg/ddt295
SN - 0964-6906
SN - 1460-2083
VL - 22
IS - 22
SP - 4460
EP - 4473
PB - Oxford Univ. Press
CY - Oxford
ER -
TY - JOUR
A1 - Mahro, Martin
A1 - Bras, Natercia F.
A1 - Cerqueira, Nuno M. F. S. A.
A1 - Teutloff, Christian
A1 - Coelho, Catarina
A1 - Romao, Maria Joao
A1 - Leimkühler, Silke
T1 - Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity
JF - PLoS one
N2 - In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.
Y1 - 2013
U6 - https://doi.org/10.1371/journal.pone.0082285
SN - 1932-6203
VL - 8
IS - 12
PB - PLoS
CY - San Fransisco
ER -
TY - JOUR
A1 - Mahro, Martin
A1 - Coelho, Catarina
A1 - Trincao, Jose
A1 - Rodrigues, David
A1 - Terao, Mineko
A1 - Garattini, Enrico
A1 - Saggu, Miguel
A1 - Lendzian, Friedhelm
A1 - Hildebrandt, Peter
A1 - Romao, Maria Joao
A1 - Leimkühler, Silke
T1 - Characterization and crystallization of mouse aldehyde oxidase 3 - from mouse liver to escherichia coli heterologous protein expression
JF - Drug metabolism and disposition : the biological fate of chemicals
N2 - Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N(1)-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the metabolism of drugs through its complex cofactor content, tissue distribution, and substrate recognition. In humans, only one AOX gene (AOX1) is present, but in mouse and other mammals different AOX homologs were identified. The multiple AOX isoforms are expressed tissue-specifically in different organisms, and it is believed that they recognize distinct substrates and carry out different physiological tasks. AOX is a dimer with a molecular mass of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofactor, two distinct [2Fe-2S] clusters, and one FAD. We purified the AOX homolog from mouse liver (mAOX3) and established a system for the heterologous expression of mAOX3 in Escherichia coli. The purified enzymes were compared. Both proteins show the same characteristics and catalytic properties, with the difference that the recombinant protein was expressed and purified in a 30% active form, whereas the native protein is 100% active. Spectroscopic characterization showed that FeSII is not assembled completely in mAOX3. In addition, both proteins were crystallized. The best crystals were from native mAOX3 and diffracted beyond 2.9 angstrom. The crystals belong to space group P1, and two dimers are present in the unit cell.
Y1 - 2011
U6 - https://doi.org/10.1124/dmd.111.040873
SN - 0090-9556
VL - 39
IS - 10
SP - 1939
EP - 1945
PB - American Society for Pharmacology and Experimental Therapeutics
CY - Bethesda
ER -
TY - JOUR
A1 - Marelja, Zvonimir
A1 - Chowdhury, Mita Mullick
A1 - Dosche, Carsten
A1 - Hille, Carsten
A1 - Baumann, Otto
A1 - Löhmannsröben, Hans-Gerd
A1 - Leimkühler, Silke
T1 - The L-cysteine desulfurase NFS1 is localized in the cytosol where it provides the sulfur for molybdenum cofactor biosynthesis in humans
JF - PLoS one
N2 - In humans, the L-cysteine desulfurase NFS1 plays a crucial role in the mitochondrial iron-sulfur cluster biosynthesis and in the thiomodification of mitochondrial and cytosolic tRNAs. We have previously demonstrated that purified NFS1 is able to transfer sulfur to the C-terminal domain of MOCS3, a cytosolic protein involved in molybdenum cofactor biosynthesis and tRNA thiolation. However, no direct evidence existed so far for the interaction of NFS1 and MOCS3 in the cytosol of human cells. Here, we present direct data to show the interaction of NFS1 and MOCS3 in the cytosol of human cells using Forster resonance energy transfer and a split-EGFP system. The colocalization of NFS1 and MOCS3 in the cytosol was confirmed by immunodetection of fractionated cells and localization studies using confocal fluorescence microscopy. Purified NFS1 was used to reconstitute the lacking molybdoenzyme activity of the Neurospora crassa nit-1 mutant, giving additional evidence that NFS1 is the sulfur donor for Moco biosynthesis in eukaryotes in general.
Y1 - 2013
U6 - https://doi.org/10.1371/journal.pone.0060869
SN - 1932-6203
VL - 8
IS - 4
PB - PLoS
CY - San Fransisco
ER -
TY - JOUR
A1 - Marelja, Zvonimir
A1 - Dambowsky, Miriam
A1 - Bolis, Marco
A1 - Georgiou, Marina L.
A1 - Garattini, Enrico
A1 - Missirlis, Fanis
A1 - Leimkühler, Silke
T1 - The four aldehyde oxidases of Drosophila melanogaster have different gene expression patterns and enzyme substrate specificities
JF - The journal of experimental biology
N2 - In the genome of Drosophila melanogaster, four genes coding for aldehyde oxidases (AOX1-4) were identified on chromosome 3. Phylogenetic analysis showed that the AOX gene cluster evolved via independent duplication events in the vertebrate and invertebrate lineages. The functional role and the substrate specificity of the distinct Drosophila AOX enzymes is unknown. Two loss-of-function mutant alleles in this gene region, low pyridoxal oxidase (Po-lpo) and aldehyde oxidase-1 (Aldox-1(n1)) are associated with a phenotype characterized by undetectable AOX enzymatic activity. However, the genes involved and the corresponding mutations have not yet been identified. In this study we characterized the activities, substrate specificities and expression profiles of the four AOX enzymes in D. melanogaster. We show that the Po-lpo-associated phenotype is the consequence of a structural alteration of the AOX1 gene. We identified an 11-bp deletion in the Po-lpo allele, resulting in a frame-shift event, which removes the molybdenum cofactor domain of the encoded enzyme. Furthermore, we show that AOX2 activity is detectable only during metamorphosis and characterize a Minos-AOX2 insertion in this developmental gene that disrupts its activity. We demonstrate that the Aldox-1(n1) phenotype maps to the AOX3 gene and AOX4 activity is not detectable in our assays.
KW - Aldehyde oxidase
KW - Molybdoenzymes
KW - Drosophila melanogaster
KW - Gene duplication
KW - Substrate specificities
Y1 - 2014
U6 - https://doi.org/10.1242/jeb.102129
SN - 0022-0949
SN - 1477-9145
VL - 217
IS - 12
SP - 2201
EP - 2211
PB - Company of Biologists Limited
CY - Cambridge
ER -
TY - JOUR
A1 - Matthies, A.
A1 - Nimtz, M.
A1 - Leimkühler, Silke
T1 - Molybdenum cofactor biosynthesis in humans : Identification of a persulfide group in the rhodanese-like domain of MOCS3 by mass spectrometry
N2 - The human MOCS3 protein contains an N-terminal domain similar to the Escherichia coli MoeB protein and a C- terminal segment displaying similarities to the sulfurtransferase rhodanese. MOCS3 is proposed to catalyze both the adenylation and the subsequent generation of a thiocarboxylate group at the C-terminus of the smaller subunit of molybdopterin (MPT) synthase during Moco biosynthesis in humans. Recent studies have shown that the MOCS3 rhodanese-like domain (MOCS3-RLD) catalyzes the transfer of sulfur from thiosulfate to cyanide and is also able to provide the sulfur for the thiocarboxylation of MOCS2A in a defined in vitro system for the generation of MPT from precursor Z. MOCS3-RLD contains four cysteine residues of which only C412 in the six amino acid active loop is conserved in homologous proteins from other organisms. ESI-MS/MS studies gave direct evidence for the formation of a persulfide group that is exclusively formed on C412. Simultaneous mutagenesis of the remaining three cysteine residues showed that none of them is involved in the sulfur transfer reaction in vitro. A disulfide bridge was identified to be formed between C316 and C324, and possible roles of the three noncatalytic cysteine residues are discussed. By ESI-MS/MS a partially gluconoylated N- terminus of the His(6)-tagged MOCS3-RLD was identified (mass increment of 178 Da) which resulted in a heterogeneity of the protein but did not influence sulfurtransferase activity
Y1 - 2005
SN - 0006-2960
ER -
TY - JOUR
A1 - McKenna, Shane M.
A1 - Leimkühler, Silke
A1 - Herter, Susanne
A1 - Turner, Nicholas J.
A1 - Carnell, Andrew J.
T1 - Enzyme cascade reactions: synthesis of furandicarboxylic acid (FDCA) and carboxylic acids using oxidases in tandem
JF - Green chemistry : an international journal and green chemistry resource
N2 - A one-pot tandem enzyme reaction using galactose oxidase M3-5 and aldehyde oxidase PaoABC was used to convert hydroxymethylfurfural (HMF) to the pure bioplastics precursor FDCA in 74% isolated yield. A range of alcohols was also converted to carboxylic acids in high yield under mild conditions.
Y1 - 2015
U6 - https://doi.org/10.1039/c5gc00707k
SN - 1463-9262
SN - 1463-9270
VL - 17
IS - 6
SP - 3271
EP - 3275
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Mendel, Ralf R.
A1 - Hercher, Thomas W.
A1 - Zupok, Arkadiusz
A1 - Hasnat, Muhammad Abrar
A1 - Leimkühler, Silke
T1 - The requirement of inorganic Fe-S clusters for the biosynthesis of the organometallic molybdenum cofactor
JF - Inorganics : open access journal
N2 - Iron-sulfur (Fe-S) clusters are essential protein cofactors. In enzymes, they are present either in the rhombic [2Fe-2S] or the cubic [4Fe-4S] form, where they are involved in catalysis and electron transfer and in the biosynthesis of metal-containing prosthetic groups like the molybdenum cofactor (Moco). Here, we give an overview of the assembly of Fe-S clusters in bacteria and humans and present their connection to the Moco biosynthesis pathway. In all organisms, Fe-S cluster assembly starts with the abstraction of sulfur froml-cysteine and its transfer to a scaffold protein. After formation, Fe-S clusters are transferred to carrier proteins that insert them into recipient apo-proteins. In eukaryotes like humans and plants, Fe-S cluster assembly takes place both in mitochondria and in the cytosol. Both Moco biosynthesis and Fe-S cluster assembly are highly conserved among all kingdoms of life. Moco is a tricyclic pterin compound with molybdenum coordinated through its unique dithiolene group. Moco biosynthesis begins in the mitochondria in a Fe-S cluster dependent step involving radical/S-adenosylmethionine (SAM) chemistry. An intermediate is transferred to the cytosol where the dithiolene group is formed, to which molybdenum is finally added. Further connections between Fe-S cluster assembly and Moco biosynthesis are discussed in detail.
KW - Moco biosynthesis
KW - Fe-S cluster assembly
KW - l-cysteine desulfurase
KW - ISC
KW - SUF
KW - NIF
KW - iron
KW - molybdenum
KW - sulfur
Y1 - 2020
U6 - https://doi.org/10.3390/inorganics8070043
SN - 2304-6740
VL - 8
IS - 7
PB - MDPI
CY - Basel
ER -
TY - JOUR
A1 - Mitrova, Biljana
A1 - Tadjoung Waffo, Armel Franklin
A1 - Kaufmann, Paul
A1 - Iobbi-Nivol, Chantal
A1 - Leimkühler, Silke
A1 - Wollenberger, Ulla
T1 - Trimethylamine N-Oxide Electrochemical Biosensor with a Chimeric Enzyme
JF - ChemElectroChem
N2 - For the first time, an enzyme-based electrochemical biosensor system for determination of trimethylamine N-oxide (TMAO) is described. It employs an active chimeric variant of TorA in combination with an enzymatically deoxygenating system and a low-potential mediator for effective regeneration of the enzyme and cathodic current generation. TMAO reductase (TorA) is a molybdoenzyme found in marine and most enterobacteria that specifically catalyzes the reduction of TMAO to trimethylamine (TMA). The chimeric TorA, named TorA-FDH, corresponds to the apoform of TorA from Escherichia coli reconstituted with the molybdenum cofactor from formate dehydrogenase (FDH). Each enzyme, TorA and TorA-FDH, was immobilized on the surface of a carbon electrode and protected with a dialysis membrane. The biosensor operates at an applied potential of -0.8V [vs. Ag/AgCl (1M KCl)] under ambient air conditions thanks to an additional enzymatic O-2-scavenger system. A comparison between the two enzymatic sensors revealed a much higher sensitivity for the biosensor with immobilized TorA-FDH. This biosensor exhibits a sensitivity of 14.16nA/M TMAO in a useful measuring range of 2-110M with a detection limit of LOD=2.96nM (S/N=3), and was similar for TMAO in buffer and in spiked serum samples. With a response time of 16 +/- 2 s, the biosensor is stable over prolonged daily measurements (n=20). This electrochemical biosensor provides suitable applications in detecting TMAO levels in human serum.
KW - trimethylamine N-oxide (TMAO)
KW - TMAO reductase
KW - chimeric enzyme
KW - molybdoenzyme
KW - electrochemical biosensor
Y1 - 2018
U6 - https://doi.org/10.1002/celc.201801422
SN - 2196-0216
VL - 6
IS - 6
SP - 1732
EP - 1737
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Mota, Cristiano
A1 - Esmaeeli Moghaddam Tabalvandani, Mariam
A1 - Coelho, Catarina
A1 - Santos-Silva, Teresa
A1 - Wolff, Martin
A1 - Foti, Alessandro
A1 - Leimkühler, Silke
A1 - Romao, Maria Joao
T1 - Human aldehyde oxidase (hAOX1)
BT - structure determination of the Moco-free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms
JF - FEBS Open Bio
N2 - Human aldehyde oxidase (hAOX1) is a molybdenum enzyme with high toxicological importance, but its physiological role is still unknown. hAOX1 metabolizes different classes of xenobiotics and is one of the main drug-metabolizing enzymes in the liver, along with cytochrome P450. hAOX1 oxidizes and inactivates a large number of drug molecules and has been responsible for the failure of several phase I clinical trials. The interindividual variability of drug-metabolizing enzymes caused by single nucleotide polymorphisms (SNPs) is highly relevant in pharmaceutical treatments. In this study, we present the crystal structure of the inactive variant G1269R, revealing the first structure of a molybdenum cofactor (Moco)-free form of hAOX1. These data allowed to model, for the first time, the flexible Gate 1 that controls access to the active site. Furthermore, we inspected the thermostability of wild-type hAOX1 and hAOX1 with various SNPs (L438V, R1231H, G1269R or S1271L) by CD spectroscopy and ThermoFAD, revealing that amino acid exchanges close to the Moco site can impact protein stability up to 10 degrees C. These results correlated with biochemical and structural data and enhance our understanding of hAOX1 and the effect of SNPs in the gene encoding this enzyme in the human population. EnzymesAldehyde oxidase (); xanthine dehydrogenase (); xanthine oxidase (). DatabasesStructural data are available in the Protein Data Bank under the accession number .
KW - human aldehyde oxidase
KW - molybdenum cofactor
KW - single nucleotide polymorphism
KW - xanthine oxidase
Y1 - 2019
U6 - https://doi.org/10.1002/2211-5463.12617
SN - 2211-5463
VL - 9
IS - 5
SP - 925
EP - 934
PB - Wiley
CY - Hoboken
ER -
TY - JOUR
A1 - Neukranz, Yannika
A1 - Kotter, Annika
A1 - Beilschmidt, Lena
A1 - Marelja, Zvonimir
A1 - Helm, Mark
A1 - Graf, Ralph
A1 - Leimkühler, Silke
T1 - Analysis of the Cellular Roles of MOCS3 Identifies a MOCS3-Independent Localization of NFS1 at the Tips of the Centrosome
JF - Biochemistry
N2 - The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the L-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm(5)s(2) U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm(5)s(2)U thio-modified tRNAs were not detectable. Because the L-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.
Y1 - 2019
U6 - https://doi.org/10.1021/acs.biochem.8b01160
SN - 0006-2960
VL - 58
IS - 13
SP - 1786
EP - 1798
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Neumann, Meina
A1 - Mittelstaedt, Gerd
A1 - Iobbi-Nivol, Chantal
A1 - Saggu, Miguel
A1 - Lendzian, Friedhelm
A1 - Hildebrandt, Peter
A1 - Leimkühler, Silke
T1 - A periplasmic aldehyde oxidoreductase represents the first molybdopterin cytosine dinucleotide cofactor containing molybdo-flavoenzyme from Escherichia coli
N2 - Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (alpha beta gamma) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 x [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD(+) or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions.
Y1 - 2009
UR - http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=1742-464X
U6 - https://doi.org/10.1111/j.1742-4658.2009.07000.x
SN - 1742-464X
ER -
TY - JOUR
A1 - Neumann, Meina
A1 - Mittelstaedt, Gerd
A1 - Seduk, Farida
A1 - Iobbi-Nivol, Chantal
A1 - Leimkühler, Silke
T1 - MocA is a specific cytidylyltransferase involved in molybdopterin cytosine dinucleotide biosynthesis in Escherichia coli
N2 - We have purified and characterized a specific CTP: molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP: molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase Yag-TSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 +/- 0.01 min(-1) was obtained. A1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 +/- 0.02 mu M for CTP and 1.17 +/- 0.18 mu M for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria.
Y1 - 2009
UR - http://www.jbc.org/
U6 - https://doi.org/10.1074/jbc.M109.008565
SN - 0021-9258
ER -
TY - JOUR
A1 - Neumann, Meina
A1 - Schulte, Marc
A1 - Jünemann, Nora
A1 - Stöcklein, Walter F. M.
A1 - Leimkühler, Silke
T1 - Rhodobacter capsulatus XdhC is involved in molybdenum cofactor binding and insertion into xanthine dehydrogenase
N2 - Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (alpha beta) 2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes
Y1 - 2006
UR - http://www.jbc.org/
U6 - https://doi.org/10.1074/jbc.M601617200
ER -
TY - JOUR
A1 - Neumann, Meina
A1 - Seduk, Farida
A1 - Iobbi-Nivol, Chantal
A1 - Leimkühler, Silke
T1 - Molybdopterin Dinucleotide Biosynthesis in Escherichia coli identification of amino acid residues of molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides
JF - The journal of biological chemistry
N2 - The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP: molybdopterin guanylyltransferase MobA and the CTP: molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein.
Y1 - 2011
U6 - https://doi.org/10.1074/jbc.M110.155671
SN - 0021-9258
VL - 286
IS - 2
SP - 1400
EP - 1408
PB - American Society for Biochemistry and Molecular Biology
CY - Bethesda
ER -
TY - JOUR
A1 - Nishino, Takeshi
A1 - Okamoto, Ken
A1 - Leimkühler, Silke
T1 - Enzymes of the Xanthine Oxidase Family
JF - Molybdenum and tungsten enzymes : biochemistry
N2 - Enzymes from the xanthine oxidase (XO) family of molybdenum enzymes are generally, with some exceptions, molybdenum iron–sulfur flavin hydroxylases. Mammalian xanthine oxidoreductase and aldehyde oxidase were among the first enzymes to be studied in detail more than 100 years ago and, surprisingly, they continue to be thoroughly studied in molecular detail with many open and unresolved questions remaining. Enzymes of the XO family are characterized by a molybdenum cofactor (Moco) active site with a MoVIOS(OH) ligand sphere where substrate hydroxylation of either aromatic or aliphatic carbon centers is catalyzed. During the reaction, electrons are transferred to the oxidizing substrate, most commonly O2 or NAD+, which react at the FAD site.
Y1 - 2016
SN - 978-1-78262-391-5
SN - 978-1-78262-089-1
SN - 978-1-78262-881-1
U6 - https://doi.org/10.1039/9781782623915-00192
VL - 5
SP - 192
EP - 239
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Ogunkola, Moses Olalekan
A1 - Guiraudie-Capraz, Gaelle
A1 - Féron, François
A1 - Leimkühler, Silke
T1 - The Human Mercaptopyruvate Sulfurtransferase TUM1 Is Involved in Moco Biosynthesis, Cytosolic tRNA Thiolation and Cellular Bioenergetics in Human Embryonic Kidney Cells
JF - Biomolecules
N2 - Sulfur is an important element that is incorporated into many biomolecules in humans. The incorporation and transfer of sulfur into biomolecules is, however, facilitated by a series of different sulfurtransferases. Among these sulfurtransferases is the human mercaptopyruvate sulfurtransferase (MPST) also designated as tRNA thiouridine modification protein (TUM1). The role of the human TUM1 protein has been suggested in a wide range of physiological processes in the cell among which are but not limited to involvement in Molybdenum cofactor (Moco) biosynthesis, cytosolic tRNA thiolation and generation of H2S as signaling molecule both in mitochondria and the cytosol. Previous interaction studies showed that TUM1 interacts with the L-cysteine desulfurase NFS1 and the Molybdenum cofactor biosynthesis protein 3 (MOCS3). Here, we show the roles of TUM1 in human cells using CRISPR/Cas9 genetically modified Human Embryonic Kidney cells. Here, we show that TUM1 is involved in the sulfur transfer for Molybdenum cofactor synthesis and tRNA thiomodification by spectrophotometric measurement of the activity of sulfite oxidase and liquid chromatography quantification of the level of sulfur-modified tRNA. Further, we show that TUM1 has a role in hydrogen sulfide production and cellular bioenergetics.
KW - Moco biosynthesis
KW - sulfite oxidase
KW - cytosolic tRNA thiolation
KW - 5-methoxycarbonylmethyl-2-thiouridine
KW - H2S biosynthesis
KW - cellular bioenergetics
Y1 - 2023
U6 - https://doi.org/10.3390/biom13010144
SN - 2218-273X
VL - 13
SP - 1
EP - 23
PB - MDPI
CY - Basel, Schweiz
ET - 1
ER -
TY - JOUR
A1 - Otrelo-Cardoso, Ana Rita
A1 - da Silva Correia, Marcia Alexandra
A1 - Schwuchow, Viola
A1 - Svergun, Dmitri I.
A1 - Romao, Maria Joao
A1 - Leimkühler, Silke
A1 - Santos-Silva, Teresa
T1 - Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis
JF - International journal of molecular sciences
N2 - The periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli is a molybdenum enzyme involved in detoxification of aldehydes in the cell. It is an example of an heterotrimeric enzyme of the xanthine oxidase family of enzymes which does not dimerize via its molybdenum cofactor binding domain. In order to structurally characterize PaoABC, X-ray crystallography and small angle X-ray scattering (SAXS) have been carried out. The protein crystallizes in the presence of 20% (w/v) polyethylene glycol 3350 using the hanging-drop vapour diffusion method. Although crystals were initially twinned, several experiments were done to overcome twinning and lowering the crystallization temperature (293 K to 277 K) was the solution to the problem. The non-twinned crystals used to solve the structure diffract X-rays to beyond 1.80 angstrom and belong to the C2 space group, with cell parameters a = 109.42 angstrom, b = 78.08 angstrom, c = 151.77 angstrom, = 99.77 degrees, and one molecule in the asymmetric unit. A molecular replacement solution was found for each subunit separately, using several proteins as search models. SAXS data of PaoABC were also collected showing that, in solution, the protein is also an heterotrimer.
KW - periplasmic aldehyde oxidoreductase
KW - X-ray crystallography
KW - small angle X-ray scattering
KW - crystal twinning
Y1 - 2014
U6 - https://doi.org/10.3390/ijms15022223
SN - 1422-0067
VL - 15
IS - 2
SP - 2223
EP - 2236
PB - MDPI
CY - Basel
ER -
TY - JOUR
A1 - Otrelo-Cardoso, Ana Rita
A1 - Schwuchow, Viola
A1 - Rodrigues, David
A1 - Cabrita, Eurico J.
A1 - Leimkühler, Silke
A1 - Romao, Maria Joao
A1 - Santos-Silva, Teresa
T1 - Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes
JF - PLoS one
N2 - Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C(4)mim]Cl and [C(2)OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference - nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.
Y1 - 2014
U6 - https://doi.org/10.1371/journal.pone.0087295
SN - 1932-6203
VL - 9
IS - 1
PB - PLoS
CY - San Fransisco
ER -
TY - JOUR
A1 - Otto, Nils
A1 - Marelja, Zvonimir
A1 - Schoofs, Andreas
A1 - Kranenburg, Holger
A1 - Bittern, Jonas
A1 - Yildirim, Kerem
A1 - Berh, Dimitri
A1 - Bethke, Maria
A1 - Thomas, Silke
A1 - Rode, Sandra
A1 - Risse, Benjamin
A1 - Jiang, Xiaoyi
A1 - Pankratz, Michael
A1 - Leimkühler, Silke
A1 - Klämbt, Christian
T1 - The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia
JF - Nature Communications
N2 - Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, we screened for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, our findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity.
Y1 - 2018
U6 - https://doi.org/10.1038/s41467-018-05645-z
SN - 2041-1723
VL - 9
PB - Nature Publ. Group
CY - London
ER -
TY - JOUR
A1 - Paragas, Erickson M.
A1 - Humphreys, Sara C.
A1 - Min, Joshua
A1 - Joswig-Jones, Carolyn A.
A1 - Leimkühler, Silke
A1 - Jones, Jeffrey P.
T1 - ecoAO
BT - a simple system for the study of human aldehyde oxidases role in drug metabolism
JF - ACS OMEGA
N2 - Although aldehyde oxidase (AO) is an important hepatic drug-metabolizing enzyme, it remains understudied and is consequently often overlooked in preclinical studies, an oversight that has resulted in the failure of multiple clinical trials. AO’s preclusion to investigation stems from the following: (1) difficulties synthesizing metabolic standards due to the chemospecificity and regiospecificity of the enzyme and (2) significant inherent variability across existing in vitro systems including liver cytosol, S9 fractions, and primary hepatocytes, which lack specificity and generate discordant expression and activity profiles. Here, we describe a practical bacterial biotransformation system, ecoAO, addressing both issues simultaneously. ecoAO is a cell paste of MoCo-producing Escherichia coli strain TP1017 expressing human AO. It exhibits specific activity toward known substrates, zoniporide, 4-trans-(N,N-dimethylamino)cinnamaldehyde, O6-benzylguanine, and zaleplon; it also has utility as a biocatalyst, yielding milligram quantities of synthetically challenging metabolite standards such as 2-oxo-zoniporide. Moreover, ecoAO enables routine determination of kcat and V/K, which are essential parameters for accurate in vivo clearance predictions. Furthermore, ecoAO has potential as a preclinical in vitro screening tool for AO activity, as demonstrated by its metabolism of 3-aminoquinoline, a previously uncharacterized substrate. ecoAO promises to provide easy access to metabolites with the potential to improve pharmacokinetic clearance predictions and guide drug development.
Y1 - 2017
U6 - https://doi.org/10.1021/acsomega.7b01054
SN - 2470-1343
VL - 2
SP - 4820
EP - 4827
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Pinyou, Piyanut
A1 - Ruff, Adrian
A1 - Poeller, Sascha
A1 - Alsaoub, Sabine
A1 - Leimkühler, Silke
A1 - Wollenberger, Ursula
A1 - Schuhmann, Wolfgang
T1 - Wiring of the aldehyde oxidoreductase PaoABC to electrode surfaces via entrapment in low potential phenothiazine-modified redox polymers
JF - Bioelectrochemistry : an international journal devoted to electrochemical aspects of biology and biological aspects of electrochemistry ; official journal of the Bioelectrochemical Society
N2 - Phenothiazine-modified redox hydrogels were synthesized and used for the wiring of the aldehyde oxidoreductase PaoABC to electrode surfaces. The effects of the pH value and electrode surface modification on the biocatalytic activity of the layers were studied in the presence of vanillin as the substrate. The enzyme electrodes were successfully employed as bioanodes in vanillin/O-2 biofuel cells in combination with a high potential bilirubin oxidase biocathode. Open circuit voltages of around 700 mV could be obtained in a two compartment biofuel cell setup. Moreover, the use of a rather hydrophobic polymer with a high degree of crosslinking sites ensures the formation of stable polymer/enzyme films which were successfully used as bioanode in membrane-less biofuel cells. (C) 2015 Elsevier B.V. All rights reserved.
KW - Aldehyde oxidoreductase
KW - Enzyme electrode
KW - Redox polymer
KW - Phenothiazine
KW - Biosensor
KW - Biofuel cell
Y1 - 2016
U6 - https://doi.org/10.1016/j.bioelechem.2015.12.005
SN - 1567-5394
SN - 1878-562X
VL - 109
SP - 24
EP - 30
PB - Elsevier
CY - Lausanne
ER -
TY - JOUR
A1 - Rademacher, Corinna
A1 - Hoffmann, Marie-Christine
A1 - Lackmann, Jan-Wilm
A1 - Moser, Roman
A1 - Pfänder, Yvonne
A1 - Leimkühler, Silke
A1 - Narberhaus, Franz
A1 - Masepohl, Bernd
T1 - Tellurite resistance gene trgB confers copper tolerance to Rhodobacter capsulatus
JF - BioMetals : an international journal on the role of metal ions in biology, biochemistry and medicine
N2 - To identify copper homeostasis genes in Rhodobacter capsulatus, we performed random transposon Tn5 mutagenesis. Screening of more than 10,000 Tn5 mutants identified tellurite resistance gene trgB as a so far unrecognized major copper tolerance determinant. The trgB gene is flanked by tellurite resistance gene trgA and cysteine synthase gene cysK2. While growth of trgA mutants was only moderately restricted by tellurite, trgB and cysK2 mutants were severely affected by tellurite, which implies that viability under tellurite stress requires increased cysteine levels. Mutational analyses revealed that trgB was the only gene in this chromosomal region conferring cross-tolerance towards copper. Expression of the monocistronic trgB gene required promoter elements overlapping the trgA coding region as shown by nested deletions. Neither copper nor tellurite affected trgB transcription as demonstrated by reverse transcriptase PCR and trgB-lacZ fusions. Addition of tellurite or copper gave rise to increased cellular tellurium and copper concentrations, respectively, as determined by inductively coupled plasma-optical emission spectroscopy. By contrast, cellular iron concentrations remained fairly constant irrespective of tellurite or copper addition. This is the first study demonstrating a direct link between copper and tellurite response in bacteria.
KW - Copper
KW - Tellurite
KW - Nudix hydrolase
KW - Metal homeostasis
KW - Rhodobacter
Y1 - 2012
U6 - https://doi.org/10.1007/s10534-012-9566-2
SN - 0966-0844
VL - 25
IS - 5
SP - 995
EP - 1008
PB - Springer
CY - Dordrecht
ER -
TY - JOUR
A1 - Redelberger, David
A1 - Seduk, Farida
A1 - Genest, Olivier
A1 - Mejean, Vincent
A1 - Leimkühler, Silke
A1 - Iobbi-Nivol, Chantal
T1 - YcdY Protein of Escherichia coli, an Atypical Member of the TorD Chaperone Family
JF - Journal of bacteriology
N2 - The TorD family of specific chaperones is divided into four subfamilies dedicated to molybdoenzyme biogenesis and a fifth one, exemplified by YcdY of Escherichia coli, for which no defined partner has been identified so far. We propose that YcdY is the chaperone of YcdX, a zinc protein involved in the swarming motility process of E. coli, since YcdY interacts with YcdX and increases its activity in vitro.
Y1 - 2011
U6 - https://doi.org/10.1128/JB.05927-11
SN - 0021-9193
VL - 193
IS - 23
SP - 6512
EP - 6516
PB - American Society for Microbiology
CY - Washington
ER -
TY - JOUR
A1 - Reeve, Holly A.
A1 - Nicholson, Jake
A1 - Altaf, Farieha
A1 - Lonsdale, Thomas H.
A1 - Preissler, Janina
A1 - Lauterbach, Lars
A1 - Lenz, Oliver
A1 - Leimkühler, Silke
A1 - Hollmann, Frank
A1 - Paul, Caroline E.
A1 - Vincent, Kylie A.
T1 - A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H
JF - Chemical communications : ChemComm
N2 - We demonstrate a recycling system for synthetic nicotinamide cofactor analogues using a soluble hydrogenase with turnover number of >1000 for reduction of the cofactor analogues by H-2.
Coupling this system to an ene reductase, we show quantitative conversion of N-ethylmaleimide to N-ethylsuccinimide.
The biocatalyst system retained >50% activity after 7 h.
Y1 - 2022
U6 - https://doi.org/10.1039/d2cc02411j
SN - 1359-7345
SN - 1364-548X
VL - 58
IS - 75
SP - 10540
EP - 10543
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Reschke, Stefan
A1 - Duffus, Benjamin R.
A1 - Schrapers, Peer
A1 - Mebs, Stefan
A1 - Teutloff, Christian
A1 - Dau, Holger
A1 - Haumann, Michael
A1 - Leimkühler, Silke
T1 - Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli
JF - Biochemistry
N2 - The oxidoreductase YdhV in Escherichia coli has been predicted to belong to the family of molybdenum/tungsten cofactor (Moco/Wco)-containing enzymes. In this study, we characterized the YdhV protein in detail, which shares amino acid sequence homology with a tungsten-containing benzoyl-CoA reductase binding the bis-W-MPT (for metal-binding pterin) cofactor. The cofactor was identified to be of a bis-Mo-MPT type with no guanine nucleotides present, which represents a form of Moco that has not been found previously in any molybdoenzyme. Our studies showed that YdhV has a preference for bis-Mo-MPT over bis-W-MPT to be inserted into the enzyme. In-depth characterization of YdhV by X-ray absorption and electron paramagnetic resonance spectroscopies revealed that the bis-Mo-MPT cofactor in YdhV is redox active. The bis-Mo-MPT and bis-W-MPT cofactors include metal centers that bind the four sulfurs from the two dithiolene groups in addition to a cysteine and likely a sulfido ligand. The unexpected presence of a bis-Mo-MPT cofactor opens an additional route for cofactor biosynthesis in E. coli and expands the canon of the structurally highly versatile molybdenum and tungsten cofactors.
Y1 - 2019
U6 - https://doi.org/10.1021/acs.biochem.9b00078
SN - 0006-2960
VL - 58
IS - 17
SP - 2228
EP - 2242
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Reschke, Stefan
A1 - Niks, Dimitri
A1 - Wilson, Heather
A1 - Sigfridsson, Kajsa G. V.
A1 - Haumann, Michael
A1 - Rajagopalan, K. V.
A1 - Hine, Russ
A1 - Leimkühler, Silke
T1 - Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase
JF - Biochemistry
N2 - Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (k(cat)/K-M) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt) X-ray absorption spectroscopy revealed a Mow Se bond length of 2.51 A, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)(O2MoS2)-S-VI(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOM4Ser was altered compared to those of SOMD4Ser and SOMD4cy,(,), with g, in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo Se bond.
Y1 - 2013
U6 - https://doi.org/10.1021/bi4008512
SN - 0006-2960
VL - 52
IS - 46
SP - 8295
EP - 8303
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Reschke, Stefan
A1 - Sigfridsson, Kajsa G. V.
A1 - Kaufmann, Paul
A1 - Leidel, Nils
A1 - Horn, Sebastian
A1 - Gast, Klaus
A1 - Schulzke, Carola
A1 - Haumann, Michael
A1 - Leimkühler, Silke
T1 - Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in escherichia coli
JF - The journal of biological chemistry
N2 - The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.
Y1 - 2013
U6 - https://doi.org/10.1074/jbc.M113.497453
SN - 0021-9258
SN - 1083-351X
VL - 288
IS - 41
SP - 29736
EP - 29745
PB - American Society for Biochemistry and Molecular Biology
CY - Bethesda
ER -
TY - JOUR
A1 - Riedel, Simona
A1 - Siemiatkowska, Beata
A1 - Watanabe, Mutsumi
A1 - Müller, Christina S.
A1 - Schünemann, Volker
A1 - Hoefgen, Rainer
A1 - Leimkühler, Silke
T1 - The ABCB7-Like Transporter PexA in Rhodobacter capsulatus Is Involved in the Translocation of Reactive Sulfur Species
JF - Frontiers in Microbiology
N2 - The mitochondrial ATP-binding cassette (ABC) transporters ABCB7 in humans, Atm1 in yeast and ATM3 in plants, are highly conserved in their overall architecture and particularly in their glutathione binding pocket located within the transmembrane spanning domains. These transporters have attracted interest in the last two decades based on their proposed role in connecting the mitochondrial iron sulfur (Fe–S) cluster assembly with its cytosolic Fe–S cluster assembly (CIA) counterpart. So far, the specific compound that is transported across the membrane remains unknown. In this report we characterized the ABCB7-like transporter Rcc02305 in Rhodobacter capsulatus, which shares 47% amino acid sequence identity with its mitochondrial counterpart. The constructed interposon mutant strain in R. capsulatus displayed increased levels of intracellular reactive oxygen species without a simultaneous accumulation of the cellular iron levels. The inhibition of endogenous glutathione biosynthesis resulted in an increase of total glutathione levels in the mutant strain. Bioinformatic analysis of the amino acid sequence motifs revealed a potential aminotransferase class-V pyridoxal-50-phosphate (PLP) binding site that overlaps with the Walker A motif within the nucleotide binding domains of the transporter. PLP is a well characterized cofactor of L-cysteine desulfurases like IscS and NFS1 which has a role in the formation of a protein-bound persulfide group within these proteins. We therefore suggest renaming the ABCB7-like transporter Rcc02305 in R. capsulatus to PexA for PLP binding exporter. We further suggest that this ABC-transporter in R. capsulatus is involved in the formation and export of polysulfide species to the periplasm.
KW - ABCB7
KW - persulfide
KW - polysulfide
KW - glutathione
KW - ABC transporter
KW - Walker A motif
KW - pyridoxal-50-phosphate
Y1 - 2019
U6 - https://doi.org/10.3389/fmicb.2019.00406
SN - 1664-302X
VL - 10
PB - Frontiers Media
CY - Lausanne
ER -
TY - JOUR
A1 - Samuel, Prinson P.
A1 - Horn, Sebastian
A1 - Döring, Alexander
A1 - Havelius, Kajsa G. V.
A1 - Reschke, Stefan
A1 - Leimkühler, Silke
A1 - Haumann, Michael
A1 - Schulzke, Carola
T1 - A Crystallographic and Mo K-Edge XAS Study of Molybdenum Oxo Bis-,Mono-, and Non-Dithiolene Complexes - First-Sphere Coordination Geometry and Noninnocence of Ligands
JF - European journal of inorganic chemistry : a journal of ChemPubSoc Europe
N2 - Ten square-based pyramidal molybdenum complexes with different sulfur donor ligands, that is, a variety of dithiolenes and sulfides, were prepared, which mimic coordination motifs of the molybdenum cofactors of molybdenum-dependent oxidoreductases. The model compounds were investigated by Mo K-edge X-ray absorption spectroscopy (XAS) and (with one exception) their molecular structures were analyzed by X-ray diffraction to derive detailed information on bond lengths and geometries of the first coordination shell of molybdenum. Only small variations in Mo=O and Mo-S bond lengths and their respective coordination angles were observed for all complexes including those containing Mo(CO)(2) or Mo(mu-S)(2)Mo motifs. XAS analysis (edge energy) revealed higher relative oxidation levels in the molybdenum ion in compounds with innocent sulfur-based ligands relative to those in dithiolene complexes, which are known to exhibit noninnocence, that is, donation of substantial electron density from ligand to metal. In addition, longer average Mo-S and Mo=O bonds and consequently lower.(Mo=O) stretching frequencies in the IR spectra were observed for complexes with dithiolene-derived ligands. The results emphasize that the noninnocent character of the dithiolene ligand influences the electronic structure of the model compounds, but does not significantly affect their metal coordination geometry, which is largely determined by the Mo(IV) or (V) ion itself. The latter conclusion also holds for the molybdenum site geometries in the oxidized Mo-VI cofactor of DMSO reductase and the reduced Mo-IV cofactor of arsenite oxidase. The innocent behavior of the dithiolene molybdopterin ligands observed in the enzymes is likely to be related to cofactor-protein interactions.
KW - Molybdenum
KW - Enzyme models
KW - X-ray absorption spectroscopy
KW - Noninnocence
KW - Bioinorganic chemistry
Y1 - 2011
U6 - https://doi.org/10.1002/ejic.201100331
SN - 1434-1948
IS - 28
SP - 4387
EP - 4399
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Sarauli, David
A1 - Borowski, Anja
A1 - Peters, Kristina
A1 - Schulz, Burkhard
A1 - Fattakhova-Rohlfing, Dina
A1 - Leimkühler, Silke
A1 - Lisdat, Fred
T1 - Investigation of the pH-Dependent Impact of Sulfonated Polyaniline on Bioelectrocatalytic Activity of Xanthine Dehydrogenase
JF - ACS catalysis
N2 - We report on the pH-dependent bioelectrocatalytic activity of the redox enzyme xanthine dehydrogenase (XDH) in the presence of sulfonated polyaniline PMSA1 (poly(2-methoxyaniline-5-sulfonic acid)-co-aniline). Ultraviolet-visible (UV-vis) spectroscopic measurements with both components in solution reveal electron transfer from the hypoxanthine (HX)-reduced enzyme to the polymer. The enzyme shows bioelectrocatalytic activity on indium tin oxide (ITO) electrodes, when the polymer is present. Depending on solution pH, different processes can be identified. It can be demonstrated that not only product-based communication with the electrode but also efficient polymer-supported bioelectrocatalysis occur. Interestingly, substrate dependent catalytic currents can be obtained in acidic and neutral solutions, although the highest activity of XDH with natural reaction partners is in the alkaline region. Furthermore, operation of the enzyme electrode without addition of the natural cofactor of XDH is feasible. Finally, macroporous ITO electrodes have been used as an immobilization platform for the fabrication of HX-sensitive electrodes. The study shows that the efficient polymer/enzyme interaction can be advantageously combined with the open structure of an electrode material of controlled pore size, resulting in good processability, stability, and defined signal transfer in the presence of a substrate.
KW - enzyme bioelectrocatalysis
KW - sulfonated polyanilines
KW - xanthine dehydrogenase
KW - pH-dependent electrochemistry
KW - macroporous ITO electrodes
Y1 - 2016
U6 - https://doi.org/10.1021/acscatal.6b02011
SN - 2155-5435
VL - 6
SP - 7152
EP - 7159
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Sarauli, David
A1 - Riedel, Marc
A1 - Wettstein, Christoph
A1 - Hahn, Robert
A1 - Stiba, Konstanze
A1 - Wollenberger, Ursula
A1 - Leimkühler, Silke
A1 - Schmuki, Patrik
A1 - Lisdat, Fred
T1 - Semimetallic TiO2 nanotubes new interfaces for bioelectrochemical enzymatic catalysis
JF - Journal of materials chemistry
N2 - Different self-organized TiO2 nanotube structures are shown to represent new interfaces for the achievement of bioelectrochemical enzymatic catalysis involving redox proteins and enzymes without further surface modification or the presence of mediators.
Y1 - 2012
U6 - https://doi.org/10.1039/c2jm16427b
SN - 0959-9428
VL - 22
IS - 11
SP - 4615
EP - 4618
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Sarauli, David
A1 - Xu, Chenggang
A1 - Dietzel, Birgit
A1 - Stiba, Konstanze
A1 - Leimkühler, Silke
A1 - Schulz, Burkhard
A1 - Lisdat, Fred
T1 - Thin films of substituted polyanilines interactions with biomolecular systems
JF - Soft matter
N2 - We use substituted polyanilines for the construction of new polymer electrodes for interaction studies with the redox protein cytochrome c (cyt c) and the enzyme sulfite oxidase (SO). For these purposes four different polyaniline copolymers are chemically synthesized. Three of them are copolymers, containing 2-methoxyaniline-5-sulfonic acid with variable ratios of aniline; the fourth copolymer consists of 3-amino-benzoic acid and aniline. The results show that all polymers are suitable for being immobilized as thin stable films on gold wire and indium tin oxide (ITO) electrode surfaces from DMSO solution. This can be demonstrated by cyclic voltammetry and UV-Vis spectroscopy measurements. Moreover, cyt c can be electrochemically detected not only in solution, but also immobilized on top of the polymer films. Furthermore, the appearance of a significant catalytic current has been demonstrated for the sulfonated polyanilines, when the polymer-coated protein electrode is being measured upon addition of sulfite oxidase, confirming the establishment of a bioanalytical signal chain. Best results have been obtained for the polymer with highest sulfonation grade. The redox switching of the polymer by the enzymatic reaction can also be analyzed by following the spectral properties of the polymer electrode.
Y1 - 2012
U6 - https://doi.org/10.1039/c2sm07261k
SN - 1744-683X
VL - 8
IS - 14
SP - 3848
EP - 3855
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Schrapers, Peer
A1 - Hartmann, Tobias
A1 - Kositzki, Ramona
A1 - Dau, Holger
A1 - Reschke, Stefan
A1 - Schulzke, Carola
A1 - Leimkühler, Silke
A1 - Haumann, Michael
T1 - 'Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus
JF - Inorganic chemistry
N2 - Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO-) to CO2 oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Moligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH.
Y1 - 2015
U6 - https://doi.org/10.1021/ic502880y
SN - 0020-1669
SN - 1520-510X
VL - 54
IS - 7
SP - 3260
EP - 3271
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Schwanhold, Nadine
A1 - Iobbi-Nivol, Chantal
A1 - Lehmann, Angelika
A1 - Leimkühler, Silke
T1 - Same but different
BT - Comparison of two system-specific molecular chaperones for the maturation of formate dehydrogenases
JF - PLoS one
N2 - The maturation of bacterial molybdoenzymes is a complex process leading to the insertion of the bulky bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor into the apoenzyme. Most molybdoenzymes were shown to contain a specific chaperone for the insertion of the bis-MGD cofactor. Formate dehydrogenases (FDH) together with their molecular chaperone partner seem to display an exception to this specificity rule, since the chaperone FdhD has been proven to be involved in the maturation of all three FDH enzymes present in Escherichia colt. Multiple roles have been suggested for FdhD-like chaperones in the past, including the involvement in a sulfur transfer reaction from the L-cysteine desulfurase IscS to bis-MGD by the action of two cysteine residues present in a conserved CXXC motif of the chaperones. However, in this study we show by phylogenetic analyses that the CXXC motif is not conserved among FdhD-like chaperones. We compared in detail the FdhD-like homologues from Rhodobacter capsulatus and E. colt and show that their roles in the maturation of FDH enzymes from different subgroups can be exchanged. We reveal that bis-MGDbinding is a common characteristic of FdhD-like proteins and that the cofactor is bound with a sulfido-ligand at the molybdenum atom to the chaperone. Generally, we reveal that the cysteine residues in the motif CXXC of the chaperone are not essential for the production of active FDH enzymes.
Y1 - 2018
U6 - https://doi.org/10.1371/journal.pone.0201935
SN - 1932-6203
VL - 13
IS - 11
PB - PLoS
CY - San Fransisco
ER -
TY - JOUR
A1 - Sezer, Murat
A1 - Spricigo, Roberto
A1 - Utesch, Tillmann
A1 - Millo, Diego
A1 - Leimkühler, Silke
A1 - Mroginski, Maria A.
A1 - Wollenberger, Ursula
A1 - Hildebrandt, Peter
A1 - Weidinger, Inez M.
T1 - Redox properties and catalytic activity of surface-bound human sulfite oxidase studied by a combined surface enhanced resonance Raman spectroscopic and electrochemical approach
N2 - Human sulfite oxidase (hSO) was immobilised on SAM-coated silver electrodes under preservation of the native heme pocket structure of the cytochrome b5 (Cyt b5) domain and the functionality of the enzyme. The redox properties and catalytic activity of the entire enzyme were studied by surface enhanced resonance Raman (SERR) spectroscopy and cyclic voltammetry (CV) and compared to the isolated heme domain when possible. It is shown that heterogeneous electron transfer and catalytic activity of hSO sensitively depend on the local environment of the enzyme. Increasing the ionic strength of the buffer solution leads to an increase of the heterogeneous electron transfer rate from 17 s(-1) to 440 s(- 1) for hSO as determined by SERR spectroscopy. CV measurements demonstrate an increase of the apparent turnover rate for the immobilised hSO from 0.85 s(-1) in 100 mM buffer to 5.26 s(-1) in 750 mM buffer. We suggest that both effects originate from the increased mobility of the surface-bound enzyme with increasing ionic strength. In agreement with surface potential calculations we propose that at high ionic strength the enzyme is immobilised via the dimerisation domain to the SAM surface. The flexible loop region connecting the Moco and the Cyt b5 domain allows alternating contact with the Moco interaction site and the SAM surface, thereby promoting the sequential intramolecular and heterogeneous electron transfer from Moco via Cyt b5 to the electrode. At lower ionic strength, the contact time of the Cyt b5 domain with the SAM surface is longer, corresponding to a slower overall electron transfer process.
Y1 - 2010
UR - http://www.rsc.org/Publishing/Journals/CP/index.asp
U6 - https://doi.org/10.1039/B927226g
SN - 1463-9076
ER -
TY - JOUR
A1 - Sivanesan, Arumugam
A1 - Kalaivani, Govindasamy
A1 - Fischer, Anna
A1 - Stiba, Konstanze
A1 - Leimkühler, Silke
A1 - Weidinger, Inez M.
T1 - Complementary surface-enhanced resonance raman Spectroscopic Biodetection of mixed protein solutions by Chitosan- and Silica-Coated Plasmon-Tuned Silver Nanoparticles
JF - Analytical chemistry
N2 - Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b(5), whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.
Y1 - 2012
U6 - https://doi.org/10.1021/ac301001a
SN - 0003-2700
VL - 84
IS - 13
SP - 5759
EP - 5764
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Sivanesan, Arumugam
A1 - Ly, Khoa H.
A1 - Adamkiewicz, Witold
A1 - Stiba, Konstanze
A1 - Leimkühler, Silke
A1 - Weidinger, Inez M.
T1 - Tunable electric field enhancement and redox chemistry on TiO2 Island films via covalent attachment to Ag or Au nanostructures
JF - The journal of physical chemistry : C, Nanomaterials and interfaces
N2 - Ag-TiO2 and Au-TiO2 hybrid electrodes were designed by covalent attachment of TiO2 nanoparticles to Ag or Au electrodes via an organic linker. The optical and electronic properties of these systems were investigated using the cytochrome b(5) (Cyt b(5)) domain of sulfite oxidase, exclusively attached to the TiO2 surface, as a Raman marker and model redox enzyme. Very strong SERR signals of Cyt b(5) were obtained for Ag-supported systems due to plasmonic field enhancement of Ag. Time-resolved surface-enhanced resonance Raman spectroscopic measurements yielded a remarkably fast electron transfer kinetic (k = 60 s(-1)) of Cyt b(5) to Ag. A much lower Raman intensity was observed for Au-supported systems with undefined and slow redox behavior. We explain this phenomenon on the basis of the different potential of zero charge of the two metals that largely influence the electronic properties of the TiO2 island film.
Y1 - 2013
U6 - https://doi.org/10.1021/jp4032578
SN - 1932-7447
VL - 117
IS - 22
SP - 11866
EP - 11872
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Spricigo, Roberto
A1 - Dronov, Roman
A1 - Lisdat, Fred
A1 - Leimkühler, Silke
A1 - Scheller, Frieder W.
A1 - Wollenberger, Ursula
T1 - Electrocatalytic sulfite biosensor with human sulfite oxidase co-immobilized with cytochrome c in a polyelectrolyte-containing multilayer
N2 - An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
Y1 - 2009
UR - http://www.springerlink.com/content/100417
U6 - https://doi.org/10.1007/s00216-008-2432-y
SN - 1618-2642
ER -
TY - JOUR
A1 - Spricigo, Roberto
A1 - Leimkühler, Silke
A1 - Gorton, Lo
A1 - Scheller, Frieder W.
A1 - Wollenberger, Ursula
T1 - The Electrically Wired Molybdenum Domain of Human Sulfite Oxidase is Bioelectrocatalytically Active
JF - European journal of inorganic chemistry : a journal of ChemPubSoc Europe
N2 - We report electron transfer between the catalytic molybdenum cofactor (Moco) domain of human sulfite oxidase (hSO) and electrodes through a poly(vinylpyridine)-bound [osmium(N,N'-methyl-2,2'-biimidazole)(3)](2+/3+) complex as the electron-transfer mediator. The biocatalyst was immobilized in this low-potential redox polymer on a carbon electrode. Upon the addition of sulfite to the immobilized separate Moco domain, the generation of a significant catalytic current demonstrated that the catalytic center is effectively wired and active. The bioelectrocatalytic current of the wired separate catalytic domain reached 25% of the signal of the wired full molybdoheme enzyme hSO, in which the heme b(5) is involved in the electron-transfer pathway. This is the first report on a catalytically active wired molybdenum cofactor domain. The formal potential of this electrochemical mediator is between the potentials of the two cofactors of hSO, and as hSO can occupy several conformations in the polymer matrix, it is imaginable that electron transfer from the catalytic site to the electrode through the osmium center occurs for the hSO molecules in which the Moco domain is sufficiently accessible. The observation of catalytic oxidation currents at low potentials is favorable for applications in bioelectronic devices.
KW - Metalloenzymes
KW - Enzyme catalysis
KW - Immobilization
KW - Osmium
Y1 - 2015
U6 - https://doi.org/10.1002/ejic.201500034
SN - 1434-1948
SN - 1099-0682
IS - 21
SP - 3526
EP - 3531
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Spricigo, Roberto
A1 - Richter, Claudia
A1 - Leimkühler, Silke
A1 - Gorton, Lo
A1 - Scheller, Frieder W.
A1 - Wollenberger, Ursula
T1 - Sulfite biosensor based on osmium redox polymer wired sulfite oxidase
N2 - A biosensor, based on a redoxactive osmium polymer and sulfite oxidase on screen-printed electrodes, is presented here as a promising method for the detection of sulfite. A catalytic oxidative current was generated when a sample containing sulfite was pumped over the carbon screen-printed electrode modified with osmium redox polymer wired sulfite oxidase. A stationary value was reached after approximately 50 s and a complete measurement lasted no more than 3 min. The electrode polarized at -0.1 V (vs. Ag vertical bar AgCl 1M KCl) permits minimizing the influence of interfering substances, since these compounds can be unspecific oxidized at higher potentials. Because of the good stability of the protein film on the electrode surface, a well functioning biosensor-flow system was possible to construct. The working stability and reproducibility were further enhanced by the addition of bovine serum albumin generating a more long-term stable and biocompatible protein environment. The optimized biosensor showed a stable signal for more than a week of operation and a coefficient of variation of 4.8% for 12 successive measurements. The lower limit of detection of the sensor was 0.5 mu M sulfite and the response was linear until 100 mu M. The high sensitivity permitted a 1:500 dilution of wine samples. The immobilization procedure and the operational conditions granted minimized interferences. Additionally, repeating the immobilization procedure to form several layers of wired SO further increased the sensitivity of such a sensor. Finally. the applicability of the developed sulfite biosensor was tested on real samples, such as white and red wines.
Y1 - 2010
UR - http://www.sciencedirect.com/science/journal/09277757
U6 - https://doi.org/10.1016/j.colsurfa.2009.09.001
SN - 0927-7757
ER -
TY - JOUR
A1 - Stripp, Sven T.
A1 - Duffus, Benjamin R.
A1 - Fourmond, Vincent
A1 - Leger, Christophe
A1 - Leimkühler, Silke
A1 - Hirota, Shun
A1 - Hu, Yilin
A1 - Jasniewski, Andrew
A1 - Ogata, Hideaki
A1 - Ribbe, Markus W.
T1 - Second and outer coordination sphere effects in nitrogenase, hydrogenase, formate dehydrogenase, and CO dehydrogenase
JF - Chemical reviews : CR
N2 - Gases like H-2, N-2, CO2, and CO are increasingly recognized as critical feedstock in "green" energy conversion and as sources of nitrogen and carbon for the agricultural and chemical sectors. However, the industrial transformation of N-2, CO2, and CO and the production of H-2 require significant energy input, which renders processes like steam reforming and the Haber-Bosch reaction economically and environmentally unviable. Nature, on the other hand, performs similar tasks efficiently at ambient temperature and pressure, exploiting gas-processing metalloenzymes (GPMs) that bind low-valent metal cofactors based on iron, nickel, molybdenum, tungsten, and sulfur. Such systems are studied to understand the biocatalytic principles of gas conversion including N-2 fixation by nitrogenase and H-2 production by hydrogenase as well as CO2 and CO conversion by formate dehydrogenase, carbon monoxide dehydrogenase, and nitrogenase. In this review, we emphasize the importance of the cofactor/protein interface, discussing how second and outer coordination sphere effects determine, modulate, and optimize the catalytic activity of GPMs. These may comprise ionic interactions in the second coordination sphere that shape the electron density distribution across the cofactor, hydrogen bonding changes, and allosteric effects. In the outer coordination sphere, proton transfer and electron transfer are discussed, alongside the role of hydrophobic substrate channels and protein structural changes. Combining the information gained from structural biology, enzyme kinetics, and various spectroscopic techniques, we aim toward a comprehensive understanding of catalysis beyond the first coordination sphere.
Y1 - 2022
U6 - https://doi.org/10.1021/acs.chemrev.1c00914
SN - 0009-2665
SN - 1520-6890
VL - 122
IS - 14
SP - 11900
EP - 11973
PB - American Chemical Society
CY - Washington, DC
ER -
TY - JOUR
A1 - Tadjoung Waffo, Armel Franklin
A1 - Mitrova, Biljana
A1 - Tiedemann, Kim
A1 - Iobbi-Nivol, Chantal
A1 - Leimkühler, Silke
A1 - Wollenberger, Ulla
T1 - Electrochemical trimethylamine n-oxide biosensor with enzyme-based oxygen-scavenging membrane for long-term operation under ambient air
JF - Biosensors : open access journal
N2 - An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O-2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O-2 scavenging membrane. The TMAO sensor operates at a potential of -0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 mu M and 15 mM, with a sensitivity of 2.75 +/- 1.7 mu A/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 mu M TMAO.
KW - trimethylamine N-oxide
KW - biosensor
KW - TMAO-reductase
KW - oxygen scavenger
KW - immobilized enzyme
KW - multienzyme electrode
KW - viologen
Y1 - 2021
U6 - https://doi.org/10.3390/bios11040098
SN - 2079-6374
VL - 11
IS - 4
PB - MDPI
CY - Basel
ER -
TY - JOUR
A1 - Tanabe, Tomohisa Sebastian
A1 - Leimkühler, Silke
A1 - Dahl, Christiane
ED - Poole, RK
T1 - The functional diversity of the prokaryotic sulfur carrier protein TusA
JF - Advances in microbial physiology
N2 - Persulfide groups participate in a wide array of biochemical pathways and are chemically very versatile. The TusA protein has been identified as a central element supplying and transferring sulfur as persulfide to a number of important biosynthetic pathways, like molybdenum cofactor biosynthesis or thiomodifications in nucleosides of tRNAs. In recent years, it has furthermore become obvious that this protein is indispensable for the oxidation of sulfur compounds in the cytoplasm. Phylogenetic analyses revealed that different TusA protein variants exists in certain organisms, that have evolved to pursue specific roles in cellular pathways. The specific TusA-like proteins thereby cannot replace each other in their specific roles and are rather specific to one sulfur transfer pathway or shared between two pathways. While certain bacteria like Escherichia coli contain several copies of TusA-like proteins, in other bacteria like Allochromatium vinosum a single copy of TusA is present with an essential role for this organism. Here, we give an overview on the multiple roles of the various TusA-like proteins in sulfur transfer pathways in different organisms to shed light on the remaining mysteries of this versatile protein.
Y1 - 2019
SN - 978-0-12-817715-0
SN - 978-0-12-817714-3
U6 - https://doi.org/10.1016/bs.ampbs.2019.07.004
SN - 0065-2911
VL - 75
SP - 233
EP - 277
PB - Elsevier Acad. Press
CY - Amsterdam
ER -
TY - JOUR
A1 - Tang, Jing
A1 - Werchmeister, Rebecka Maria Larsen
A1 - Preda, Loredana
A1 - Huang, Wei
A1 - Zheng, Zhiyong
A1 - Leimkühler, Silke
A1 - Wollenberger, Ulla
A1 - Xiao, Xinxin
A1 - Engelbrekt, Christian
A1 - Ulstrup, Jens
A1 - Zhang, Jingdong
T1 - Three-dimensional sulfite oxidase bioanodes based on graphene functionalized carbon paper for sulfite/O-2 biofuel cells
JF - ACS catalysis
N2 - We have developed a three-dimensional (3D) graphene electrode suitable for the immobilization of human sulfite oxidase (hSO), which catalyzes the electrochemical oxidation of sulfite via direct electron transfer (DET). The electrode is fabricated by drop-casting graphene-polyethylenimine (G-P) composites on carbon papers (CPs) precoated with graphene oxide (GO). The negatively charged hSO can be adsorbed electrostatically on the positively charged matrix (G-P) on CP electrodes coated with GO (CPG), with a proper orientation for accelerated DET. Notably, further electrochemical reduction of G-P on CPG electrodes leads to a 9-fold increase of the saturation catalytic current density (j(m)) for sulfite oxidation reaching 24.4 +/- 0.3 mu A to cm(-2), the highest value among reported DET-based hSO bioelectrodes. The increased electron transfer rate plays a dominating role in the enhancement of direct enzymatic current because of the improved electric contact of hSO with the electrode, The optimized hSO bioelectrode shows a significant catalytic rate (k(cat): 25.6 +/- 0.3 s(-1)) and efficiency (k(cat)/K-m: 0.231 +/- 0.003 s(-1) mu M-1) compared to the reported hSO bioelectrodes. The assembly of the hSO bioanode and a commercial platinum biocathode allows the construction of sulfite/O-2 enzymatic biofuel cells (EBFCs) with flowing fuels. The optimized EBFC displays an open-circuit voltage (OCV) of 0.64 +/- 0.01 V and a maximum power density of 61 +/- 6 mu W cm(-2) (122 +/- 12 mW m(-3)) at 30 degrees C, which exceeds the best reported value by more than 6 times.
KW - enzymatic biofuel cell
KW - reduced graphene oxide
KW - sulfite oxidase
KW - carbon paper
KW - direct electron transfer
Y1 - 2019
U6 - https://doi.org/10.1021/acscatal.9b01715
SN - 2155-5435
VL - 9
IS - 7
SP - 6543
EP - 6554
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Terao, Mineko
A1 - Garattini, Enrico
A1 - Romão, Maria João
A1 - Leimkühler, Silke
T1 - Evolution, expression, and substrate specificities of aldehyde oxidase enzymes in eukaryotes
JF - The journal of biological chemistry
N2 - Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene (AOX1), those of rodents, such as mice, are endowed with four genes (Aox1-4), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles.
KW - metalloenzyme
KW - molybdenum
KW - mouse
KW - drug metabolism
KW - flavoprotein
KW - xenobiotic
KW - oxidase
KW - oxygen radicals
KW - iron-sulfur protein
KW - aldehyde oxidase (AOX)
KW - enzyme evolution
KW - metal-containing enzyme
KW - molybdenum cofactor (Moco)
KW - molybdo-flavoenzyme
KW - 2Fe-2S cluster
KW - flavin adenine dinucleotide (FAD)
Y1 - 2020
U6 - https://doi.org/10.1074/jbc.REV119.007741
SN - 0021-9258
SN - 1083-351X
VL - 295
IS - 16
SP - 5377
EP - 5389
PB - American Society for Biochemistry and Molecular Biology
CY - Rockville
ER -
TY - JOUR
A1 - Tiedemann, Kim
A1 - Iobbi-Nivol, Chantal
A1 - Leimkühler, Silke
T1 - The Role of the Nucleotides in the Insertion of the bis-Molybdopterin Guanine Dinucleotide Cofactor into apo-Molybdoenzymes
JF - Molecules
N2 - The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA.
KW - bis-MGD
KW - chaperone
KW - molybdenum cofactor
KW - TMAO reductase
Y1 - 2022
U6 - https://doi.org/10.3390/molecules27092993
SN - 1420-3049
VL - 27
SP - 1
EP - 15
PB - MDPI
CY - Basel, Schweiz
ET - 9
ER -
TY - JOUR
A1 - Voss, Martin
A1 - Nimtz, Manfred
A1 - Leimkühler, Silke
T1 - Elucidation of the dual role of Mycobacterial MoeZR in Molybdenum Cofactor Biosynthesis and Cysteine Biosynthesis
JF - PLoS one
N2 - The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo, while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria.
Y1 - 2011
U6 - https://doi.org/10.1371/journal.pone.0028170
SN - 1932-6203
VL - 6
IS - 11
PB - PLoS
CY - San Fransisco
ER -
TY - JOUR
A1 - Wiethaus, Jessica
A1 - Mueller, Alexandra
A1 - Neumann, Meina
A1 - Neumann, Sandra
A1 - Leimkühler, Silke
A1 - Narberhaus, Franz
A1 - Masepohl, Bernd
T1 - Specific interactions between four Molybdenum-binding proteins contribute to Mo-dependent gene regulation in Rhodobacter capsulatus
N2 - The phototrophic purple bacterium Rhodobacter capsulatus encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA- binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the R. capsulatus Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent mop-lacZ fusion in mutant strains defective for either mopA, mopB, or both suggested that MopB negatively modulates expression of the mop promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling mop gene expression.
Y1 - 2009
UR - http://jb.asm.org/
U6 - https://doi.org/10.1128/Jb.00526-09
SN - 0021-9193
ER -
TY - JOUR
A1 - Yan, Jiawei
A1 - Frøkjær, Emil Egede
A1 - Engelbrekt, Christian
A1 - Leimkühler, Silke
A1 - Ulstrup, Jens
A1 - Wollenberger, Ulla
A1 - Xiao, Xinxin
A1 - Zhang, Jingdong
T1 - Voltammetry and single-molecule in situ scanning tunnelling microscopy of the redox metalloenzyme human sulfite oxidase
JF - ChemElectroChem
N2 - Human sulfite oxidase (hSO) is a homodimeric two-domain enzyme central in the biological sulfur cycle. A pyranopterin molybdenum cofactor (Moco) is the catalytic site and a heme b(5) group located in the N-terminal domain. The two domains are connected by a flexible linker region. Electrons produced at the Moco in sulfite oxidation, are relayed via heme b(5) to electron acceptors or an electrode surface. Inter-domain conformational changes between an open and a closed enzyme conformation, allowing "gated" electron transfer has been suggested. We first recorded cyclic voltammetry (CV) of hSO on single-crystal Au(111)-electrode surfaces modified by self-assembled monolayers (SAMs) both of a short rigid thiol, cysteamine and of a longer structurally flexible thiol, omega-amino-octanethiol (AOT). hSO on cysteamine SAMs displays a well-defined pair of voltammetric peaks around -0.207 V vs. SCE in the absence of sulfite substrate, but no electrocatalysis. hSO on AOT SAMs displays well-defined electrocatalysis, but only "fair" quality voltammetry in the absence of sulfite. We recorded next in situ scanning tunnelling spectroscopy (STS) of hSO on AOT modified Au(111)-electrodes, disclosing, a 2-5 % surface coverage of strong molecular scale contrasts, assigned to single hSO molecules, notably with no contrast difference in the absence and presence of sulfite. In situ STS corroborated this observation with a sigmoidal tunnelling current/overpotential correlation.
KW - cyclic voltammetry
KW - human sulfite oxidase
KW - in situ scanning
KW - tunnelling spectroscopy
KW - self-assembled molecular monolayers
KW - single-crystal gold electrodes
Y1 - 2021
U6 - https://doi.org/10.1002/celc.202001258
SN - 2196-0216
VL - 8
IS - 1
SP - 164
EP - 171
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Yan, Robert
A1 - Friemel, Martin
A1 - Aloisi, Claudia
A1 - Huynen, Martijn
A1 - Taylor, Ian A.
A1 - Leimkühler, Silke
A1 - Pastore, Annalisa
T1 - The Eukaryotic-Specific ISD11 Is a Complex-Orphan Protein with Ability to Bind the Prokaryotic IscS
JF - PLoS one
N2 - The eukaryotic protein Isd11 is a chaperone that binds and stabilizes the central component of the essential metabolic pathway responsible for formation of iron-sulfur clusters in mitochondria, the desulfurase Nfs1. Little is known about the exact role of Isd11. Here, we show that human Isd11 (ISD11) is a helical protein which exists in solution as an equilibrium between monomer, dimeric and tetrameric species when in the absence of human Nfs1 (NFS1). We also show that, surprisingly, recombinant ISD11 expressed in E. coli co-purifies with the bacterial orthologue of NFS1, IscS. Binding is weak but specific suggesting that, despite the absence of Isd11 sequences in bacteria, there is enough conservation between the two desulfurases to retain a similar mode of interaction. This knowledge may inform us on the conservation of the mode of binding of Isd11 to the desulfurase. We used evolutionary evidence to suggest Isd11 residues involved in the interaction.
Y1 - 2016
U6 - https://doi.org/10.1371/journal.pone.0157895
SN - 1932-6203
VL - 11
SP - 383
EP - 395
PB - PLoS
CY - San Fransisco
ER -
TY - JOUR
A1 - Yildiz, Tugba
A1 - Leimkühler, Silke
T1 - TusA is a versatile protein that links translation efficiency to cell division in Escherichia coli
JF - Journal of bacteriology
N2 - To enable accurate and efficient translation, sulfur modifications are introduced posttranscriptionally into nucleosides in tRNAs. The biosynthesis of tRNA sulfur modifications involves unique sulfur trafficking systems for the incorporation of sulfur atoms in different nucleosides of tRNA. One of the proteins that is involved in inserting the sulfur for 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modifications in tRNAs is the TusA protein. TusA, however, is a versatile protein that is also involved in numerous other cellular pathways. Despite its role as a sulfur transfer protein for the 2-thiouridine formation in tRNA, a fundamental role of TusA in the general physiology of Escherichia coli has also been discovered. Poor viability, a defect in cell division, and a filamentous cell morphology have been described previously for tusA-deficient cells. In this report, we aimed to dissect the role of TusA for cell viability. We were able to show that the lack of the thiolation status of wobble uridine (U-34) nucleotides present on Lys, Gln, or Glu in tRNAs has a major consequence on the translation efficiency of proteins; among the affected targets are the proteins RpoS and Fis. Both proteins are major regulatory factors, and the deregulation of their abundance consequently has a major effect on the cellular regulatory network, with one consequence being a defect in cell division by regulating the FtsZ ring formation.
IMPORTANCE More than 100 different modifications are found in RNAs. One of these modifications is the mnm(5)s(2)U modification at the wobble position 34 of tRNAs for Lys, Gln, and Glu. The functional significance of U34 modifications is substantial since it restricts the conformational flexibility of the anticodon, thus providing translational fidelity. We show that in an Escherichia coli TusA mutant strain, involved in sulfur transfer for the mnm(5)s(2)U34 thio modifications, the translation efficiency of RpoS and Fis, two major cellular regulatory proteins, is altered. Therefore, in addition to the transcriptional regulation and the factors that influence protein stability, tRNA modifications that ensure the translational efficiency provide an additional crucial regulatory factor for protein synthesis.
KW - iron-sulfur clusters
KW - tRNA thio modifications
KW - FtsZ ring formation
KW - cell
KW - division
KW - TusA
KW - RpoS
KW - Fis
KW - FtsZ
Y1 - 2021
U6 - https://doi.org/10.1128/JB.00659-20
SN - 1098-5530
VL - 203
IS - 7
PB - American Society for Microbiology
CY - Washington
ER -
TY - JOUR
A1 - Zeng, Ting
A1 - Frasca, Stefano
A1 - Rumschöttel, Jens
A1 - Koetz, Joachim
A1 - Leimkühler, Silke
A1 - Wollenberger, Ursula
T1 - Role of Conductive Nanoparticles in the Direct Unmediated Bioelectrocatalysis of Immobilized Sulfite Oxidase
JF - Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis
KW - Direct electron transfer
KW - Protein voltammetry
KW - Human sulfite oxidase
KW - Bioelectrocatalysis
KW - Nanoparticles
Y1 - 2016
U6 - https://doi.org/10.1002/elan.201600246
SN - 1040-0397
SN - 1521-4109
VL - 28
SP - 2303
EP - 2310
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Zeng, Ting
A1 - Leimkühler, Silke
A1 - Koetz, Joachim
A1 - Wollenberger, Ursula
T1 - Effective Electrochemistry of Human Sulfite Oxidase Immobilized on Quantum-Dots-Modified Indium Tin Oxide Electrode
JF - ACS applied materials & interfaces
N2 - The bioelectrocatalytic sulfite oxidation by human sulfite oxidase (hSO) on indium tin oxide (ITO) is reported, which is facilitated by functionalizing of the electrode surface with polyethylenimine (PEI)-entrapped CdS nanoparticles and enzyme. hSO was assembled onto the electrode with a high surface loading of electroactive enzyme. In the presence of sulfite but without additional mediators, a high bioelectrocatalytic current was generated. Reference experiments with only PEI showed direct electron transfer and catalytic activity of hSO, but these were less pronounced. The application of the polyelectrolyte-entrapped quantum dots (QDs) on ITO electrodes provides a compatible surface for enzyme binding with promotion of electron transfer. Variations of the buffer solution conditions, e.g., ionic strength, pH, viscosity, and the effect of oxygen, were studied in order to understand intramolecular and heterogeneous electron transfer from hSO to the electrode. The results are consistent with a model derived for the enzyme by using flash photolysis in solution and spectroelectrochemistry and molecular dynamic simulations of hSO on monolayer-modified gold electrodes. Moreover, for the first time a photoelectrochemical electrode involving immobilized hSO is demonstrated where photoexcitation of the CdS/hSO-modified electrode lead to an enhanced generation of bioelectrocatalytic currents upon sulfite addition. Oxidation starts already at the redox potential of the electron transfer domain of hSO and is greatly increased by application of a small overpotential to the CdS/hSO-modified ITO.
KW - human sulfite oxidase
KW - direct electrochemistry
KW - bioelectrocatalysis
KW - photocurrent
KW - CdS quantum dots
Y1 - 2015
U6 - https://doi.org/10.1021/acsami.5b06665
SN - 1944-8244
VL - 7
IS - 38
SP - 21487
EP - 21494
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Zeng, Ting
A1 - Leimkühler, Silke
A1 - Wollenberger, Ulla
A1 - Fourmond, Vincent
T1 - Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes
JF - Journal of the American Chemical Society
N2 - Sulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a “closed” conformation, suitable for internal electron transfer, and an “open” conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization leads to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modeled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization.
Y1 - 2017
U6 - https://doi.org/10.1021/jacs.7b05480
SN - 0002-7863
VL - 139
SP - 11559
EP - 11567
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Zeng, Ting
A1 - Pankratov, Dmitry
A1 - Falk, Magnus
A1 - Leimkühler, Silke
A1 - Shleev, Sergey
A1 - Wollenberger, Ursula
T1 - Miniature direct electron transfer based sulphite/oxygen enzymatic fuel cells
JF - Biosensors and bioelectronics : the principal international journal devoted to research, design development and application of biosensors and bioelectronics
N2 - A direct electron transfer (DET) based sulphite/oxygen biofuel cell is reported that utilises human sulphite oxidase (hSOx) and Myrothecium verrucaria bilirubin oxidase (MvBOx) and nanostructured gold electrodes. For bioanode construction, the nanostructured gold microelectrodes were further modified with 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester) to which polyethylene imine was covalently attached. hSOx was adsorbed onto this chemically modified nanostructured electrode with high surface loading of electroactive enzyme and in presence of sulphite high anodic bioelectrocatalytic currents were generated with an onset potential of 0.05 V vs. NHE. The biocathode contained MyBOx directly adsorbed to the deposited gold nanoparticles for cathodic oxygen reduction starting at 0.71 V vs. NHE. Both enzyme electrodes were integrated to a DET-type biofuel cell. Power densities of 8 and 1 mu W cm(-2) were achieved at 0.15 V and 0.45 V of cell voltages, respectively, with the membrane based biodevices under aerobic conditions. (C) 2014 Elsevier B.V. All rights reserved.
KW - Enzymatic fuel cell
KW - Microscale electrode
KW - Direct electron transfer
KW - Sulphite oxidase
KW - Bilirubin oxidase
Y1 - 2015
U6 - https://doi.org/10.1016/j.bios.2014.10.080
SN - 0956-5663
SN - 1873-4235
VL - 66
SP - 39
EP - 42
PB - Elsevier
CY - Oxford
ER -