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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - GEN A1 - Moga, A. A1 - Robinson, T. A1 - Leimkühler, Silke T1 - Towards reconstituting a biosynthetic pathway within compartmentalized GUVs T2 - European biophysics journal : with biophysics letters ; an international journal of biophysics Y1 - 2019 SN - 0175-7571 SN - 1432-1017 VL - 48 SP - S218 EP - S218 PB - Springer CY - New York 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 - 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 - 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 - 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 - GEN 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 T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 975 KW - molybdenum cofactor deficiency KW - blood-brain-barrier KW - larval locomotion KW - energy-metabolism KW - cerebral-cortex KW - astrocytes KW - behavior KW - cells KW - transmission KW - disease KW - Diseases of the nervous system KW - Glial biology KW - Glial development KW - Neurotransmitters Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-426205 SN - 1866-8372 IS - 975 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 - GEN 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 T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1268 KW - bis-MGD KW - chaperone KW - molybdenum cofactor KW - TMAO reductase Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-561728 SN - 1866-8372 SP - 1 EP - 15 PB - Universitätsverlag Potsdam CY - Potsdam ER - TY - JOUR A1 - Bühning, Martin A1 - Valleriani, Angelo A1 - Leimkühler, Silke T1 - The role of SufS is restricted to Fe-S cluster biosynthesis in escherichia coli JF - Biochemistry N2 - In Escherichia coli, two different systems that are important for the coordinate formation of Fe–S clusters have been identified, namely, the ISC and SUF systems. The ISC system is the housekeeping Fe–S machinery, which provides Fe–S clusters for numerous cellular proteins. The IscS protein of this system was additionally revealed to be the primary sulfur donor for several sulfur-containing molecules with important biological functions, among which are the molybdenum cofactor (Moco) and thiolated nucleosides in tRNA. Here, we show that deletion of central components of the ISC system in addition to IscS leads to an overall decrease in Fe–S cluster enzyme and molybdoenzyme activity in addition to a decrease in the number of Fe–S-dependent thiomodifications of tRNA, based on the fact that some proteins involved in Moco biosynthesis and tRNA thiolation are Fe–S-dependent. Complementation of the ISC deficient strains with the suf operon restored the activity of Fe–S-containing proteins, including the MoaA protein, which is involved in the conversion of 5′GTP to cyclic pyranopterin monophosphate in the fist step of Moco biosynthesis. While both systems share a high degree of similarity, we show that the function of their respective l-cysteine desulfurase IscS or SufS is specific for each cellular pathway. It is revealed that SufS cannot play the role of IscS in sulfur transfer for the formation of 2-thiouridine, 4-thiouridine, or the dithiolene group of molybdopterin, being unable to interact with TusA or ThiI. The results demonstrate that the role of the SUF system is exclusively restricted to Fe–S cluster assembly in the cell. Y1 - 2017 U6 - https://doi.org/10.1021/acs.biochem.7b00040 SN - 0006-2960 VL - 56 SP - 1987 EP - 2000 PB - American Chemical Society CY - Washington ER - TY - JOUR A1 - Yokoyama, Kenichi A1 - Leimkühler, Silke T1 - The role of FeS clusters for molybdenum cofactor biosynthesis and molybdoenzymes in bacteria JF - Biochimica et biophysica acta : Molecular cell research N2 - The biosynthesis of the molybdenum cofactor (Moco) has been intensively studied, in addition to its insertion into molybdoenzymes. In particular, a link between the assembly of molybdoenzymes and the biosynthesis of FeS clusters has been identified in the recent years: 1) the synthesis of the first intermediate in Moco biosynthesis requires an FeS-cluster containing protein, 2) the sulfurtransferase for the dithiolene group in Moco is also involved in the synthesis of FeS clusters, thiamin and thiolated tRNAs, 3) the addition of a sulfido-ligand to the molybdenum atom in the active site additionally involves a sulfurtransferase, and 4) most molybdoenzymes in bacteria require FeS clusters as redox active cofactors. In this review we will focus on the biosynthesis of the molybdenum cofactor in bacteria, its modification and insertion into molybdoenzymes, with an emphasis to its link to FeS cluster biosynthesis and sulfur transfer. (C) 2014 Elsevier B.V. All rights reserved. KW - Molybdenum-iron-iron-sulfur cluster KW - Molybdenum cofactor KW - tRNA KW - Sulfur transfer KW - L-Cysteine desulfurase Y1 - 2015 U6 - https://doi.org/10.1016/j.bbamcr.2014.09.021 SN - 0167-4889 SN - 0006-3002 VL - 1853 IS - 6 SP - 1335 EP - 1349 PB - Elsevier CY - Amsterdam 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 - Zupok, Arkadiusz A1 - Iobbi-Nivol, Chantal A1 - Mejean, Vincent A1 - Leimkühler, Silke T1 - The regulation of Moco biosynthesis and molybdoenzyme gene expression by molybdenum and iron in bacteria JF - Metallomics : integrated biometal science N2 - Bacterial molybdoenzymes are key enzymes involved in the global sulphur, nitrogen and carbon cycles. These enzymes require the insertion of the molybdenum cofactor (Moco) into their active sites and are able to catalyse a large range of redox-reactions. Escherichia coli harbours nineteen different molybdoenzymes that require a tight regulation of their synthesis according to substrate availability, oxygen availability and the cellular concentration of molybdenum and iron. The synthesis and assembly of active molybdoenzymes are regulated at the level of transcription of the structural genes and of translation in addition to the genes involved in Moco biosynthesis. The action of global transcriptional regulators like FNR, NarXL/QP, Fur and ArcA and their roles on the expression of these genes is described in detail. In this review we focus on what is known about the molybdenum- and iron-dependent regulation of molybdoenzyme and Moco biosynthesis genes in the model organism E. coli. The gene regulation in E. coli is compared to two other well studied model organisms Rhodobacter capsulatus and Shewanella oneidensis. Y1 - 2019 U6 - https://doi.org/10.1039/c9mt00186g SN - 1756-5901 SN - 1756-591X VL - 11 IS - 10 SP - 1602 EP - 1624 PB - Royal Society of Chemistry CY - Cambridge 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - GEN 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 T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1307 KW - Moco biosynthesis KW - sulfite oxidase KW - cytosolic tRNA thiolation KW - 5-methoxycarbonylmethyl-2-thiouridine KW - H2S biosynthesis KW - cellular bioenergetics Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-579580 SN - 1866-8372 IS - 1307 ER - TY - JOUR A1 - Leimkühler, Silke A1 - Wuebbens, Margot M. A1 - Rajagopalan, K. V. T1 - The history of the discovery of the molybdenum cofactor and novel aspects of its biosynthesis in bacteria JF - Coordination chemistry reviews N2 - The biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to molybdopterin phosphate group or the replacement of ligands at the molybdenum center. KW - Molybdenum cofactor KW - Molybdopterin KW - Precursor Z KW - Molybdopterin guanine dinucleotide cofactor KW - Urothione KW - Dithiolene group Y1 - 2011 U6 - https://doi.org/10.1016/j.ccr.2010.12.003 SN - 0010-8545 VL - 255 IS - 9-10 SP - 1129 EP - 1144 PB - Elsevier CY - Lausanne 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 - 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 - 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 - 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 - 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 - 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 - 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 - Boehmer, Nadine A1 - Hartmann, Tobias A1 - Leimkühler, Silke T1 - The chaperone FdsC for Rhodobacter capsulatus formate dehydrogenase binds the bis-molybdopterin guanine dinucleotide cofactor JF - FEBS letters : the journal for rapid publication of short reports in molecular biosciences N2 - Molybdoenzymes are complex enzymes in which the molybdenum cofactor (Moco) is deeply buried in the enzyme. Most molybdoenzymes contain a specific chaperone for the insertion of Moco. For the formate dehydrogenase FdsGBA from Rhodobacter capsulatus the two chaperones FdsC and FdsD were identified to be essential for enzyme activity, but are not a subunit of the mature enzyme. Here, we purified and characterized the FdsC protein after heterologous expression in Escherichia coli. We were able to copurify FdsC with the bound Moco derivate bis-molybdopterin guanine dinucleotide. This cofactor successfully was used as a source to reconstitute the activity of molybdoenzymes. Structured summary of protein interactions: FdsC and FdsC bind by molecular sieving (View interaction) FdsD binds to RcMobA by surface plasmon resonance (View interaction) FdsC binds to RcMobA by surface plasmon resonance (View interaction) FdsC binds to FdsA by surface plasmon resonance (View interaction) KW - Molybdenum cofactor KW - L-cysteine desulfurase KW - Formate dehydrogenase KW - Chaperone KW - bis-MGD Y1 - 2014 U6 - https://doi.org/10.1016/j.febslet.2013.12.033 SN - 0014-5793 SN - 1873-3468 VL - 588 IS - 4 SP - 531 EP - 537 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Leimkühler, Silke T1 - The biosynthesis of the molybdenum cofactors in Escherichia coli JF - Environmental microbiology N2 - The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes containing Moco, 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 four steps in in bacteria: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5 '-GTP, (ii) in the second step the two sulfur atoms are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into MPT to form Moco and (iv) in the fourth step bis-Mo-MPT is formed and an additional modification of Moco is possible with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review presents an update on the well-characterized Moco biosynthesis in the model organism Escherichia coli including novel discoveries from the recent years. KW - periplasmic nitrate reductase KW - biotin sulfoxide reductase KW - in-vitro-synthesis KW - n-oxide reductase KW - crystal-structure KW - molybdopterin synthase KW - formate dehydrogenase KW - rhodobacter-capsulatus KW - xanthine dehydrogenase KW - converting factor Y1 - 2020 U6 - https://doi.org/10.1111/1462-2920.15003 SN - 1462-2912 SN - 1462-2920 VL - 22 IS - 6 SP - 2007 EP - 2026 PB - Wiley CY - Hoboken ER - TY - GEN A1 - Leimkühler, Silke T1 - The biosynthesis of the molybdenum cofactors in Escherichia coli T2 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes containing Moco, 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 four steps in in bacteria: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5 '-GTP, (ii) in the second step the two sulfur atoms are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into MPT to form Moco and (iv) in the fourth step bis-Mo-MPT is formed and an additional modification of Moco is possible with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review presents an update on the well-characterized Moco biosynthesis in the model organism Escherichia coli including novel discoveries from the recent years. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1433 KW - periplasmic nitrate reductase KW - biotin sulfoxide reductase KW - in-vitro-synthesis KW - n-oxide reductase KW - crystal-structure KW - molybdopterin synthase KW - formate dehydrogenase KW - rhodobacter-capsulatus KW - xanthine dehydrogenase KW - converting factor Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-516559 SN - 1866-8372 IS - 6 ER - TY - JOUR A1 - Mendel, Ralf R. A1 - Leimkühler, Silke T1 - The biosynthesis of the molybdenum cofactors JF - Journal of biological inorganic chemistry N2 - The biosynthesis of the molybdenum cofactors (Moco) is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified to date. In all molybdoenzymes except nitrogenase, molybdenum is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into three general steps, with a fourth one present only in bacteria and archaea: (1) formation of the cyclic pyranopterin monophosphate, (2) formation of MPT, (3) insertion of molybdenum into molybdopterin to form Moco, and (4) additional modification of Moco in bacteria with the attachment of a nucleotide to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on the biosynthesis of Moco in bacteria, humans and plants. KW - Molybdenum KW - Molybdenum cofactor KW - cPMP KW - bis-MGD KW - Sulfuration KW - Sulfite oxidase Y1 - 2015 U6 - https://doi.org/10.1007/s00775-014-1173-y SN - 0949-8257 SN - 1432-1327 VL - 20 IS - 2 SP - 337 EP - 347 PB - Springer CY - New York ER - TY - GEN 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 T2 - Postprints der Universität Potsdam Mathematisch-Naturwissenschaftliche Reihe 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. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 740 KW - ABCB7 KW - persulfide KW - polysulfide KW - glutathione KW - ABC transporter KW - Walker A motif KW - pyridoxal-50-phosphate Y1 - 1019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-434975 SN - 1866-8372 IS - 740 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 - Brietzke, Thomas Martin A1 - Dietz, Thomas A1 - Kelling, Alexandra A1 - Schilde, Uwe A1 - Bois, Juliana A1 - Kelm, Harald A1 - Reh, Manuel A1 - Schmitz, Markus A1 - Koerzdoerfer, Thomas A1 - Leimkühler, Silke A1 - Wollenberger, Ulla A1 - Krueger, Hans-Joerg A1 - Holdt, Hans-Jürgen T1 - The 1,6,7,12-Tetraazaperylene Bridging Ligand as an Electron Reservoir and Its Disulfonato Derivative as Redox Mediator in an Enzyme-Electrode Process JF - Chemistry - a European journal N2 - The homodinuclear ruthenium(II) complex [{Ru(l-N4Me2)}(2)(-tape)](PF6)(4) {[1](PF6)(4)} (l-N4Me2=N,N-dimethyl-2,11-diaza[3.3](2,6)-pyridinophane, tape=1,6,7,12-tetraazaperylene) can store one or two electrons in the energetically low-lying * orbital of the bridging ligand tape. The corresponding singly and doubly reduced complexes [{Ru(l-N4Me2)}(2)(-tape(.-))](PF6)(3) {[2](PF6)(3)} and [{Ru(l-N4Me2)}(2)(-tape(2-))](PF6)(2) {[3](PF6)(2)}, respectively, were electrochemically generated, successfully isolated and fully characterized by single-crystal X-ray crystallography, spectroscopic methods and magnetic susceptibility measurements. The singly reduced complex [2](PF6)(3) contains the -radical tape(.-) and the doubly reduced [3](PF6)(2) the diamagnetic dianion tape(2-) as bridging ligand, respectively. Nucleophilic aromatic substitution at the bridging tape in [1](4+) by two sulfite units gave the complex [{Ru(l-N4Me2)}(2){-tape-(SO3)(2)}](2+) ([4](2+)). Complex dication [4](2+) was exploited as a redox mediator between an anaerobic homogenous reaction solution of an enzyme system (sulfite/sulfite oxidase) and the electrode via participation of the low-energy *-orbital of the disulfonato-substituted bridging ligand tape-(SO3)(2)(2-) (E-red1=-0.1V versus Ag/AgCl/1m KCl in water). KW - electrochemistry KW - enzyme catalysis KW - N-ligands KW - redox-active ligands KW - ruthenium Y1 - 2017 U6 - https://doi.org/10.1002/chem.201703639 SN - 0947-6539 SN - 1521-3765 VL - 23 SP - 15583 EP - 15587 PB - Wiley-VCH CY - Weinheim 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 - 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 - 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 - CHAP A1 - Leimkühler, Silke T1 - Studies on the Oxygen tolerant formate deyhdrogenase from rhodobacter capsulatus T2 - Journal of biological inorganic chemistry Y1 - 2014 SN - 0949-8257 SN - 1432-1327 VL - 19 SP - S72 EP - S72 PB - Springer CY - New York ER - TY - CHAP A1 - Leimkühler, Silke A1 - Hartmann, Tobias A1 - Garattini, Enrico A1 - Jones, Jeffrey P. T1 - Structure-function studies on human aldehyde oxidase and the impact of polymorphisms on enzyme activity T2 - Drug metabolism reviews : biotransformation and disposition of xenobiotics ; official journal of the International Society for the Study of Xenobiotics Y1 - 2011 SN - 0360-2532 VL - 43 IS - 6 SP - 13 EP - 13 PB - Taylor & Francis Group CY - London ER - TY - JOUR A1 - Terao, Mineko A1 - Romao, Maria Joao A1 - Leimkühler, Silke A1 - Bolis, Marco A1 - Fratelli, Maddalena A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Garattini, Enrico T1 - Structure and function of mammalian aldehyde oxidases JF - Archives of toxicology : official journal of EUROTOX N2 - Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX. KW - Aldehyde oxidase KW - Molybdo-flavoenzymes KW - Xanthine oxidoreductase KW - Drug metabolism Y1 - 2016 U6 - https://doi.org/10.1007/s00204-016-1683-1 SN - 0340-5761 SN - 1432-0738 VL - 90 SP - 753 EP - 780 PB - Springer CY - Heidelberg 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 - 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 - 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 - Romao, Maria Joao A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Foti, Alessandro A1 - Terao, Mineko A1 - Garattini, Enrico A1 - Leimkühler, Silke T1 - Structural basis for the role of mammalian aldehyde oxidases in the metabolism of drugs and xenobiotics JF - Current Opinion in Chemical Biology 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. Mammals are characterized by a complement of species specific AOX isoenzymes, that varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, AOX3 and AOX4). The physiological function of mammalian AOX isoenzymes is unknown, although human AOX1 is an emerging enzyme in phase-I drug metabolism. Indeed, the number of therapeutic molecules under development which act as AOX substrates is increasing. The recent crystallization and structure determination of human AOX1 as well as mouse AOX3 has brought new insights into the mechanisms underlying substrate/inhibitor binding as well as the catalytic activity of this class of enzymes. Y1 - 2017 U6 - https://doi.org/10.1016/j.cbpa.2017.01.005 SN - 1367-5931 SN - 1879-0402 VL - 37 SP - 39 EP - 47 PB - Elsevier CY - Oxford 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 - GEN A1 - Schumann, Silvia A1 - Terao, Mineko A1 - Garattini, Enrico A1 - Saggu, Miguel A1 - Lendzian, Friedhelm A1 - Hildebrandt, Peter A1 - Leimkühler, Silke T1 - Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1 N2 - Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - paper 134 Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-45030 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 - GEN A1 - Leimkühler, Silke A1 - Bühning, Martin A1 - Beilschmidt, Lena T1 - Shared sulfur mobilization routes for tRNA thiolation and molybdenum cofactor biosynthesis in prokaryotes and eukaryotes T2 - Postprints der Universität Potsdam : Mathematisch Naturwissenschaftliche Reihe N2 - Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the thiomodifications xm(5)s(2)U at the wobble position 34 in tRNAs for Lys, Gln and Glu, were suggested to have an important role during the translation process by ensuring accurate deciphering of the genetic code and by stabilization of the tRNA structure. The trafficking and delivery of sulfur nucleosides is a complex process carried out by sulfur relay systems involving numerous proteins, which not only deliver sulfur to the specific tRNAs but also to other sulfur-containing molecules including iron-sulfur clusters, thiamin, biotin, lipoic acid and molybdopterin (MPT). Among the biosynthesis of these sulfur-containing molecules, the biosynthesis of the molybdenum cofactor (Moco) and the synthesis of thio-modified tRNAs in particular show a surprising link by sharing protein components for sulfur mobilization in pro- and eukaryotes. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1015 KW - tRNA KW - molybdenum cofactor KW - persulfide KW - thiocarboxylate KW - thionucleosides KW - sulfurtransferase KW - l-cysteine desulfurase Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-475011 SN - 1866-8372 IS - 1015 ER - TY - JOUR A1 - Leimkühler, Silke A1 - Bühning, Martin A1 - Beilschmidt, Lena T1 - Shared sulfur mobilization routes for tRNA thiolation and molybdenum cofactor biosynthesis in prokaryotes and eukaryotes JF - Biomolecules N2 - Modifications of transfer RNA (tRNA) have been shown to play critical roles in the biogenesis, metabolism, structural stability and function of RNA molecules, and the specific modifications of nucleobases with sulfur atoms in tRNA are present in pro- and eukaryotes. Here, especially the thiomodifications xm(5)s(2)U at the wobble position 34 in tRNAs for Lys, Gln and Glu, were suggested to have an important role during the translation process by ensuring accurate deciphering of the genetic code and by stabilization of the tRNA structure. The trafficking and delivery of sulfur nucleosides is a complex process carried out by sulfur relay systems involving numerous proteins, which not only deliver sulfur to the specific tRNAs but also to other sulfur-containing molecules including iron-sulfur clusters, thiamin, biotin, lipoic acid and molybdopterin (MPT). Among the biosynthesis of these sulfur-containing molecules, the biosynthesis of the molybdenum cofactor (Moco) and the synthesis of thio-modified tRNAs in particular show a surprising link by sharing protein components for sulfur mobilization in pro- and eukaryotes. KW - tRNA KW - molybdenum cofactor KW - persulfide KW - thiocarboxylate KW - thionucleosides KW - sulfurtransferase KW - l-cysteine desulfurase Y1 - 2017 U6 - https://doi.org/10.3390/biom7010005 SN - 2218-273X VL - 7 IS - 1 PB - MDPI CY - Basel ER - TY - JOUR A1 - Leimkühler, Silke T1 - Shared function and moonlighting proteins in molybdenum cofactor biosynthesis JF - Biological chemistry N2 - The biosynthesis of the molybdenum cofactor (Moco) is a highly conserved pathway in bacteria, archaea and eukaryotes. The molybdenum atom in Moco-containing enzymes is coordinated to the dithiolene group of a tricyclic pyranopterin monophosphate cofactor. The biosynthesis of Moco can be divided into three conserved steps, with a fourth present only in bacteria and archaea: (1) formation of cyclic pyranopterin monophosphate, (2) formation of molybdopterin (MPT), (3) insertion of molybdenum into MPT to form Mo-MPT, and (4) additional modification of Mo-MPT in bacteria with the attachment of a GMP or CMP nucleotide, forming the dinucleotide variants of Moco. While the proteins involved in the catalytic reaction of each step of Moco biosynthesis are highly conserved among the Phyla, a surprising link to other cellular pathways has been identified by recent discoveries. In particular, the pathways for FeS cluster assembly and thio-modifications of tRNA are connected to Moco biosynthesis by sharing the same protein components. Further, proteins involved in Moco biosynthesis are not only shared with other pathways, but additionally have moonlighting roles. This review gives an overview of Moco biosynthesis in bacteria and humans and highlights the shared function and moonlighting roles of the participating proteins. KW - FeS cluster KW - molybdenum cofactor KW - molybdo-enzymes KW - moonlighting KW - sulfur transfer KW - tRNA thiolation Y1 - 2017 U6 - https://doi.org/10.1515/hsz-2017-0110 SN - 1431-6730 SN - 1437-4315 VL - 398 SP - 1009 EP - 1026 PB - De Gruyter CY - Berlin 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 - 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 - 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 - 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 - 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 - CHAP A1 - Duffus, Benjamin R. A1 - Hartmann, Tobias A1 - Teutloff, Christian A1 - Leimkühler, Silke T1 - Refining catalytic insights toward the chemical mechanism of R. capsulatus formate dehydrogenase via EPR spectroscopy T2 - Abstracts of papers : joint conference / The Chemical Institute of Cananda, CIC, American Chemical Society, ACS Y1 - 2019 SN - 0065-7727 VL - 257 PB - American Chemical Society CY - Washington 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 - Cazelles, R. A1 - Lalaoui, N. A1 - Hartmann, Tobias A1 - Leimkühler, Silke A1 - Wollenberger, Ursula A1 - Antonietti, Markus A1 - Cosnier, S. T1 - Ready to use bioinformatics analysis as a tool to predict immobilisation strategies for protein direct electron transfer (DET) JF - Polymer : the international journal for the science and technology of polymers KW - Bioinformatic KW - Bioelectrocatalysis KW - Electron transfer KW - Dehydrogenase KW - Nicotinamide Y1 - 2016 U6 - https://doi.org/10.1016/j.bios.2016.04.078 SN - 0956-5663 SN - 1873-4235 VL - 85 SP - 90 EP - 95 PB - Elsevier CY - Oxford 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 - 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 - 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 - 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 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke T1 - Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli JF - Biochimica et biophysica acta : Bioenergetics N2 - Molybdenum cofactor (Moco) biosynthesis is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified in bacteria to date. In molybdoenzymes Mo is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into four general steps in bacteria: I) formation of the cyclic pyranopterin monophosphate, 2) formation of MPT, 3) insertion of molybdenum into molybdopterin to form Moco, and 4) additional modification of Moco with the attachment of GMP or CMP to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on molybdoenzymes, the biosynthesis of Moco, and its incorporation into specific target proteins focusing on Escherichia coli. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. KW - Molybdenum cofactor KW - Molybdenum KW - Dithiolene KW - Molybdopterin KW - Bis-MGD KW - Moco Y1 - 2013 U6 - https://doi.org/10.1016/j.bbabio.2012.11.007 SN - 0005-2728 VL - 1827 IS - 8-9 SP - 1086 EP - 1101 PB - Elsevier CY - Amsterdam 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 - 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 - 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 - 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 - 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 - 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 - 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 - Zhang, Wanjiao A1 - Urban, Alexander A1 - Mihara, Hisaaki A1 - Leimkühler, Silke A1 - Kurihara, Tatsuo A1 - Esaki, Nobuyoshi T1 - IscS functions as a primary sulfur-donating enzyme by interacting specifically with MoeB and MoaD in the biosynthesis of molybdopterin in escherichia coli N2 - The persulfide sulfur formed on an active site cysteine residue of pyridoxal 5'-phosphate-dependent cysteine desulfurases is subsequently incorporated into the biosynthetic pathways of a variety of sulfur-containing cofactors and thionucleosides. In molybdenum cofactor biosynthesis, MoeB activates the C terminus of the MoaD subunit of molybdopterin (MPT) synthase to form MoaD-adenylate, which is subsequently converted to a thiocarboxylate for the generation of the dithiolene group of MPT. It has been shown that three cysteine desulfurases (CsdA, SufS, and IscS) of Escherichia coli can transfer sulfur from L-cysteine to the thiocarboxylate of MoaD in vitro. Here, we demonstrate by surface plasmon resonance analyses that IscS, but not CsdA or SufS, interacts with MoeB and MoaD. MoeB and MoaD can stimulate the IscS activity up to 1.6-fold. Analysis of the sulfuration level of MoaD isolated from strains defective in cysteine desulfurases shows a largely decreased sulfuration level of the protein in an iscS deletion strain but not in a csdA/sufS deletion strain. We also show that another iscS deletion strain of E. coli accumulates compound Z, a direct oxidation product of the immediate precursor of MPT, to the same extent as an MPT synthase-deficient strain. In contrast, analysis of the content of compound Z in Delta csdA and Delta sufS strains revealed no such accumulation. These findings indicate that IscS is the primary physiological sulfur-donating enzyme for the generation of the thiocarboxylate of MPT synthase in MPT biosynthesis. Y1 - 2010 UR - http://www.jbc.org/ U6 - https://doi.org/10.1074/jbc.M109.082172 SN - 0021-9258 ER - TY - JOUR A1 - Burschel, Sabrina A1 - Decovic, Doris Kreuzer A1 - Nuber, Franziska A1 - Stiller, Marie A1 - Hofmann, Maud A1 - Zupok, Arkadiusz A1 - Siemiatkowska, Beata A1 - Gorka, Michal Jakub A1 - Leimkühler, Silke A1 - Friedrich, Thorsten T1 - Iron-sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH BT - ubiquinone oxidoreductase (complex I) JF - Molecular microbiology N2 - The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron-sulfur clusters. To identify iron-sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b. Y1 - 2018 U6 - https://doi.org/10.1111/mmi.14137 SN - 0950-382X SN - 1365-2958 VL - 111 IS - 1 SP - 31 EP - 45 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Zupok, Arkadiusz A1 - Górka, Michał Jakub A1 - Siemiatkowska, Beata A1 - Skirycz, Aleksandra A1 - Leimkühler, Silke T1 - Iron-Dependent Regulation of Molybdenum Cofactor Biosynthesis Genes in Escherichia coli JF - Journal of bacteriology N2 - Molybdenum cofactor (Moco) biosynthesis is a complex process that involves the coordinated function of several proteins. In recent years it has become obvious that the availability of iron plays an important role in the biosynthesis of Moco. First, the MoaA protein binds two (4Fe-4S] clusters per monomer. Second, the expression of the moaABCDE and moeAB operons is regulated by FNR, which senses the availability of oxygen via a functional NFe-4S) cluster. Finally, the conversion of cyclic pyranopterin monophosphate to molybdopterin requires the availability of the L-cysteine desulfurase IscS, which is a shared protein with a main role in the assembly of Fe-S clusters. In this report, we investigated the transcriptional regulation of the moaABCDE operon by focusing on its dependence on cellular iron availability. While the abundance of selected molybdoenzymes is largely decreased under iron-limiting conditions, our data show that the regulation of the moaABCDE operon at the level of transcription is only marginally influenced by the availability of iron. Nevertheless, intracellular levels of Moco were decreased under iron-limiting conditions, likely based on an inactive MoaA protein in addition to lower levels of the L-cysteine desulfurase IscS, which simultaneously reduces the sulfur availability for Moco production. IMPORTANCE FNR is a very important transcriptional factor that represents the master switch for the expression of target genes in response to anaerobiosis. Among the FNR-regulated operons in Escherichia coli is the moaABCDE operon, involved in Moco biosynthesis. Molybdoenzymes have essential roles in eukaryotic and prokaryotic organisms. In bacteria, molybdoenzymes are crucial for anaerobic respiration using alternative electron acceptors. This work investigates the connection of iron availability to the biosynthesis of Moco and the production of active molybdoenzymes. KW - Escherichia coli KW - FNR KW - iron regulation KW - iron-sulfur cluster KW - anaerobic respiration KW - molybdenum cofactor Y1 - 2019 U6 - https://doi.org/10.1128/JB.00382-19 SN - 0021-9193 SN - 1098-5530 VL - 201 IS - 17 PB - American Society for Microbiology CY - Washington 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - Bühning, Martin A1 - Friemel, Martin A1 - Leimkühler, Silke T1 - Functional Complementation Studies Reveal Different Interaction Partners of Escherichia coil IscS and Human NFS1 JF - Biochemistry N2 - The trafficking and delivery of sulfur to cofactors and nucleosides is a highly regulated and conserved process among all organisms. All sulfur transfer pathways generally have an L-cysteine desulfurase as an initial sulfur mobilizing enzyme in common, which serves as a sulfur donor for the biosynthesis of sulfur-containing biomolecules like iron sulfur (Fe-S) clusters, thiamine, biotin, lipoic acid, the molybdenum cofactor (Moco), and thiolated nucleosides in tRNA. The human L-cysteine desulfurase NFS1 and the Escherichia coli homologue IscS share a level of amino acid sequence identity of similar to 60%. While E. coli IscS has a versatile role in the cell and was shown to have numerous interaction partners, NFS1 is mainly localized in mitochondria with a crucial role in the biosynthesis of Fe-S clusters. Additionally, NFS1 is also located in smaller amounts in the cytosol with a role in Moco biosynthesis and mcm(5)s(2)U34 thio modifications of nucleosides in tRNA. NFS1 and IscS were conclusively shown to have different interaction partners in their respective organisms. Here, we used functional complementation studies of an E. coli iscS deletion strain with human NFS1 to dissect their conserved roles in the transfer of sulfur to a specific target protein. Our results show that human NFS1 and E. coli IscS share conserved binding sites for proteins involved in Fe-S cluster assembly like IscU, but not with proteins for tRNA thio modifications or Moco biosynthesis. In addition, we show that human NFS1 was almost fully able to complement the role of IscS in Moco biosynthesis when its specific interaction partner protein MOCS3 from humans was also present. Y1 - 2017 U6 - https://doi.org/10.1021/acs.biochem.7b00627 SN - 0006-2960 VL - 56 SP - 4592 EP - 4605 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 - 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 - 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 -