TY - JOUR
A1 - Reeve, Holly A.
A1 - Nicholson, Jake
A1 - Altaf, Farieha
A1 - Lonsdale, Thomas H.
A1 - Preissler, Janina
A1 - Lauterbach, Lars
A1 - Lenz, Oliver
A1 - Leimkühler, Silke
A1 - Hollmann, Frank
A1 - Paul, Caroline E.
A1 - Vincent, Kylie A.
T1 - A hydrogen-driven biocatalytic approach to recycling synthetic analogues of NAD(P)H
JF - Chemical communications : ChemComm
N2 - We demonstrate a recycling system for synthetic nicotinamide cofactor analogues using a soluble hydrogenase with turnover number of >1000 for reduction of the cofactor analogues by H-2.
Coupling this system to an ene reductase, we show quantitative conversion of N-ethylmaleimide to N-ethylsuccinimide.
The biocatalyst system retained >50% activity after 7 h.
Y1 - 2022
U6 - https://doi.org/10.1039/d2cc02411j
SN - 1359-7345
SN - 1364-548X
VL - 58
IS - 75
SP - 10540
EP - 10543
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - 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 - 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 - 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 - Tadjoung Waffo, Armel Franklin
A1 - Mitrova, Biljana
A1 - Tiedemann, Kim
A1 - Iobbi-Nivol, Chantal
A1 - Leimkühler, Silke
A1 - Wollenberger, Ulla
T1 - Electrochemical trimethylamine n-oxide biosensor with enzyme-based oxygen-scavenging membrane for long-term operation under ambient air
JF - Biosensors : open access journal
N2 - An amperometric trimethylamine N-oxide (TMAO) biosensor is reported, where TMAO reductase (TorA) and glucose oxidase (GOD) and catalase (Cat) were immobilized on the electrode surface, enabling measurements of mediated enzymatic TMAO reduction at low potential under ambient air conditions. The oxygen anti-interference membrane composed of GOD, Cat and polyvinyl alcohol (PVA) hydrogel, together with glucose concentration, was optimized until the O-2 reduction current of a Clark-type electrode was completely suppressed for at least 3 h. For the preparation of the TMAO biosensor, Escherichia coli TorA was purified under anaerobic conditions and immobilized on the surface of a carbon electrode and covered by the optimized O-2 scavenging membrane. The TMAO sensor operates at a potential of -0.8 V vs. Ag/AgCl (1 M KCl), where the reduction of methylviologen (MV) is recorded. The sensor signal depends linearly on TMAO concentrations between 2 mu M and 15 mM, with a sensitivity of 2.75 +/- 1.7 mu A/mM. The developed biosensor is characterized by a response time of about 33 s and an operational stability over 3 weeks. Furthermore, measurements of TMAO concentration were performed in 10% human serum, where the lowest detectable concentration is of 10 mu M TMAO.
KW - trimethylamine N-oxide
KW - biosensor
KW - TMAO-reductase
KW - oxygen scavenger
KW - immobilized enzyme
KW - multienzyme electrode
KW - viologen
Y1 - 2021
U6 - https://doi.org/10.3390/bios11040098
SN - 2079-6374
VL - 11
IS - 4
PB - MDPI
CY - Basel
ER -
TY - JOUR
A1 - 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 - 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 - 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 - 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 - 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 - Duffus, Benjamin R.
A1 - Schrapers, Peer
A1 - Schuth, Nils
A1 - Mebs, Stefan
A1 - Dau, Holger
A1 - Leimkühler, Silke
A1 - Haumann, Michael
T1 - Anion binding and oxidative modification at the molybdenum cofactor of formate dehydrogenase from Rhodobacter capsulatus studied by X-ray absorption spectroscopy
JF - Inorganic chemistry
N2 - Formate dehydrogenase (FDH) enzymes are versatile catalysts for CO2 conversion. The FDH from Rhodobacter capsulatus contains a molybdenum cofactor with the dithiolene functions of two pyranopterin guanine dinucleotide molecules, a conserved cysteine, and a sulfido group bound at Mo(VI). In this study, we focused on metal oxidation state and coordination changes in response to exposure to O-2, inhibitory anions, and redox agents using X-ray absorption spectroscopy (XAS) at the Mo K-edge. Differences in the oxidative modification of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor relative to samples prepared aerobically without inhibitor, such as variations in the relative numbers of sulfido (Mo=S) and oxo (Mo=O) bonds, were observed in the presence of azide (N-3(-)) or cyanate (OCN-). Azide provided best protection against O-2, resulting in a quantitatively sulfurated cofactor with a displaced cysteine ligand and optimized formate oxidation activity. Replacement of the cysteine ligand by a formate (HCO2-) ligand at the molybdenum in active enzyme is compatible with our XAS data. Cyanide (CN-) inactivated the enzyme by replacing the sulfido ligand at Mo(VI) with an oxo ligand. Evidence that the sulfido group may become protonated upon molybdenum reduction was obtained. Our results emphasize the role of coordination flexibility at the molybdenum center during inhibitory and catalytic processes of FDH enzymes.
Y1 - 2020
U6 - https://doi.org/10.1021/acs.inorgchem.9b01613
SN - 0020-1669
SN - 1520-510X
VL - 59
IS - 1
SP - 214
EP - 225
PB - American Chemical Society
CY - Washington, DC
ER -
TY - JOUR
A1 - 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 - 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 - Ogunkola, Moses Olalekan
A1 - Guiraudie-Capraz, Gaelle
A1 - Féron, François
A1 - Leimkühler, Silke
T1 - The Human Mercaptopyruvate Sulfurtransferase TUM1 Is Involved in Moco Biosynthesis, Cytosolic tRNA Thiolation and Cellular Bioenergetics in Human Embryonic Kidney Cells
JF - Biomolecules
N2 - Sulfur is an important element that is incorporated into many biomolecules in humans. The incorporation and transfer of sulfur into biomolecules is, however, facilitated by a series of different sulfurtransferases. Among these sulfurtransferases is the human mercaptopyruvate sulfurtransferase (MPST) also designated as tRNA thiouridine modification protein (TUM1). The role of the human TUM1 protein has been suggested in a wide range of physiological processes in the cell among which are but not limited to involvement in Molybdenum cofactor (Moco) biosynthesis, cytosolic tRNA thiolation and generation of H2S as signaling molecule both in mitochondria and the cytosol. Previous interaction studies showed that TUM1 interacts with the L-cysteine desulfurase NFS1 and the Molybdenum cofactor biosynthesis protein 3 (MOCS3). Here, we show the roles of TUM1 in human cells using CRISPR/Cas9 genetically modified Human Embryonic Kidney cells. Here, we show that TUM1 is involved in the sulfur transfer for Molybdenum cofactor synthesis and tRNA thiomodification by spectrophotometric measurement of the activity of sulfite oxidase and liquid chromatography quantification of the level of sulfur-modified tRNA. Further, we show that TUM1 has a role in hydrogen sulfide production and cellular bioenergetics.
KW - Moco biosynthesis
KW - sulfite oxidase
KW - cytosolic tRNA thiolation
KW - 5-methoxycarbonylmethyl-2-thiouridine
KW - H2S biosynthesis
KW - cellular bioenergetics
Y1 - 2023
U6 - https://doi.org/10.3390/biom13010144
SN - 2218-273X
VL - 13
SP - 1
EP - 23
PB - MDPI
CY - Basel, Schweiz
ET - 1
ER -
TY - JOUR
A1 - 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 - 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 - Hasnat, Muhammad Abrar
A1 - Zupok, Arkadiusz
A1 - Olas-Apelt, Justyna Jadwiga
A1 - Müller-Röber, Bernd
A1 - Leimkühler, Silke
T1 - A-type carrier proteins are involved in [4Fe-4S] cluster insertion into the radical S-adenosylmethionine protein MoaA for the synthesis of active molybdoenzymes
JF - Journal of bacteriology
N2 - Iron sulfur (Fe-S) clusters are important biological cofactors present in proteins with crucial biological functions, from photosynthesis to DNA repair, gene expression, and bioenergetic processes. For the insertion of Fe-S clusters into proteins, A-type carrier proteins have been identified. So far, three of them have been characterized in detail in Escherichia coli, namely, IscA, SufA, and ErpA, which were shown to partially replace each other in their roles in [4Fe-4S] cluster insertion into specific target proteins. To further expand the knowledge of [4Fe-4S] cluster insertion into proteins, we analyzed the complex Fe-S cluster-dependent network for the synthesis of the molybdenum cofactor (Moco) and the expression of genes encoding nitrate reductase in E. coli. Our studies include the identification of the A-type carrier proteins ErpA and IscA, involved in [4Fe-4S] cluster insertion into the radical Sadenosyl-methionine (SAM) enzyme MoaA. We show that ErpA and IscA can partially replace each other in their role to provide [4Fe-4S] clusters for MoaA. Since most genes expressing molybdoenzymes are regulated by the transcriptional regulator for fumarate and nitrate reduction (FNR) under anaerobic conditions, we also identified the proteins that are crucial to obtain an active FNR under conditions of nitrate respiration. We show that ErpA is essential for the FNR-dependent expression of the narGHJI operon, a role that cannot be compensated by IscA under the growth conditions tested. SufA does not appear to have a role in Fe-S cluster insertion into MoaA or FNR under anaerobic growth employing nitrate respiration, based on the low level of gene expression.
IMPORTANCE Understanding the assembly of iron-sulfur (Fe-S) proteins is relevant to many fields, including nitrogen fixation, photosynthesis, bioenergetics, and gene regulation. Remaining critical gaps in our knowledge include how Fe-S clusters are transferred to their target proteins and how the specificity in this process is achieved, since different forms of Fe-S clusters need to be delivered to structurally highly diverse target proteins. Numerous Fe-S carrier proteins have been identified in prokaryotes like Escherichia coli, including ErpA, IscA, SufA, and NfuA. In addition, the diverse Fe-S cluster delivery proteins and their target proteins underlie a complex regulatory network of expression, to ensure that both proteins are synthesized under particular growth conditions.
KW - iron-sulfur clusters
KW - Moco biosynthesis
KW - MoaA
KW - A-type carrier protein
KW - FNR
KW - nitrate reductase
KW - molybdenum cofactor
Y1 - 2021
U6 - https://doi.org/10.1128/JB.00086-21
SN - 1098-5530
VL - 203
IS - 12
PB - American Society for Microbiology
CY - Washington
ER -
TY - JOUR
A1 - 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 - JOUR
A1 - Nishino, Takeshi
A1 - Okamoto, Ken
A1 - Leimkühler, Silke
T1 - Enzymes of the Xanthine Oxidase Family
JF - Molybdenum and tungsten enzymes : biochemistry
N2 - Enzymes from the xanthine oxidase (XO) family of molybdenum enzymes are generally, with some exceptions, molybdenum iron–sulfur flavin hydroxylases. Mammalian xanthine oxidoreductase and aldehyde oxidase were among the first enzymes to be studied in detail more than 100 years ago and, surprisingly, they continue to be thoroughly studied in molecular detail with many open and unresolved questions remaining. Enzymes of the XO family are characterized by a molybdenum cofactor (Moco) active site with a MoVIOS(OH) ligand sphere where substrate hydroxylation of either aromatic or aliphatic carbon centers is catalyzed. During the reaction, electrons are transferred to the oxidizing substrate, most commonly O2 or NAD+, which react at the FAD site.
Y1 - 2016
SN - 978-1-78262-391-5
SN - 978-1-78262-089-1
SN - 978-1-78262-881-1
U6 - https://doi.org/10.1039/9781782623915-00192
VL - 5
SP - 192
EP - 239
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Leimkühler, Silke
A1 - Lemaire, Olivier N.
A1 - Iobbi-Nivol, Chantal
T1 - Bacterial Molybdoenzymes
BT - Chaperones, Assembly and Insertion
JF - Molybdenum and tungsten enzymes : biochemistry
N2 - The biogenesis of molybdoenzymes is a cytoplasmic event requiring both the folded apoenzymes and the matured molybdenum cofactor. The structure and the complexity of the molybdenum cofactor varies in each molybdoenzyme family and consequently different accessory proteins are required for the maturation of the respective enzymes. Thus, for enzymes of both the DMSO reductase and xanthine oxidase families, specific chaperones exist which are dedicated to increase the stability and the folding of specific members of each family. In this review, we describe the role of these chaperones for molybdoenzyme maturation. We present a model which describes step by step the mechanism of the maturation of representative molybdoenzymes from each family.
Y1 - 2016
SN - 978-1-78262-391-5
SN - 978-1-78262-089-1
U6 - https://doi.org/10.1039/9781782623915-00117
VL - 5
SP - 117
EP - 142
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Leimkühler, Silke
A1 - Mendel, Ralf-Rainer
T1 - Molybdenum Cofactor Biosynthesis
JF - Molybdenum and tungsten enzymes: biochemistry
N2 - The biosynthesis of the molybdenum cofactor (Moco) is highly conserved among all kingdoms of life. In all molybdoenzymes with the exception of nitrogenase, the molybdenum atom is coordinated to a dithiolene group present in the pterin-based 6-alkyl side chain of molybdopterin (MPT). In general, the biosynthesis of Moco can be divided into three steps in eukaryotes, and four steps in bacteria and archaea: (i) the starting point is the formation of the cyclic pyranopterin monophosphate (cPMP) from 5′GTP, (ii) in the second step the two sulfur molecules are inserted into cPMP leading to the formation of MPT, (iii) in the third step the molybdenum atom is inserted into molybdopterin to form Moco and (iv) additional modification of Moco occurs in bacteria and archaea with the attachment of a nucleotide (CMP or GMP) to the phosphate group of MPT, forming the dinucleotide variants of Moco. This review will focus on the biosynthesis of Moco in bacteria, humans and plants.
Y1 - 2016
SN - 978-1-78262-391-5
SN - 978-1-78262-089-1
SN - 978-1-78262-881-1
U6 - https://doi.org/10.1039/9781782623915
VL - 5
SP - 100
EP - 116
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - 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 - Tiedemann, Kim
A1 - Iobbi-Nivol, Chantal
A1 - Leimkühler, Silke
T1 - The Role of the Nucleotides in the Insertion of the bis-Molybdopterin Guanine Dinucleotide Cofactor into apo-Molybdoenzymes
JF - Molecules
N2 - The role of the GMP nucleotides of the bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor of the DMSO reductase family has long been a subject of discussion. The recent characterization of the bis-molybdopterin (bis-Mo-MPT) cofactor present in the E. coli YdhV protein, which differs from bis-MGD solely by the absence of the nucleotides, now enables studying the role of the nucleotides of bis-MGD and bis-MPT cofactors in Moco insertion and the activity of molybdoenzymes in direct comparison. Using the well-known E. coli TMAO reductase TorA as a model enzyme for cofactor insertion, we were able to show that the GMP nucleotides of bis-MGD are crucial for the insertion of the bis-MGD cofactor into apo-TorA.
KW - bis-MGD
KW - chaperone
KW - molybdenum cofactor
KW - TMAO reductase
Y1 - 2022
U6 - https://doi.org/10.3390/molecules27092993
SN - 1420-3049
VL - 27
SP - 1
EP - 15
PB - MDPI
CY - Basel, Schweiz
ET - 9
ER -
TY - JOUR
A1 - Reschke, Stefan
A1 - Mebs, Stefan
A1 - Sigfridsson-Clauss, Kajsa G. V.
A1 - Kositzki, Ramona
A1 - Leimkühler, Silke
A1 - Haumann, Michael
T1 - Protonation and Sulfido versus Oxo Ligation Changes at the Molybdenum Cofactor in Xanthine Dehydrogenase (XDH) Variants Studied by X-ray Absorption Spectroscopy
JF - Inorganic chemistry
N2 - Enzymes of the xanthine oxidase family are among the best characterized mononuclear molybdenum enzymes. Open questions about their mechanism of transfer of an oxygen atom to the substrate remain. The enzymes share a molybdenum cofactor (Moco) with the metal ion binding a molybdopterin (MPT) molecule via its dithiolene function and terminal sulfur and oxygen groups. For xanthine dehydrogenase (XDH) from the bacterium Rhodobacter capsulatus, we used X-ray absorption spectroscopy to determine the Mo site structure, its changes in a pH range of 5-10, and the influence of amino acids (Glu730 and Gln179) close to Moco in wild-type (WT), Q179A, and E730A variants, complemented by enzyme kinetics and quantum chemical studies. Oxidized WT and Q179A revealed a similar Mo (VI) ion with each one MPT, Mo=O, Mo-O-, and Mo=S ligand, and a weak Mo-O(E730) bond at alkaline pH. Protonation of an oxo to a hydroxo (OH) ligand (pK similar to 6.8) causes inhibition of XDH at acidic pH, whereas deprotonated xanthine (pK similar to 8.8) is an inhibitor at alkaline pH. A similar acidic pK for the WT and Q179A. variants, as well as the metrical parameters of the Mo site and density functional theory calculations, suggested protonation at the equatorial oxo group. The sulfido was replaced with an oxo ligand in the inactive E730A variant, further showing another oxo and one Mo OH ligand at Mo, which are independent of pH. Our findings suggest a reaction mechanism for XDH in which an initial oxo rather than a hydroxo group and the sulfido ligand are essential for xanthine oxidation.
Y1 - 2017
U6 - https://doi.org/10.1021/acs.inorgchem.6b02846
SN - 0020-1669
SN - 1520-510X
VL - 56
IS - 4
SP - 2165
EP - 2176
PB - American Chemical Society
CY - Washington
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 - 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 - 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 - 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 - Paragas, Erickson M.
A1 - Humphreys, Sara C.
A1 - Min, Joshua
A1 - Joswig-Jones, Carolyn A.
A1 - Leimkühler, Silke
A1 - Jones, Jeffrey P.
T1 - ecoAO
BT - a simple system for the study of human aldehyde oxidases role in drug metabolism
JF - ACS OMEGA
N2 - Although aldehyde oxidase (AO) is an important hepatic drug-metabolizing enzyme, it remains understudied and is consequently often overlooked in preclinical studies, an oversight that has resulted in the failure of multiple clinical trials. AO’s preclusion to investigation stems from the following: (1) difficulties synthesizing metabolic standards due to the chemospecificity and regiospecificity of the enzyme and (2) significant inherent variability across existing in vitro systems including liver cytosol, S9 fractions, and primary hepatocytes, which lack specificity and generate discordant expression and activity profiles. Here, we describe a practical bacterial biotransformation system, ecoAO, addressing both issues simultaneously. ecoAO is a cell paste of MoCo-producing Escherichia coli strain TP1017 expressing human AO. It exhibits specific activity toward known substrates, zoniporide, 4-trans-(N,N-dimethylamino)cinnamaldehyde, O6-benzylguanine, and zaleplon; it also has utility as a biocatalyst, yielding milligram quantities of synthetically challenging metabolite standards such as 2-oxo-zoniporide. Moreover, ecoAO enables routine determination of kcat and V/K, which are essential parameters for accurate in vivo clearance predictions. Furthermore, ecoAO has potential as a preclinical in vitro screening tool for AO activity, as demonstrated by its metabolism of 3-aminoquinoline, a previously uncharacterized substrate. ecoAO promises to provide easy access to metabolites with the potential to improve pharmacokinetic clearance predictions and guide drug development.
Y1 - 2017
U6 - https://doi.org/10.1021/acsomega.7b01054
SN - 2470-1343
VL - 2
SP - 4820
EP - 4827
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Küçükgöze, Gökhan
A1 - Leimkühler, Silke
T1 - Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities
JF - PLOS ONE
N2 - Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. In mouse, the isoforms mAOX1, mAOX3, mAOX4 and mAOX2 are present. We previously established a codon-optimized heterologous expression systems for the mAOX1-4 isoforms in Escherichia coli that gives yield to sufficient amounts of active protein for kinetic characterizations and sets the basis in this study for site-directed mutagenesis and structure-function studies. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes on a larger number of substrates has never been performed. Here, thirty different structurally related aromatic, aliphatic and N-heterocyclic compounds were used as substrates, and the kinetic parameters of all four mAOX enzymes were directly compared. The results show that especially mAOX4 displays a higher substrate selectivity, while no major differences between mAOX1, mAOX2 and mAOX3 were identified. Generally, mAOX1 was the enzyme with the highest catalytic turnover for most substrates. To understand the factors that contribute to the substrate specificity of mAOX4, site-directed mutagenesis was applied to substitute amino acids in the substrate-binding funnel by the ones present in mAOX1, mAOX3, and mAOX2. An increase in activity was obtained by the amino acid exchange M1088V in the active site identified to be specific for mAOX4, to the amino acid identified in mAOX3.
Y1 - 2018
U6 - https://doi.org/10.1371/journal.pone.0191819
SN - 1932-6203
VL - 13
IS - 1
PB - Public Library of Science
CY - San Fransisco
ER -
TY - JOUR
A1 - Marelja, Zvonimir
A1 - Leimkühler, Silke
A1 - Missirlis, Fanis
T1 - Iron sulfur and molybdenum cofactor enzymes regulate the drosophila life cycle by controlling cell metabolism
JF - Frontiers in physiology
N2 - Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.
KW - aldehyde oxidase
KW - DNA polymerase
KW - electron transport chain
KW - ecdysone
KW - iron regulatory protein
KW - quiescent mitochondria
KW - magnetoreceptor
KW - mitoflashes
Y1 - 2018
U6 - https://doi.org/10.3389/fphys.2018.00050
SN - 1664-042X
VL - 9
PB - Frontiers Research Foundation
CY - Lausanne
ER -
TY - JOUR
A1 - Kaufmann, Hans Paul
A1 - Duffus, Benjamin R.
A1 - Mitrova, Biljana
A1 - Iobbi-Nivol, Chantal
A1 - Teutloff, Christian
A1 - Nimtz, Manfred
A1 - Jaensch, Lothar
A1 - Wollenberger, Ulla
A1 - Leimkühler, Silke
T1 - Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamie N-Oxide Reductase
JF - Biochemistry
N2 - The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.
Y1 - 2018
U6 - https://doi.org/10.1021/acs.biochem.7b01108
SN - 0006-2960
VL - 57
IS - 7
SP - 1130
EP - 1143
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Kaufmann, Paul
A1 - Duffus, Benjamin R.
A1 - Teutloff, Christian
A1 - Leimkühler, Silke
T1 - Functional Studies on Oligotropha carboxidovorans Molybdenum-Copper CO Dehydrogenase Produced in Escherichia coli
JF - Biochemistry
N2 - The Mo/Cu-dependent CO dehydrogenase (CODH) from Oligotropha carboxidovorans is an enzyme that is able to catalyze both the oxidation of CO to CO2 and the oxidation of H-2 to protons and electrons. Despite the close to atomic resolution structure (1.1 angstrom), significant uncertainties have remained with regard to the reaction mechanism of substrate oxidation at the unique Mo/Cu center, as well as the nature of intermediates formed during the catalytic cycle. So far, the investigation of the role of amino acids at the active site was hampered by the lack of a suitable expression system that allowed for detailed site-directed mutagenesis studies at the active site. Here, we report on the establishment of a functional heterologous expression system of O. carboxidovorans CODH in Escherichia coli. We characterize the purified enzyme in detail by a combination of kinetic and spectroscopic studies and show that it was purified in a form with characteristics comparable to those of the native enzyme purified from O. carboxidovorans. With this expression system in hand, we were for the first time able to generate active-site variants of this enzyme. Our work presents the basis for more detailed studies of the reaction mechanism for CO and H-2 oxidation of Mo/Cu-dependent CODHs in the future.
Y1 - 2018
U6 - https://doi.org/10.1021/acs.biochem.8b00128
SN - 0006-2960
VL - 57
IS - 19
SP - 2889
EP - 2901
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - Öner, Ibrahim Halil
A1 - Querebillo, Christine Joy
A1 - David, Christin
A1 - Gernert, Ulrich
A1 - Walter, Carsten
A1 - Driess, Matthias
A1 - Leimkühler, Silke
A1 - Ly, Khoa Hoang
A1 - Weidinger, Inez M.
T1 - High electromagnetic field enhancement of TiO2 nanotube electrodes
JF - Angewandte Chemie : a journal of the Gesellschaft Deutscher Chemiker ; International edition
N2 - We present the fabrication of TiO2 nanotube electrodes with high biocompatibility and extraordinary spectroscopic properties. Intense surface-enhanced resonance Raman signals of the heme unit of the redox enzyme Cytochromeb(5) were observed upon covalent immobilization of the protein matrix on the TiO2 surface, revealing overall preserved structural integrity and redox behavior. The enhancement factor could be rationally controlled by varying the electrode annealing temperature, reaching a record maximum value of over 70 at 475 degrees C. For the first time, such high values are reported for non-directly surface-interacting probes, for which the involvement of charge-transfer processes in signal amplification can be excluded. The origin of the surface enhancement is exclusively attributed to enhanced localized electric fields resulting from the specific optical properties of the nanotubular geometry of the electrode.
KW - electromagnetic field enhancement
KW - photonic crystals
KW - spectro-electrochemistry
KW - surface-enhanced Raman spectroscopy
KW - TiO2 nanotubes
Y1 - 2018
U6 - https://doi.org/10.1002/anie.201802597
SN - 1433-7851
SN - 1521-3773
VL - 57
IS - 24
SP - 7225
EP - 7229
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Mota, Cristiano
A1 - Coelho, Catarina
A1 - Leimkühler, Silke
A1 - Garattini, Enrico
A1 - Terao, Mineko
A1 - Santos-Silva, Teresa
A1 - Romao, Maria Joao
T1 - Critical overview on the structure and metabolism of human aldehyde oxidase and its role in pharmacokinetics
JF - Coordination chemistry reviews
N2 - Aldehyde oxidases are molybdenum and flavin dependent enzymes characterized by a very wide substrate specificity and performing diverse reactions that include oxidations (e.g., aldehydes and azaheterocycles), hydrolysis of amide bonds, and reductions (e.g., nitro, S-oxides and N-oxides). Oxidation reactions and amide hydrolysis occur at the molybdenum site while the reductions are proposed to occur at the flavin site. AOX activity affects the metabolism of different drugs and xenobiotics, some of which designed to resist other liver metabolizing enzymes (e.g., cytochrome P450 monooxygenase isoenzymes), raising its importance in drug development. This work consists of a comprehensive overview on aldehyde oxidases, concerning the genetic evolution of AOX, its diversity among the human population, the crystal structures available, the known catalytic reactions and the consequences in pre-clinical pharmacokinetic and pharmacodynamic studies. Analysis of the different animal models generally used for pre-clinical trials and comparison between the human (hAOX1), mouse homologs as well as the related xanthine oxidase (XOR) are extensively considered. The data reviewed also include a systematic analysis of representative classes of molecules that are hAOX1 substrates as well as of typical and well characterized hAOX1 inhibitors. The considerations made on the basis of a structural and functional analysis are correlated with reported kinetic and metabolic data for typical classes of drugs, searching for potential structural determinants that may dictate substrate and/or inhibitor specificities.
KW - Drug metabolism
KW - Aldehyde oxidase
KW - Xenobiotics
KW - Molybdoenzymes
KW - Non-CYP enzymes
KW - Hepatic clearance
Y1 - 2018
U6 - https://doi.org/10.1016/j.ccr.2018.04.006
SN - 0010-8545
SN - 1873-3840
VL - 368
SP - 35
EP - 59
PB - Elsevier
CY - Lausanne
ER -
TY - JOUR
A1 - Otto, Nils
A1 - Marelja, Zvonimir
A1 - Schoofs, Andreas
A1 - Kranenburg, Holger
A1 - Bittern, Jonas
A1 - Yildirim, Kerem
A1 - Berh, Dimitri
A1 - Bethke, Maria
A1 - Thomas, Silke
A1 - Rode, Sandra
A1 - Risse, Benjamin
A1 - Jiang, Xiaoyi
A1 - Pankratz, Michael
A1 - Leimkühler, Silke
A1 - Klämbt, Christian
T1 - The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia
JF - Nature Communications
N2 - Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, we screened for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, our findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity.
Y1 - 2018
U6 - https://doi.org/10.1038/s41467-018-05645-z
SN - 2041-1723
VL - 9
PB - Nature Publ. Group
CY - London
ER -
TY - JOUR
A1 - Lemaire, Olivier N.
A1 - Infossi, Pascale
A1 - Chaouche, Amine Ali
A1 - Espinosa, Leon
A1 - Leimkühler, Silke
A1 - Giudici-Orticoni, Marie-Therese
A1 - Mejean, Vincent
A1 - Iobbi-Nivol, Chantal
T1 - Small membranous proteins of the TorE/NapE family, crutches for cognate respiratory systems in Proteobacteria
JF - Scientific reports
N2 - In this report, we investigate small proteins involved in bacterial alternative respiratory systems that improve the enzymatic efficiency through better anchorage and multimerization of membrane components. Using the small protein TorE of the respiratory TMAO reductase system as a model, we discovered that TorE is part of a subfamily of small proteins that are present in proteobacteria in which they play a similar role for bacterial respiratory systems. We reveal by microscopy that, in Shewanella oneidensis MR1, alternative respiratory systems are evenly distributed in the membrane contrary to what has been described for Escherichia coli. Thus, the better efficiency of the respiratory systems observed in the presence of the small proteins is not due to a specific localization in the membrane, but rather to the formation of membranous complexes formed by TorE homologs with their c-type cytochrome partner protein. By an in vivo approach combining Clear Native electrophoresis and fluorescent translational fusions, we determined the 4: 4 stoichiometry of the complexes. In addition, mild solubilization of the cytochrome indicates that the presence of the small protein reinforces its anchoring to the membrane. Therefore, assembly of the complex induced by this small protein improves the efficiency of the respiratory system.
Y1 - 2018
U6 - https://doi.org/10.1038/s41598-018-31851-2
SN - 2045-2322
VL - 8
PB - Nature Publ. Group
CY - London
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 - 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 - 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 - 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 - 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 - Neukranz, Yannika
A1 - Kotter, Annika
A1 - Beilschmidt, Lena
A1 - Marelja, Zvonimir
A1 - Helm, Mark
A1 - Graf, Ralph
A1 - Leimkühler, Silke
T1 - Analysis of the Cellular Roles of MOCS3 Identifies a MOCS3-Independent Localization of NFS1 at the Tips of the Centrosome
JF - Biochemistry
N2 - The deficiency of the molybdenum cofactor (Moco) is an autosomal recessive disease, which leads to the loss of activity of all molybdoenzymes in humans with sulfite oxidase being the essential protein. Moco deficiency generally results in death in early childhood. Moco is a sulfur-containing cofactor synthesized in the cytosol with the sulfur being provided by a sulfur relay system composed of the L-cysteine desulfurase NFS1, MOCS3, and MOCS2A. Human MOCS3 is a dual-function protein that was shown to play an important role in Moco biosynthesis and in the mcm(5)s(2) U thio modifications of nucleosides in cytosolic tRNAs for Lys, Gln, and Glu. In this study, we constructed a homozygous MOCS3 knockout in HEK293T cells using the CRISPR/Cas9 system. The effects caused by the absence of MOCS3 were analyzed in detail. We show that sulfite oxidase activity was almost completely abolished, on the basis of the absence of Moco in these cells. In addition, mcm(5)s(2)U thio-modified tRNAs were not detectable. Because the L-cysteine desulfurase NFS1 was shown to act as a sulfur donor for MOCS3 in the cytosol, we additionally investigated the impact of a MOCS3 knockout on the cellular localization of NFS1. By different methods, we identified a MOCS3-independent novel localization of NFS1 at the centrosome.
Y1 - 2019
U6 - https://doi.org/10.1021/acs.biochem.8b01160
SN - 0006-2960
VL - 58
IS - 13
SP - 1786
EP - 1798
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - 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 - 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 - Badalyan, Artavazd
A1 - Dierich, Marlen
A1 - Stiba, Konstanze
A1 - Schwuchow, Viola
A1 - Leimkühler, Silke
A1 - Wollenberger, Ulla
T1 - Electrical wiring of the aldehyde oxidoreductase PaoABC with a polymer containing osmium redox centers
BT - biosensors for benzaldehyde and GABA
JF - Biosensors
N2 - Biosensors for the detection of benzaldehyde and g-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below −0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer.
KW - redox polymer
KW - aldehyde oxidoreductase
KW - ionic strength
KW - benzaldehyde
KW - GABA
KW - biosensor
Y1 - 2014
U6 - https://doi.org/10.3390/bios4040403
VL - 4
IS - 4
SP - 403
EP - 421
PB - MDPI
CY - Basel
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 - GEN
A1 - Badalyan, Artavazd
A1 - Dierich, Marlen
A1 - Stiba, Konstanze
A1 - Schwuchow, Viola
A1 - Leimkühler, Silke
A1 - Wollenberger, Ulla
T1 - Electrical wiring of the aldehyde oxidoreductase PaoABC with a polymer containing osmium redox centers
BT - biosensors for benzaldehyde and GABA
T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
N2 - Biosensors for the detection of benzaldehyde and g-aminobutyric acid (GABA) are reported using aldehyde oxidoreductase PaoABC from Escherichia coli immobilized in a polymer containing bound low potential osmium redox complexes. The electrically connected enzyme already electrooxidizes benzaldehyde at potentials below −0.15 V (vs. Ag|AgCl, 1 M KCl). The pH-dependence of benzaldehyde oxidation can be strongly influenced by the ionic strength. The effect is similar with the soluble osmium redox complex and therefore indicates a clear electrostatic effect on the bioelectrocatalytic efficiency of PaoABC in the osmium containing redox polymer. At lower ionic strength, the pH-optimum is high and can be switched to low pH-values at high ionic strength. This offers biosensing at high and low pH-values. A “reagentless” biosensor has been formed with enzyme wired onto a screen-printed electrode in a flow cell device. The response time to addition of benzaldehyde is 30 s, and the measuring range is between 10–150 µM and the detection limit of 5 µM (signal to noise ratio 3:1) of benzaldehyde. The relative standard deviation in a series (n = 13) for 200 µM benzaldehyde is 1.9%. For the biosensor, a response to succinic semialdehyde was also identified. Based on this response and the ability to work at high pH a biosensor for GABA is proposed by coimmobilizing GABA-aminotransferase (GABA-T) and PaoABC in the osmium containing redox polymer.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1082
KW - redox polymer
KW - aldehyde oxidoreductase
KW - ionic strength
KW - benzaldehyde
KW - GABA
KW - biosensor
Y1 - 2021
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-475070
SN - 1866-8372
IS - 1082
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 - 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 - 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 - 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 - GEN
A1 - Spricigo, Roberto
A1 - Dronov, Roman
A1 - Lisdat, Fred
A1 - Leimkühler, Silke
A1 - Scheller, Frieder W.
A1 - Wollenberger, Ursula
T1 - Electrocatalytic sulfite biosensor with human sulfite oxidase co-immobilized with cytochrome c in a polyelectrolyte-containing multilayer
T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
N2 - An efficient electrocatalytic biosensor for sulfite detection was developed by co-immobilizing sulfite oxidase and cytochrome c with polyaniline sulfonic acid in a layer-by-layer assembly. QCM, UV-Vis spectroscopy and cyclic voltammetry revealed increasing loading of electrochemically active protein with the formation of multilayers. The sensor operates reagentless at low working potential. A catalytic oxidation current was detected in the presence of sulfite at the modified gold electrode, polarized at +0.1 V ( vs. Ag/AgCl 1 M KCl). The stability of the biosensor performance was characterized and optimized. A 17-bilayer electrode has a linear range between 1 and 60 mu M sulfite with a sensitivity of 2.19 mA M-1 sulfite and a response time of 2 min. The electrode retained a stable response for 3 days with a serial reproducibility of 3.8% and lost 20% of sensitivity after 5 days of operation. It is possible to store the sensor in a dry state for more than 2 months. The multilayer electrode was used for determination of sulfite in unspiked and spiked samples of red and white wine. The recovery and the specificity of the signals were evaluated for each sample.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 945
KW - bioelectrocatalysis
KW - sulfite
KW - sulfite oxidase
KW - cytochrome c
KW - multilayer
Y1 - 2020
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-431176
SN - 1866-8372
IS - 945
SP - 225
EP - 233
ER -
TY - GEN
A1 - Lemaire, Olivier N.
A1 - Infossi, Pascale
A1 - Chaouche, Amine Ali
A1 - Espinosa, Leon
A1 - Leimkühler, Silke
A1 - Giudici-Orticoni, Marie-Thérèse
A1 - Méjean, Vincent
A1 - Iobbi-Nivol, Chantal
T1 - Small membranous proteins of the TorE/NapE family, crutches for cognate respiratory systems in Proteobacteria
T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
N2 - In this report, we investigate small proteins involved in bacterial alternative respiratory systems that improve the enzymatic efficiency through better anchorage and multimerization of membrane components. Using the small protein TorE of the respiratory TMAO reductase system as a model, we discovered that TorE is part of a subfamily of small proteins that are present in proteobacteria in which they play a similar role for bacterial respiratory systems. We reveal by microscopy that, in Shewanella oneidensis MR1, alternative respiratory systems are evenly distributed in the membrane contrary to what has been described for Escherichia coli. Thus, the better efficiency of the respiratory systems observed in the presence of the small proteins is not due to a specific localization in the membrane, but rather to the formation of membranous complexes formed by TorE homologs with their c-type cytochrome partner protein. By an in vivo approach combining Clear Native electrophoresis and fluorescent translational fusions, we determined the 4: 4 stoichiometry of the complexes. In addition, mild solubilization of the cytochrome indicates that the presence of the small protein reinforces its anchoring to the membrane. Therefore, assembly of the complex induced by this small protein improves the efficiency of the respiratory system.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 933
KW - trimethylamine n-oxide
KW - molybdenum cofactor biosynthesis
KW - cytochrome bd oxidase
KW - c-type cytochromes
KW - escherichia-coli
KW - swiss-model
KW - native electrophoresis
KW - mutational analysis
KW - reductase
KW - nitrate
KW - microbiology
KW - microbiology techniques
Y1 - 2020
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-459208
SN - 1866-8372
IS - 933
ER -
TY - GEN
A1 - Marelja, Zvonimir
A1 - Leimkühler, Silke
A1 - Missirlis, Fanis
T1 - Iron sulfur and molybdenum cofactor enzymes regulate the Drosophila life cycle by controlling cell metabolism
T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
N2 - Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 925
KW - aldehyde oxidase
KW - DNA polymerase
KW - electron transport chain
KW - ecdysone
KW - iron regulatory protein
KW - quiescent mitochondria
KW - magnetoreceptor
KW - mitoflashes
Y1 - 2020
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-445670
SN - 1866-8372
IS - 925
ER -
TY - JOUR
A1 - Hahnewald, Rita
A1 - Leimkühler, Silke
A1 - Vilaseca, Antonia
A1 - Acquaviva-Bourdain, Cecile
A1 - Lenz, Ulrike
A1 - Reiss, Jochen
T1 - A novel MOCS2 mutation reveals coordinated expression of the small and large subunit of molybdopterin synthase
JF - Molecular genetics and metabolism
N2 - The small and large subunits of molybdopterin (MPT) synthase (MOCS2A and MOCS2B), are both encoded by the MOCS2 gene in overlapping and shifted open reading frames (ORFs), which is a highly unusual structure for eukaryotes. Theoretical analysis of genomic sequences suggested that the expression of these overlapping ORFs is facilitated by the use of alternate first exons leading to alternative transcripts. Here, we confirm the existence of these overlapping transcripts experimentally. Further, we identified a deletion in a molybdenum cofactor deficient patient, which removes the start codon for the small subunit (MOCS2A). We observed undisturbed production of both transcripts, while Western blot analysis demonstrated that MOCS2B, the large subunit, is unstable in the absence of MOCS2A. This reveals new insights into the expression of this evolutionary ancient anabolic system.
KW - molybdenum cofactor deficiency
KW - MOCS2
KW - overlapping reading frames
Y1 - 2006
U6 - https://doi.org/10.1016/j.ymgme.2006.04.008
SN - 1096-7192
VL - 89
IS - 3
SP - 210
EP - 213
PB - Elsevier
CY - San Diego
ER -
TY - JOUR
A1 - 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 - Foti, Alessandro
A1 - Dorendorf, Frank
A1 - Leimkühler, Silke
T1 - A single nucleotide polymorphism causes enhanced radical oxygen species production by human aldehyde oxidase
JF - PLoS one
N2 - Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. The physiological function of mammalian AOX isoenzymes is still unclear, however, human AOX (hAOX1) is an emerging enzyme in phase-I drug metabolism. Indeed, the number of xenobiotics acting as hAOX1 substrates is increasing. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified within the hAOX1 gene. SNPs are a major source of inter-individual variability in the human population, and SNP-based amino acid exchanges in hAOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. In this report we selected ten novel SNPs resulting in amino acid exchanges in proximity to the FAD site of hAOX1 and characterized the purified enzymes after heterologous expression in Escherichia coli. The hAOX1 variants were characterized carefully by quantitative differences in their ability to produce superoxide radical. ROS represent prominent key molecules in physiological and pathological conditions in the cell. Our data reveal significant alterations in superoxide anion production among the variants. In particular the SNP-based amino acid exchange L438V in proximity to the isoalloxanzine ring of the FAD cofactor resulted in increased rate of superoxide radical production of 75%. Considering the high toxicity of the superoxide in the cell, the hAOX1-L438V SNP variant is an eventual candidate for critical or pathological roles of this natural variant within the human population.
Y1 - 2017
U6 - https://doi.org/10.1371/journal.pone.0182061
SN - 1932-6203
VL - 12
SP - 18338
EP - 18347
PB - PLoS
CY - San Fransisco
ER -
TY - JOUR
A1 - Kuecuekgoeze, Goekhan
A1 - Terao, Mineko
A1 - Garattini, Enrico
A1 - Leimkühler, Silke
T1 - Direct Comparison of the Enzymatic Characteristics and Superoxide Production of the Four Aldehyde Oxidase Enzymes Present in Mouse
JF - Drug metabolism and disposition : the biological fate of chemicals
N2 - Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4, and mAOX2, which are the products of distinct genes, are present in the mouse. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes has never been performed. In this report, the four catalytically active mAOX enzymes were purified after heterologous expression in Escherichia coli. The kinetic parameters of the four mouse AOX enzymes were determined and compared with the use of six predicted substrates of physiologic and toxicological interest, i.e., retinaldehyde, N1-methylnicotinamide, pyridoxal, vanillin, 4-(dimethylamino) cinnamaldehyde (p-DMAC), and salicylaldehyde. While retinaldehyde, vanillin, p-DMAC, and salycilaldehyde are efficient substrates for the four mouse AOX enzymes, N1-methylnicotinamide is not a substrate of mAOX1 or mAOX4, and pyridoxal is notmetabolized by any of the purified enzymes. Overall, mAOX1, mAOX2, mAOX3, and mAOX4 are characterized by significantly different KM and kcat values for the active substrates. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40% in relation to moles of substrate converted, and mAOX1, the homolog to the human enzyme, produces a rate of approximately 30% of superoxide radicals with the same substrate.
Y1 - 2017
U6 - https://doi.org/10.1124/dmd.117.075937
SN - 0090-9556
SN - 1521-009X
VL - 45
SP - 947
EP - 955
PB - American Society for Pharmacology and Experimental Therapeutics
CY - Bethesda
ER -
TY - JOUR
A1 - Zeng, Ting
A1 - Leimkühler, Silke
A1 - Wollenberger, Ulla
A1 - Fourmond, Vincent
T1 - Transient Catalytic Voltammetry of Sulfite Oxidase Reveals Rate Limiting Conformational Changes
JF - Journal of the American Chemical Society
N2 - Sulfite oxidases are metalloenzymes that oxidize sulfite to sulfate at a molybdenum active site. In vertebrate sulfite oxidases, the electrons generated at the Mo center are transferred to an external electron acceptor via a heme domain, which can adopt two conformations: a “closed” conformation, suitable for internal electron transfer, and an “open” conformation suitable for intermolecular electron transfer. This conformational change is an integral part of the catalytic cycle. Sulfite oxidases have been wired to electrode surfaces, but their immobilization leads to a significant decrease in their catalytic activity, raising the question of the occurrence of the conformational change when the enzyme is on an electrode. We recorded and quantitatively modeled for the first time the transient response of the catalytic cycle of human sulfite oxidase immobilized on an electrode. We show that conformational changes still occur on the electrode, but at a lower rate than in solution, which is the reason for the decrease in activity of sulfite oxidases upon immobilization.
Y1 - 2017
U6 - https://doi.org/10.1021/jacs.7b05480
SN - 0002-7863
VL - 139
SP - 11559
EP - 11567
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - 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 - 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 - 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 - Iobbi-Nivol, Chantal
T1 - Bacterial molybdoenzymes: old enzymes for new purposes
JF - FEMS microbiology reviews
N2 - Molybdoenzymes are widespread in eukaryotic and prokaryotic organisms where they play crucial functions in detoxification reactions in the metabolism of humans and bacteria, in nitrate assimilation in plants and in anaerobic respiration in bacteria. To be fully active, these enzymes require complex molybdenum-containing cofactors, which are inserted into the apoenzymes after folding. For almost all the bacterial molybdoenzymes, molybdenum cofactor insertion requires the involvement of specific chaperones. In this review, an overview on the molybdenum cofactor biosynthetic pathway is given together with the role of specific chaperones dedicated for molybdenum cofactor insertion and maturation. Many bacteria are involved in geochemical cycles on earth and therefore have an environmental impact. The roles of molybdoenzymes in bioremediation and for environmental applications are presented.This review gives an overview of the diverse mechanisms leading to the insertion of the different forms of the molybdenum cofactor into the respective target enzymes and summarizes the roles of different molybdoenzymes in the environment.This review gives an overview of the diverse mechanisms leading to the insertion of the different forms of the molybdenum cofactor into the respective target enzymes and summarizes the roles of different molybdoenzymes in the environment.
KW - molybdenum cofactor
KW - specific chaperons
KW - TorD family
KW - XdhC
KW - molybdoenzyme maturation
KW - bioremediation
Y1 - 2016
U6 - https://doi.org/10.1093/femsre/fuv043
SN - 0168-6445
SN - 1574-6976
VL - 40
SP - 1
EP - 18
PB - Oxford Univ. Press
CY - Oxford
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 - 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 - 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, 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 - 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 - 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 - 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 - 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 - 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 - 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 - 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 - GEN
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
T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe
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.
T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 551
KW - sulfur cluster formation
KW - Escherichia coli
KW - cysteine desulfurase
KW - interacting protein
KW - bacterial frataxin
KW - statistical-model
KW - biogenesis
KW - biosynthesis
KW - NFS1
KW - deficiency
Y1 - 2019
U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-411906
SN - 1866-8372
IS - 551
ER -
TY - JOUR
A1 - McKenna, Shane M.
A1 - Leimkühler, Silke
A1 - Herter, Susanne
A1 - Turner, Nicholas J.
A1 - Carnell, Andrew J.
T1 - Enzyme cascade reactions: synthesis of furandicarboxylic acid (FDCA) and carboxylic acids using oxidases in tandem
JF - Green chemistry : an international journal and green chemistry resource
N2 - A one-pot tandem enzyme reaction using galactose oxidase M3-5 and aldehyde oxidase PaoABC was used to convert hydroxymethylfurfural (HMF) to the pure bioplastics precursor FDCA in 74% isolated yield. A range of alcohols was also converted to carboxylic acids in high yield under mild conditions.
Y1 - 2015
U6 - https://doi.org/10.1039/c5gc00707k
SN - 1463-9262
SN - 1463-9270
VL - 17
IS - 6
SP - 3271
EP - 3275
PB - Royal Society of Chemistry
CY - Cambridge
ER -
TY - JOUR
A1 - Mendel, Ralf R.
A1 - 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 - JOUR
A1 - Contin, Andrea
A1 - Frasca, Stefano
A1 - Vivekananthan, Jeevanthi
A1 - Leimkühler, Silke
A1 - Wollenberger, Ursula
A1 - Plumere, Nicolas
A1 - Schuhmann, Wolfgang
T1 - A pH Responsive Redox Hydrogel for Electrochemical Detection of Redox Silent Biocatalytic Processes. Control of Hydrogel Solvation
JF - Electroanalysis : an international journal devoted to fundamental and practical aspects of electroanalysis
N2 - The control of bioelectrocatalytic processes by external stimuli for the indirect detection of non-redox active species was achieved using an esterase and a redox enzyme both integrated within a redox hydrogel. The poly( vinyl) imidazole Os(bpy)(2)Cl hydrogel displays pH-responsive properties. The esterase catalysed reaction leads to a local pH decrease causing protonation of imidazole moieties thus increasing hydrogel solvation and mobility of the tethered Os-complexes. This is the key step to enable improved electron transfer between an aldehyde oxidoreductase and the polymer-bound Os-complexes. The off-on switch is further integrated in a biofuel cell system for self-powered signal generation.
KW - pH responsive hydrogel
KW - External stimuli
KW - Biofuel cell
KW - Self-powered biosensor
KW - Solvation
Y1 - 2015
U6 - https://doi.org/10.1002/elan.201400621
SN - 1040-0397
SN - 1521-4109
VL - 27
IS - 4
SP - 938
EP - 944
PB - Wiley-VCH
CY - Weinheim
ER -
TY - JOUR
A1 - Schrapers, Peer
A1 - Hartmann, Tobias
A1 - Kositzki, Ramona
A1 - Dau, Holger
A1 - Reschke, Stefan
A1 - Schulzke, Carola
A1 - Leimkühler, Silke
A1 - Haumann, Michael
T1 - 'Sulfido and Cysteine Ligation Changes at the Molybdenum Cofactor during Substrate Conversion by Formate Dehydrogenase (FDH) from Rhodobacter capsulatus
JF - Inorganic chemistry
N2 - Formate dehydrogenase (FDH) enzymes are attractive catalysts for potential carbon dioxide conversion applications. The FDH from Rhodobacter capsulatus (RcFDH) binds a bis-molybdopterin-guanine-dinucleotide (bis-MGD) cofactor, facilitating reversible formate (HCOO-) to CO2 oxidation. We characterized the molecular structure of the active site of wildtype RcFDH and protein variants using X-ray absorption spectroscopy (XAS) at the Mo K-edge. This approach has revealed concomitant binding of a sulfido ligand (Mo=S) and a conserved cysteine residue (S(Cys386)) to Mo(VI) in the active oxidized molybdenum cofactor (Moco), retention of such a coordination motif at Mo(V) in a chemically reduced enzyme, and replacement of only the S(Cys386) ligand by an oxygen of formate upon Mo(IV) formation. The lack of a Mo=S bond in RcFDH expressed in the absence of FdsC implies specific metal sulfuration by this bis-MGD binding chaperone. This process still functioned in the Cys386Ser variant, showing no Mo-S(Cys386) ligand, but retaining a Mo=S bond. The C386S variant and the protein expressed without FdsC were inactive in formate oxidation, supporting that both Moligands are essential for catalysis. Low-pH inhibition of RcFDH was attributed to protonation at the conserved His387, supported by the enhanced activity of the His387Met variant at low pH, whereas inactive cofactor species showed sulfido-to-oxo group exchange at the Mo ion. Our results support that the sulfido and S(Cys386) ligands at Mo and a hydrogen-bonded network including His387 are crucial for positioning, deprotonation, and oxidation of formate during the reaction cycle of RcFDH.
Y1 - 2015
U6 - https://doi.org/10.1021/ic502880y
SN - 0020-1669
SN - 1520-510X
VL - 54
IS - 7
SP - 3260
EP - 3271
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - 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 - 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 - 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 - 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 - Hartmann, Tobias
A1 - Schwanhold, Nadine
A1 - Leimkühler, Silke
T1 - Assembly and catalysis of molybdenum or tungsten-containing formate dehydrogenases from bacteria
JF - Biochimica et biophysica acta : Proteins and proteomics
N2 - The global carbon cycle depends on the biological transformations of C-1 compounds, which include the reductive incorporation of CO2 into organic molecules (e.g. in photosynthesis and other autotrophic pathways), in addition to the production of CO2 from formate, a reaction that is catalyzed by formate dehydrogenases (FDHs). FDHs catalyze, in general, the oxidation of formate to CO2 and H+. However, selected enzymes were identified to act as CO2 reductases, which are able to reduce CO2 to formate under physiological conditions. This reaction is of interest for the generation of formate as a convenient storage form of H-2 for future applications. Cofactor-containing FDHs are found in anaerobic bacteria and archaea, in addition to facultative anaerobic or aerobic bacteria. These enzymes are highly diverse and employ different cofactors such as the molybdenum cofactor (Moco), FeS clusters and flavins, or cytochromes. Some enzymes include tungsten (W) in place of molybdenum (Mo) at the active site. For catalytic activity, a selenocysteine (SeCys) or cysteine (Cys) ligand at the Mo atom in the active site is essential for the reaction. This review will focus on the characterization of Mo- and W-containing FDHs from bacteria, their active site structure, subunit compositions and its proposed catalytic mechanism. We will give an overview on the different mechanisms of substrate conversion available so far, in addition to providing an outlook on bio-applications of FDHs. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications. (C) 2014 Elsevier B.V. All rights reserved.
KW - Molybdenum cofactor
KW - L-Cysteine desulfurase
KW - Formate dehydrogenase
KW - Chaperone
KW - Bis-MGD
Y1 - 2015
U6 - https://doi.org/10.1016/j.bbapap.2014.12.006
SN - 1570-9639
SN - 0006-3002
VL - 1854
IS - 9
SP - 1090
EP - 1100
PB - Elsevier
CY - Amsterdam
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 - Zeng, Ting
A1 - Leimkühler, Silke
A1 - Koetz, Joachim
A1 - Wollenberger, Ursula
T1 - Effective Electrochemistry of Human Sulfite Oxidase Immobilized on Quantum-Dots-Modified Indium Tin Oxide Electrode
JF - ACS applied materials & interfaces
N2 - The bioelectrocatalytic sulfite oxidation by human sulfite oxidase (hSO) on indium tin oxide (ITO) is reported, which is facilitated by functionalizing of the electrode surface with polyethylenimine (PEI)-entrapped CdS nanoparticles and enzyme. hSO was assembled onto the electrode with a high surface loading of electroactive enzyme. In the presence of sulfite but without additional mediators, a high bioelectrocatalytic current was generated. Reference experiments with only PEI showed direct electron transfer and catalytic activity of hSO, but these were less pronounced. The application of the polyelectrolyte-entrapped quantum dots (QDs) on ITO electrodes provides a compatible surface for enzyme binding with promotion of electron transfer. Variations of the buffer solution conditions, e.g., ionic strength, pH, viscosity, and the effect of oxygen, were studied in order to understand intramolecular and heterogeneous electron transfer from hSO to the electrode. The results are consistent with a model derived for the enzyme by using flash photolysis in solution and spectroelectrochemistry and molecular dynamic simulations of hSO on monolayer-modified gold electrodes. Moreover, for the first time a photoelectrochemical electrode involving immobilized hSO is demonstrated where photoexcitation of the CdS/hSO-modified electrode lead to an enhanced generation of bioelectrocatalytic currents upon sulfite addition. Oxidation starts already at the redox potential of the electron transfer domain of hSO and is greatly increased by application of a small overpotential to the CdS/hSO-modified ITO.
KW - human sulfite oxidase
KW - direct electrochemistry
KW - bioelectrocatalysis
KW - photocurrent
KW - CdS quantum dots
Y1 - 2015
U6 - https://doi.org/10.1021/acsami.5b06665
SN - 1944-8244
VL - 7
IS - 38
SP - 21487
EP - 21494
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - 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 - Otrelo-Cardoso, Ana Rita
A1 - Schwuchow, Viola
A1 - Rodrigues, David
A1 - Cabrita, Eurico J.
A1 - Leimkühler, Silke
A1 - Romao, Maria Joao
A1 - Santos-Silva, Teresa
T1 - Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes
JF - PLoS one
N2 - Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C(4)mim]Cl and [C(2)OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference - nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.
Y1 - 2014
U6 - https://doi.org/10.1371/journal.pone.0087295
SN - 1932-6203
VL - 9
IS - 1
PB - PLoS
CY - San Fransisco
ER -
TY - JOUR
A1 - 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 - 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 - 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 - 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 - Dey, Pradip
A1 - Adamovski, Miriam
A1 - Friebe, Simon
A1 - Badalyan, Artavazd
A1 - Mutihac, Radu-Cristian
A1 - Paulus, Florian
A1 - Leimkühler, Silke
A1 - Wollenberger, Ursula
A1 - Haag, Rainer
T1 - Dendritic polyglycerol-poly(ethylene glycol)-based polymer networks for biosensing application
JF - ACS applied materials & interfaces
N2 - This work describes the formation of a new dendritic polyglycerol-poly(ethylene glycol)-based 3D polymer network as a matrix for immobilization of the redox enzyme periplasmatic aldehyde oxidoreductase to create an electrochemical biosensor. The novel network is built directly on the gold surface, where it simultaneously stabilizes the enzyme for up to 4 days. The prepared biosensors can be used for amperometric detection of benzaldehyde in the range of 0.8-400 mu M.
KW - biosensors
KW - hydrogel
KW - amperometry
KW - dendritic
Y1 - 2014
U6 - https://doi.org/10.1021/am502018x
SN - 1944-8244
VL - 6
IS - 12
SP - 8937
EP - 8941
PB - American Chemical Society
CY - Washington
ER -
TY - JOUR
A1 - 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 - Bechi, Beatrice
A1 - Herter, Susanne
A1 - McKenna, Shane
A1 - Riley, Christopher
A1 - Leimkühler, Silke
A1 - Turner, Nicholas J.
A1 - Carnell, Andrew J.
T1 - Catalytic bio-chemo and bio-bio tandem oxidation reactions for amide and carboxylic acid synthesis
JF - Green chemistry : an international journal and green chemistry resource
N2 - A catalytic toolbox for three different water-based one-pot cascades to convert aryl alcohols to amides and acids and cyclic amines to lactams, involving combination of oxidative enzymes (monoamine oxidase, xanthine dehydrogenase, galactose oxidase and laccase) and chemical oxidants (TBHP or Cul(cat)/H2O2) at mild temperatures, is presented. Mutually compatible conditions were found to afford products in good to excellent yields.
Y1 - 2014
U6 - https://doi.org/10.1039/c4gc01321b
SN - 1463-9262
SN - 1463-9270
VL - 16
IS - 10
SP - 4524
EP - 4529
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 -