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. <br /> 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. <br /> 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  -