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 - 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 - 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 - 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 - 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 - 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 - Lemaire, Olivier N. A1 - Honore, Flora A. A1 - Tempel, Sebastien A1 - Fortier, Emma M. A1 - Leimkühler, Silke A1 - Mejean, Vincent A1 - Iobbi-Nivol, Chantal T1 - Shewanella decolorationis LDS1 Chromate Resistance JF - Applied and environmental microbiology N2 - The genus Shewanella is well known for its genetic diversity, its outstanding respiratory capacity, and its high potential for bioremediation. Here, a novel strain isolated from sediments of the Indian Ocean was characterized. A 16S rRNA analysis indicated that it belongs to the species Shewanella decolorationis. It was named Shewanella decolorationis LDS1. This strain presented an unusual ability to grow efficiently at temperatures from 24 degrees C to 40 degrees C without apparent modifications of its metabolism, as shown by testing respiratory activities or carbon assimilation, and in a wide range of salt concentrations. Moreover, S. decolorationis LDS1 tolerates high chromate concentrations. Indeed, it was able to grow in the presence of 4 mM chromate at 28 degrees C and 3 mM chromate at 40 degrees C. Interestingly, whatever the temperature, when the culture reached the stationary phase, the strain reduced the chromate present in the growth medium. In addition, S. decolorationis LDS1 degrades different toxic dyes, including anthraquinone, triarylmethane, and azo dyes. Thus, compared to Shewanella oneidensis, this strain presented better capacity to cope with various abiotic stresses, particularly at high temperatures. The analysis of genome sequence preliminary data indicated that, in contrast to S. oneidensis and S. decolorationis S12, S. decolorationis LDS1 possesses the phosphorothioate modification machinery that has been described as participating in survival against various abiotic stresses by protecting DNA. We demonstrate that its heterologous production in S. oneidensis allows it to resist higher concentrations of chromate. IMPORTANCE Shewanella species have long been described as interesting microorganisms in regard to their ability to reduce many organic and inorganic compounds, including metals. However, members of the Shewanella genus are often depicted as cold-water microorganisms, although their optimal growth temperature usually ranges from 25 to 28 degrees C under laboratory growth conditions. Shewanella decolorationis LDS1 is highly attractive, since its metabolism allows it to develop efficiently at temperatures from 24 to 40 degrees C, conserving its ability to respire alternative substrates and to reduce toxic compounds such as chromate or toxic dyes. Our results clearly indicate that this novel strain has the potential to be a powerful tool for bioremediation and unveil one of the mechanisms involved in its chromate resistance. KW - Shewanella KW - bioremediation KW - chromium KW - decolorization KW - dndBCDE KW - dyes KW - temperature Y1 - 2019 U6 - https://doi.org/10.1128/AEM.00777-19 SN - 0099-2240 SN - 1098-5336 VL - 85 IS - 18 PB - American Society for Microbiology CY - Washington ER - TY - JOUR A1 - 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 - 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 - 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 - Neumann, Meina A1 - Seduk, Farida A1 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke T1 - Molybdopterin Dinucleotide Biosynthesis in Escherichia coli identification of amino acid residues of molybdopterin dinucleotide transferases that determine specificity for binding of guanine or cytosine nucleotides JF - The journal of biological chemistry N2 - The molybdenum cofactor is modified by the addition of GMP or CMP to the C4' phosphate of molybdopterin forming the molybdopterin guanine dinucleotide or molybdopterin cytosine dinucleotide cofactor, respectively. The two reactions are catalyzed by specific enzymes as follows: the GTP: molybdopterin guanylyltransferase MobA and the CTP: molybdopterin cytidylyltransferase MocA. Both enzymes show 22% amino acid sequence identity and are specific for their respective nucleotides. Crystal structure analysis of MobA revealed two conserved motifs in the N-terminal domain of the protein involved in binding of the guanine base. Based on these motifs, we performed site-directed mutagenesis studies to exchange the amino acids to the sequence found in the paralogue MocA. Using a fully defined in vitro system, we showed that the exchange of five amino acids was enough to obtain activity with both GTP and CTP in either MocA or MobA. Exchange of the complete N-terminal domain of each protein resulted in the total inversion of nucleotide specificity activity, showing that the N-terminal domain determines nucleotide recognition and binding. Analysis of protein-protein interactions showed that the C-terminal domain of either MocA or MobA determines the specific binding to the respective acceptor protein. Y1 - 2011 U6 - https://doi.org/10.1074/jbc.M110.155671 SN - 0021-9258 VL - 286 IS - 2 SP - 1400 EP - 1408 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Redelberger, David A1 - Seduk, Farida A1 - Genest, Olivier A1 - Mejean, Vincent A1 - Leimkühler, Silke A1 - Iobbi-Nivol, Chantal T1 - YcdY Protein of Escherichia coli, an Atypical Member of the TorD Chaperone Family JF - Journal of bacteriology N2 - The TorD family of specific chaperones is divided into four subfamilies dedicated to molybdoenzyme biogenesis and a fifth one, exemplified by YcdY of Escherichia coli, for which no defined partner has been identified so far. We propose that YcdY is the chaperone of YcdX, a zinc protein involved in the swarming motility process of E. coli, since YcdY interacts with YcdX and increases its activity in vitro. Y1 - 2011 U6 - https://doi.org/10.1128/JB.05927-11 SN - 0021-9193 VL - 193 IS - 23 SP - 6512 EP - 6516 PB - American Society for Microbiology CY - Washington ER - TY - JOUR A1 - Dahl, Jan-Ulrik A1 - Radon, Christin A1 - Bühning, Martin A1 - Nimtz, Manfred A1 - Leichert, Lars I. A1 - Denis, Yann A1 - Jourlin-Castelli, Cecile A1 - Iobbi-Nivol, Chantal A1 - Mejean, Vincent A1 - Leimkühler, Silke T1 - The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis JF - JOURNAL OF BIOLOGICAL CHEMISTRY N2 - The Escherichia coli L-cysteine desulfurase IscS mobilizes sulfur from L-cysteine for the synthesis of several biomolecules such as iron-sulfur (FeS) clusters, molybdopterin, thiamin, lipoic acid, biotin, and the thiolation of tRNAs. The sulfur transfer from IscS to various biomolecules is mediated by different interaction partners (e.g. TusA for thiomodification of tRNAs, IscU for FeS cluster biogenesis, and ThiI for thiamine biosynthesis/tRNA thiolation), which bind at different sites of IscS. Transcriptomic and proteomic studies of a Delta tusA strain showed that the expression of genes of the moaABCDE operon coding for proteins involved in molybdenum cofactor biosynthesis is increased under aerobic and anaerobic conditions. Additionally, under anaerobic conditions the expression of genes encoding hydrogenase 3 and several molybdoenzymes such as nitrate reductase were also increased. On the contrary, the activity of all molydoenzymes analyzed was significantly reduced in the Delta tusA mutant. Characterization of the Delta tusA strain under aerobic conditions showed an overall low molybdopterin content and an accumulation of cyclic pyranopterin monophosphate. Under anaerobic conditions the activity of nitrate reductase was reduced by only 50%, showing that TusA is not essential for molybdenum cofactor biosynthesis. We present a model in which we propose that the direction of sulfur transfer for each sulfur-containing biomolecule is regulated by the availability of the interaction partner of IscS. We propose that in the absence of TusA, more IscS is available for FeS cluster biosynthesis and that the overproduction of FeS clusters leads to a modified expression of several genes. Y1 - 2013 U6 - https://doi.org/10.1074/jbc.M112.431569 SN - 0021-9258 VL - 288 IS - 8 SP - 5426 EP - 5442 PB - AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC CY - BETHESDA ER - TY - JOUR A1 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke T1 - Molybdenum enzymes, their maturation and molybdenum cofactor biosynthesis in Escherichia coli JF - Biochimica et biophysica acta : Bioenergetics N2 - Molybdenum cofactor (Moco) biosynthesis is an ancient, ubiquitous, and highly conserved pathway leading to the biochemical activation of molybdenum. Moco is the essential component of a group of redox enzymes, which are diverse in terms of their phylogenetic distribution and their architectures, both at the overall level and in their catalytic geometry. A wide variety of transformations are catalyzed by these enzymes at carbon, sulfur and nitrogen atoms, which include the transfer of an oxo group or two electrons to or from the substrate. More than 50 molybdoenzymes were identified in bacteria to date. In molybdoenzymes Mo is coordinated to a dithiolene group on the 6-alkyl side chain of a pterin called molybdopterin (MPT). The biosynthesis of Moco can be divided into four general steps in bacteria: I) formation of the cyclic pyranopterin monophosphate, 2) formation of MPT, 3) insertion of molybdenum into molybdopterin to form Moco, and 4) additional modification of Moco with the attachment of GMP or CMP to the phosphate group of MPT, forming the dinucleotide variant of Moco. This review will focus on molybdoenzymes, the biosynthesis of Moco, and its incorporation into specific target proteins focusing on Escherichia coli. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems. KW - Molybdenum cofactor KW - Molybdenum KW - Dithiolene KW - Molybdopterin KW - Bis-MGD KW - Moco Y1 - 2013 U6 - https://doi.org/10.1016/j.bbabio.2012.11.007 SN - 0005-2728 VL - 1827 IS - 8-9 SP - 1086 EP - 1101 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Neumann, Meina A1 - Mittelstaedt, Gerd A1 - Seduk, Farida A1 - Iobbi-Nivol, Chantal A1 - Leimkühler, Silke T1 - MocA is a specific cytidylyltransferase involved in molybdopterin cytosine dinucleotide biosynthesis in Escherichia coli N2 - We have purified and characterized a specific CTP: molybdopterin cytidylyltransferase for the biosynthesis of the molybdopterin (MPT) cytosine dinucleotide (MCD) cofactor in Escherichia coli. The protein, named MocA, shows 22% amino acid sequence identity to E. coli MobA, the specific GTP: molybdopterin guanylyltransferase for molybdopterin guanine dinucleotide biosynthesis. MocA is essential for the activity of the MCD-containing enzymes aldehyde oxidoreductase Yag-TSR and the xanthine dehydrogenases XdhABC and XdhD. Using a fully defined in vitro assay, we showed that MocA, Mo-MPT, CTP, and MgCl2 are required and sufficient for MCD biosynthesis in vitro. The activity of MocA is specific for CTP; other nucleotides such as ATP and GTP were not utilized. In the defined in vitro system a turnover number of 0.37 +/- 0.01 min(-1) was obtained. A1:1 binding ratio of MocA to Mo-MPT and CTP was determined to monomeric MocA with dissociation constants of 0.23 +/- 0.02 mu M for CTP and 1.17 +/- 0.18 mu M for Mo-MPT. We showed that MocA was also able to convert MPT to MCD in the absence of molybdate, however, with only one catalytic turnover. The addition of molybdate after one turnover gave rise to a higher MCD production, revealing that MCD remains bound to MocA in the absence of molybdate. This work presents the first characterization of a specific enzyme involved in MCD biosynthesis in bacteria. Y1 - 2009 UR - http://www.jbc.org/ U6 - https://doi.org/10.1074/jbc.M109.008565 SN - 0021-9258 ER - TY - JOUR A1 - Neumann, Meina A1 - Mittelstaedt, Gerd A1 - Iobbi-Nivol, Chantal A1 - Saggu, Miguel A1 - Lendzian, Friedhelm A1 - Hildebrandt, Peter A1 - Leimkühler, Silke T1 - A periplasmic aldehyde oxidoreductase represents the first molybdopterin cytosine dinucleotide cofactor containing molybdo-flavoenzyme from Escherichia coli N2 - Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm. The 135 kDa enzyme comprised a noncovalent (alpha beta gamma) heterotrimer with a large (78.1 kDa) molybdenum cofactor (Moco)-containing YagR subunit, a medium (33.9 kDa) FAD-containing YagS subunit, and a small (21.0 kDa) 2 x [2Fe2S]-containing YagT subunit. YagQ is not a subunit of the mature enzyme, and the protein is expected to be involved in Moco modification and insertion into YagTSR. Analysis of the form of Moco present in YagTSR revealed the presence of the molybdopterin cytosine dinucleotide cofactor. Two different [2Fe2S] clusters, typical for this class of enzyme, were identified by EPR. YagTSR represents the first example of a molybdopterin cytosine dinucleotide-containing protein in E. coli. Kinetic characterization of the enzyme revealed that YagTSR converts a broad spectrum of aldehydes, with a preference for aromatic aldehydes. Ferredoxin instead of NAD(+) or molecular oxygen was used as terminal electron acceptor. Complete growth inhibition of E. coli cells devoid of genes from the yagTSRQ operon was observed by the addition of cinnamaldehyde to a low-pH medium. This finding shows that YagTSR might have a role in the detoxification of aromatic aldehydes for E. coli under certain growth conditions. Y1 - 2009 UR - http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=1742-464X U6 - https://doi.org/10.1111/j.1742-4658.2009.07000.x SN - 1742-464X ER -