@article{KaufmannDuffusMitrovaetal.2018, author = {Kaufmann, Hans Paul and Duffus, Benjamin R. and Mitrova, Biljana and Iobbi-Nivol, Chantal and Teutloff, Christian and Nimtz, Manfred and Jaensch, Lothar and Wollenberger, Ulla and Leimk{\"u}hler, Silke}, title = {Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamie N-Oxide Reductase}, series = {Biochemistry}, volume = {57}, journal = {Biochemistry}, number = {7}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/acs.biochem.7b01108}, pages = {1130 -- 1143}, year = {2018}, abstract = {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.}, language = {en} } @article{ForlaniCeredaFreueretal.2005, author = {Forlani, Fabio and Cereda, Angelo and Freuer, Andrea and Nimtz, Manfred and Leimk{\"u}hler, Silke and Pagani, Silvia}, title = {The cysteine-desulfurase IscS promotes the production of the rhodanese RhdA in the persulfurated form}, issn = {0014-5793}, year = {2005}, abstract = {After heterologous expression in Escherichia coli, the Azotobacter vinelandii rhodanese RhdA is purified in a persulfurated form (RhdA-SSH). We identified L-cysteine as the most effective sulfur source in producing RhdA-SSH. An E. coli soluble extract was required for in vitro persulfuration of RhdA, and the addition of pyridoxal-5'-phosphate increased RhdA-SSH production, indicating a likely involvement of a cysteine desulfurase. We were able to show the formation of a covalent complex between IscS and RhdA. By combining a time-course fluorescence assay and mass spectrometry analysis, we demonstrated the transfer of sulfur from E. coli IscS to RhdA. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved}, language = {en} } @article{VossNimtzLeimkuehler2011, author = {Voss, Martin and Nimtz, Manfred and Leimk{\"u}hler, Silke}, title = {Elucidation of the dual role of Mycobacterial MoeZR in Molybdenum Cofactor Biosynthesis and Cysteine Biosynthesis}, series = {PLoS one}, volume = {6}, journal = {PLoS one}, number = {11}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0028170}, pages = {9}, year = {2011}, abstract = {The pathway of molybdenum cofactor biosynthesis has been studied in detail by using proteins from Mycobacterium species, which contain several homologs associated with the first steps of Moco biosynthesis. While all Mycobacteria species contain a MoeZR, only some strains have acquired an additional homolog, MoeBR, by horizontal gene transfer. The role of MoeBR and MoeZR was studied in detail for the interaction with the two MoaD-homologs involved in Moco biosynthesis, MoaD1 and MoaD2, in addition to the CysO protein involved in cysteine biosynthesis. We show that both proteins have a role in Moco biosynthesis, while only MoeZR, but not MoeBR, has an additional role in cysteine biosynthesis. MoeZR and MoeBR were able to complement an E. coli moeB mutant strain, but only in conjunction with the Mycobacterial MoaD1 or MoaD2 proteins. Both proteins were able to sulfurate MoaD1 and MoaD2 in vivo, while only MoeZR additionally transferred the sulfur to CysO. Our in vivo studies show that Mycobacteria have acquired several homologs to maintain Moco biosynthesis. MoeZR has a dual role in Moco- and cysteine biosynthesis and is involved in the sulfuration of MoaD and CysO, whereas MoeBR only has a role in Moco biosynthesis, which is not an essential function for Mycobacteria.}, language = {en} } @article{DahlUrbanBolteetal.2011, author = {Dahl, Jan-Ulrik and Urban, Alexander and Bolte, Andrea and Sriyabhaya, Promjit and Donahue, Janet L. and Nimtz, Manfred and Larson, Timothy J. and Leimk{\"u}hler, Silke}, title = {The identification of a novel protein involved in Molybdenum Cofactor Biosynthesis in Escherichia coli}, series = {The journal of biological chemistry}, volume = {286}, journal = {The journal of biological chemistry}, number = {41}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {0021-9258}, doi = {10.1074/jbc.M111.282368}, pages = {35801 -- 35812}, year = {2011}, abstract = {Background: In Moco biosynthesis, sulfur is transferred from L-cysteine to MPT synthase, catalyzing the conversion of cPMP to MPT. Results: The rhodanese-like protein YnjE is a novel protein involved in Moco biosynthesis. Conclusion: YnjE enhances the rate of conversion of cPMP to MPT and interacts with MoeB and IscS. S ignificance: To understand the mechanism of sulfur transfer and the role of rhodaneses in the cell.}, language = {en} } @article{HartmannSchrapersUteschetal.2016, author = {Hartmann, Tobias and Schrapers, Peer and Utesch, Tillmann and Nimtz, Manfred and Rippers, Yvonne and Dau, Holger and Mroginski, Maria Andrea and Haumann, Michael and Leimk{\"u}hler, Silke}, title = {The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C-H Bond Cleavage and Oxygen Atom Transfer Reactions}, series = {Biochemistry}, volume = {55}, journal = {Biochemistry}, publisher = {American Chemical Society}, address = {Washington}, issn = {0006-2960}, doi = {10.1021/acs.biochem.6b00002}, pages = {2381 -- 2389}, year = {2016}, abstract = {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.}, language = {en} } @article{DahlRadonBuehningetal.2013, author = {Dahl, Jan-Ulrik and Radon, Christin and B{\"u}hning, Martin and Nimtz, Manfred and Leichert, Lars I. and Denis, Yann and Jourlin-Castelli, Cecile and Iobbi-Nivol, Chantal and Mejean, Vincent and Leimk{\"u}hler, Silke}, title = {The Sulfur Carrier Protein TusA Has a Pleiotropic Role in Escherichia coli That Also Affects Molybdenum Cofactor Biosynthesis}, series = {JOURNAL OF BIOLOGICAL CHEMISTRY}, volume = {288}, journal = {JOURNAL OF BIOLOGICAL CHEMISTRY}, number = {8}, publisher = {AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC}, address = {BETHESDA}, issn = {0021-9258}, doi = {10.1074/jbc.M112.431569}, pages = {5426 -- 5442}, year = {2013}, abstract = {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.}, language = {en} }