title: Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in Escherichia coli.
abstract: The molybdenum cofactor is an important cofactor, and its biosynthesis is essential for many organisms, including humans. Its basic form comprises a single molybdopterin (MPT) unit, which binds a molybdenum ion bearing three oxygen ligands via a dithiolene function, thus forming Mo-MPT. In bacteria, this form is modified to form the bis-MPT guanine dinucleotide cofactor with two MPT units coordinated at one molybdenum atom, which additionally contains GMPs bound to the terminal phosphate group of the MPTs (bis-MGD). The MobA protein catalyzes the nucleotide addition to MPT, but the mechanism of the biosynthesis of the bis-MGD cofactor has remained enigmatic. We have established an in vitro system for studying bis-MGD assembly using purified compounds. Quantification of the MPT/molybdenum and molybdenum/phosphorus ratios, time-dependent assays for MPT and MGD detection, and determination of the numbers and lengths of Mo-S and Mo-O bonds by X-ray absorption spectroscopy enabled identification of a novel bis-Mo-MPT intermediate on MobA prior to nucleotide attachment. The addition of Mg-GTP to MobA loaded with bis-Mo-MPT resulted in formation and release of the final bis-MGD product. This cofactor was fully functional and reconstituted the catalytic activity of apo-TMAO reductase (TorA). We propose a reaction sequence for bis-MGD formation, which involves 1) the formation of bis-Mo-MPT, 2) the addition of two GMP units to form bis-MGD on MobA, and 3) the release and transfer of the mature cofactor to the target protein TorA, in a reaction that is supported by the specific chaperone TorD, resulting in an active molybdoenzyme.
authors.affiliation: From the Institute for Biochemistry and Biology, Department of Molecular Enzymology, and.
authors.forename: Stefan
authors.initials: S
authors.lastname: Reschke
authors.forename: Kajsa G V
authors.initials: KG
authors.lastname: Sigfridsson
authors.forename: Paul
authors.initials: P
authors.lastname: Kaufmann
authors.forename: Nils
authors.initials: N
authors.lastname: Leidel
authors.forename: Sebastian
authors.initials: S
authors.lastname: Horn
authors.forename: Klaus
authors.initials: K
authors.lastname: Gast
authors.forename: Carola
authors.initials: C
authors.lastname: Schulzke
authors.forename: Michael
authors.initials: M
authors.lastname: Haumann
authors.forename: Silke
authors.initials: S
authors.lastname: Leimkühler
mesh_terms: D003067:Coenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000378:metabolism; D006801:Humans; D008667:Metalloproteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D008982:Molybdenum / Q000737:chemistry / Q000378:metabolism*; D000090006:Molybdenum Cofactors; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011621:Pteridines / Q000737:chemistry / Q000378:metabolism*; D013466:Sulfurtransferases / Q000378:metabolism; D056928:X-Ray Absorption Spectroscopy
chemical_list: D003067:Coenzymes; D029968:Escherichia coli Proteins; D006157:Guanosine Monophosphate; D008667:Metalloproteins; D018832:Molecular Chaperones; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D010089:Oxidoreductases, N-Demethylating; D010710:Phosphates; D011621:Pteridines; D013466:Sulfurtransferases; C404104:Mog protein, E coli; C088083:TorD protein, E coli; C095754:mobA protein, E coli; C022261:molybdopterin; C023186:trimethylamine dehydrogenase; C111302:MoeA protein, E coli
keywords: Biosynthesis; Bis-MGD; Cofactor Assembly; Dithiolene; Escherichia coli; Molecular Chaperone; Molybdenum; Vitamins and Cofactors