- 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 MobAThe 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.…
MetadatenVerfasserangaben: | Stefan ReschkeGND, Kajsa G. V. Sigfridsson, Paul Kaufmann, Nils Leidel, Sebastian Horn, Klaus Gast, Carola Schulzke, Michael HaumannORCiD, Silke LeimkühlerORCiDGND |
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DOI: | https://doi.org/10.1074/jbc.M113.497453 |
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ISSN: | 0021-9258 |
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ISSN: | 1083-351X |
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Titel des übergeordneten Werks (Englisch): | The journal of biological chemistry |
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Verlag: | American Society for Biochemistry and Molecular Biology |
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Verlagsort: | Bethesda |
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Publikationstyp: | Wissenschaftlicher Artikel |
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Sprache: | Englisch |
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Jahr der Erstveröffentlichung: | 2013 |
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Erscheinungsjahr: | 2013 |
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Datum der Freischaltung: | 26.03.2017 |
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Band: | 288 |
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Ausgabe: | 41 |
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Seitenanzahl: | 10 |
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Erste Seite: | 29736 |
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Letzte Seite: | 29745 |
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Fördernde Institution: | Deutsche Forschungsgemeinschaft (DFG) Cluster of Excellence "Unifying
Concepts in Catalysis"; DFG [LE1171/6-1, Ha3265/3-1, Ha3265/6-1];
Heisenberg Fellowship; European Cooperation in Science and Technology
(COST) Action [CM1003]; Stiftelsen Bengt Lundqvist minne; Wenner-Gren
Foundation; European Research Council (ERC) |
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Organisationseinheiten: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
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Peer Review: | Referiert |
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