- 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.…
MetadatenAuthor details: | 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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Title of parent work (English): | The journal of biological chemistry |
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Publisher: | American Society for Biochemistry and Molecular Biology |
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Place of publishing: | Bethesda |
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Publication type: | Article |
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Language: | English |
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Year of first publication: | 2013 |
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Publication year: | 2013 |
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Release date: | 2017/03/26 |
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Volume: | 288 |
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Issue: | 41 |
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Number of pages: | 10 |
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First page: | 29736 |
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Last Page: | 29745 |
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Funding 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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Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Biochemie und Biologie |
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Peer review: | Referiert |
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