Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in escherichia coli
(2013) In Journal of Biological Chemistry 288(41). p.29736-29745- 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... (More)
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.
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- author
- Reschke, Stefan ; Sigfridsson, Kajsa G.V. LU ; Kaufmann, Paul ; Leidel, Nils ; Horn, Sebastian ; Gast, Klaus ; Schulzke, Carola ; Haumann, Michael and Leimkühler, Silke
- publishing date
- 2013-10-11
- type
- Contribution to journal
- publication status
- published
- in
- Journal of Biological Chemistry
- volume
- 288
- issue
- 41
- pages
- 10 pages
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- pmid:24003231
- scopus:84885639305
- ISSN
- 0021-9258
- DOI
- 10.1074/jbc.M113.497453
- language
- English
- LU publication?
- no
- id
- 93715e4f-b3ea-479a-ac1f-2ce9a26bfc78
- date added to LUP
- 2020-01-15 10:16:08
- date last changed
- 2024-06-26 09:45:24
@article{93715e4f-b3ea-479a-ac1f-2ce9a26bfc78,
abstract = {{<p>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.</p>}},
author = {{Reschke, Stefan and Sigfridsson, Kajsa G.V. and Kaufmann, Paul and Leidel, Nils and Horn, Sebastian and Gast, Klaus and Schulzke, Carola and Haumann, Michael and Leimkühler, Silke}},
issn = {{0021-9258}},
language = {{eng}},
month = {{10}},
number = {{41}},
pages = {{29736--29745}},
publisher = {{American Society for Biochemistry and Molecular Biology}},
series = {{Journal of Biological Chemistry}},
title = {{Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in escherichia coli}},
url = {{http://dx.doi.org/10.1074/jbc.M113.497453}},
doi = {{10.1074/jbc.M113.497453}},
volume = {{288}},
year = {{2013}},
}