TY  - JOUR
A1  - Reschke, Stefan
A1  - Sigfridsson, Kajsa G. V.
A1  - Kaufmann, Paul
A1  - Leidel, Nils
A1  - Horn, Sebastian
A1  - Gast, Klaus
A1  - Schulzke, Carola
A1  - Haumann, Michael
A1  - Leimkühler, Silke
T1  - Identification of a bis-molybdopterin intermediate in molybdenum cofactor biosynthesis in escherichia coli
JF  - The journal of biological chemistry
N2  - 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.
Y1  - 2013
U6  - https://doi.org/10.1074/jbc.M113.497453
SN  - 0021-9258
SN  - 1083-351X
VL  - 288
IS  - 41
SP  - 29736
EP  - 29745
PB  - American Society for Biochemistry and Molecular Biology
CY  - Bethesda
ER  -