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  - 
TY  - JOUR
A1  - Reschke, Stefan
A1  - Niks, Dimitri
A1  - Wilson, Heather
A1  - Sigfridsson, Kajsa G. V.
A1  - Haumann, Michael
A1  - Rajagopalan, K. V.
A1  - Hine, Russ
A1  - Leimkühler, Silke
T1  - Effect of exchange of the cysteine molybdenum ligand with selenocysteine on the structure and function of the active site in human sulfite oxidase
JF  - Biochemistry
N2  - Sulfite oxidase (SO) is an essential molybdoenzyme for humans, catalyzing the final step in the degradation of sulfur-containing amino acids and lipids, which is the oxidation of sulfite to sulfate. The catalytic site of SO consists of a molybdenum ion bound to the dithiolene sulfurs of one molybdopterin (MPT) molecule, carrying two oxygen ligands, and is further coordinated by the thiol sulfur of a conserved cysteine residue. We have exchanged four non-active site cysteines in the molybdenum cofactor (Moco) binding domain of human SO (SOMD) with serine using site-directed mutagenesis. This facilitated the specific replacement of the active site Cys207 with selenocysteine during protein expression in Escherichia coli. The sulfite oxidizing activity (k(cat)/K-M) of SeSOMD4Ser was increased at least 1.5-fold, and the pH optimum was shifted to a more acidic value compared to those of SOMD4Ser and SOMD4Cys(wt) X-ray absorption spectroscopy revealed a Mow Se bond length of 2.51 A, likely caused by the specific binding of Sec207 to the molybdenum, and otherwise rather similar square-pyramidal S/Se(Cys)(O2MoS2)-S-VI(MPT) site structures in the three constructs. The low-pH form of the Mo(V) electron paramagnetic resonance (EPR) signal of SeSOM4Ser was altered compared to those of SOMD4Ser and SOMD4cy,(,), with g, in particular shifted to a lower magnetic field, due to the Se ligation at the molybdenum. In contrast, the Mo(V) EPR signal of the high-pH form was unchanged. The substantially stronger effect of substituting selenocysteine for cysteine at low pH as compared to high pH is most likely due to the decreased covalency of the Mo Se bond.
Y1  - 2013
U6  - https://doi.org/10.1021/bi4008512
SN  - 0006-2960
VL  - 52
IS  - 46
SP  - 8295
EP  - 8303
PB  - American Chemical Society
CY  - Washington
ER  -