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  - 
TY  - JOUR
A1  - Leimkühler, Silke
A1  - Wuebbens, Margot M.
A1  - Rajagopalan, K. V.
T1  - The history of the discovery of the molybdenum cofactor and novel aspects of its biosynthesis in bacteria
JF  - Coordination chemistry reviews
N2  - The biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to molybdopterin phosphate group or the replacement of ligands at the molybdenum center.
KW  - Molybdenum cofactor
KW  - Molybdopterin
KW  - Precursor Z
KW  - Molybdopterin guanine dinucleotide cofactor
KW  - Urothione
KW  - Dithiolene group
Y1  - 2011
U6  - https://doi.org/10.1016/j.ccr.2010.12.003
SN  - 0010-8545
VL  - 255
IS  - 9-10
SP  - 1129
EP  - 1144
PB  - Elsevier
CY  - Lausanne
ER  -