Membrane topology and mutational analysis of Mycobacterium tuberculosis VKOR, a protein involved in disulfide bond formation and a homologue of human vitamin K epoxide reductase.
We have presented evidence that a homologue of vertebrate membrane protein vitamin K epoxide reductase (VKOR) is an important component of the protein disulfide bond-forming pathway in many bacteria. Bacterial VKOR appears to take the place of the nonhomologous DsbB found in Escherichia coli. We also determined the structure of a VKOR from a Cyanobacterium and showed that two or four conserved cysteines are required, according to different reductants for activity in an in vitro assay. Here we present evidence for the topologic arrangement in the cytoplasmic membrane of the VKOR from Mycobacterium tuberculosis (Mtb). The results show that Mtb VKOR is a membrane protein that spans the membrane 5 times with its N-terminus in the cytoplasm, C-terminus in the periplasm, and all four cysteines facing the periplasm. The essentiality of the four conserved cysteine residues has also been demonstrated in promoting disulfide bond formation in vivo and a mixed disulfide between a cysteine of DsbA of E. coli, and one of the cysteines (Cys(57)) of the VKOR homologue has been identified to be a likely intermediate in the disulfide bond-forming pathway. These studies may inform future resolution of issues surrounding the functioning of human VKOR.
College of Life Science, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai'an, P.R. China.
Xiaoyun
X
Wang
Rachel J
RJ
Dutton
Jon
J
Beckwith
Dana
D
Boyd
D055162:Biocatalysis; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism; D003545:Cysteine / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology* / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D064417:Vitamin K Epoxide Reductases
D004220:Disulfides; D006899:Mixed Function Oxygenases; D064417:Vitamin K Epoxide Reductases; D003545:Cysteine