Membrane Topology and Mutational Analysis ofMycobacterium tuberculosisVKOR, a Protein Involved in Disulfide Bond Formation and a Homologue of Human Vitamin K Epoxide Reductase

Abstract

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.

Altered Escherichia coli membrane protein assembly machinery allows proper membrane assembly of eukaryotic protein vitamin K epoxide reductase

“…Of VKORc1's seven cysteines, only the fourconserved cysteines, C43, C51, and the two comprising the canonical redox site CXXC, are essential for its functioning in E. coli. These findings are consistent with our current knowledge of the function of DsbB, with the reported bacterial VKOR structure, and with our previous findings on the function of mycobacterial VKOR (25,30). However, they contrast with other reports arguing that only the CXXC is required to regenerate reduced VK (31,17).…”

“…S1) is periplasmically localized because it directly receives electrons from DsbA via a thiol-disulfide exchange reaction. Thus, all tests for the mechanism of action of VKORc1 ΔAAR, G60D function in E. coli are essentially analogous to that of DsbB and bacterial VKOR and consistent with many of the studies on the functioning of VKORc1 in the endoplasmic reticulum (16,24,25). For this test, we used a previously reported construct of E. coli DsbB (MalF-DsbB), which is dependent on positively charged amino acids in cytoplasmic loops of DsbB for its proper orientation in the membrane, following the positive-inside rule (26).…”

“…We have previously reported the functional replacement of the E. coli membrane disulfide-generating enzyme DsbB by an unrelated membrane quinone reductase VKOR from M. tuberculosis (12,25). In this current study we show that the mammalian homolog, VKORc1, cannot complement ΔdsbB unless changes are made to the sequence of VKORc1 and/or to the membrane insertion and quality control machinery, exemplified by the insertase yidC and the cytoplasmic protease system hslVU.…”

Lumen Thiol Oxidoreductase1, a Disulfide Bond-Forming Catalyst, Is Required for the Assembly of Photosystem II inArabidopsis

“…Both of these methods place the functional domains in the periplasm, which is analogous to the plastid lumen (Dutton et al, 2008;Li et al, 2010;Wang et al, 2011). In proteobacteria, DsbA introduces disulfide bonds in Cyscontaining targets, while DsbB recycles DsbA to its oxidized form with transfer of electrons to a quinone (Depuydt et al, 2011;Kadokura and Beckwith, 2010).…”

“…This class has similarity to VKOR (for vitamin K epoxide reductase) and was recognized in cyanobacteria (Singh et al, 2008;Li et al, 2010), some bacterial phyla lacking the typical DsbAB components (Dutton et al, 2008Wang et al, 2011), and the green lineage (Grossman et al, 2010). The VKOR-like proteins were defined based on the presence of a redox domain containing two Cys pairs.…”