Identification of the Thioredoxin Partner of Vitamin K Epoxide Reductase in Mycobacterial Disulfide Bond Formation

Abstract

Disulfide bonds influence the stability and activity of many proteins. In <i>Escherichia coli</i>, the DsbA and DsbB enzymes promote disulfide bond formation. Other bacteria, including the <i>Actinobacteria</i>, use instead of DsbB the enzyme vitamin K epoxide reductase (VKOR), whose gene is found either fused to or in the same operon as a <i>dsbA</i>-like gene. <i>Mycobacterium tuberculosis</i> and other Gram-positive actinobacteria secrete many proteins with even numbers of cysteines to the cell envelope. These organisms have predicted oxidoreductases and VKOR orthologs. These findings indicate that such bacteria likely form disulfide bonds in the cell envelope. The <i>M. tuberculosis</i><i>vkor</i> gene complements an <i>E. coli</i><i>dsbB</i> deletion strain, restoring the oxidation of <i>E. coli</i> DsbA. While we have suggested that the <i>dsbA</i> gene linked to the <i>vkor</i> gene may express VKOR's partner in mycobacteria, others have suggested that two other extracytoplasmic oxidoreductases (DsbE or DsbF) may be catalysts of protein disulfide bond formation. However, there is no direct evidence for interactions of VKOR with either DsbA, DsbE, or DsbF. To identify the actual substrate of VKOR, we identified two additional predicted extracytoplasmic DsbA-like proteins using bioinformatics analysis of the <i>M. tuberculosis</i> genome. Using the five potential DsbAs, we attempted to reconstitute disulfide bond pathways in <i>E. coli</i> and in <i>Mycobacterium smegmatis</i>, a close relative of <i>M. tuberculosis</i> Our results show that only <i>M. tuberculosis</i> DsbA is oxidized by VKOR. Comparison of the properties of <i>dsbA</i>- and <i>vkor</i>-null mutants in <i>M. smegmatis</i> shows parallels to the properties of <i>dsb</i> mutations in <i>E. coli</i><b>IMPORTANCE</b> Disulfide bond formation has a great impact on bacterial pathogenicity. Thus, disulfide-bond-forming proteins represent new targets for the development of antibacterials, since the inhibition of disulfide bond formation would result in the simultaneous loss of the activity of several classes of virulence factors. Here, we identified five candidate proteins encoded by the <i>M. tuberculosis</i> genome as possible substrates of the <i>M. tuberculosis</i> VKOR protein involved in disulfide bond formation. We then reconstituted the mycobacterial disulfide bond formation pathway in <i>E. coli</i> and showed that of the five candidates, only <i>M. tuberculosis</i> DsbA is efficiently oxidized by VKOR in <i>E. coli</i> We also present evidence for the involvement of VKOR in DsbA oxidation in <i>M. smegmatis</i>.