Abstract
ncgl2478 gene from Corynebacterium glutamicum encodes a thiol-disulfide oxidoreductase enzyme annotated as dithiol-disulfide isomerase DsbA. It preserves a Cys-Pro-Phe-Cys active-site motif, which is presumed to be an exclusive characteristic of the novel DsbA-mycoredoxin 1 (Mrx1) cluster. However, the real mode of action, the nature of the electron donor pathway and biological functions of NCgl2478 in C. glutamicum have remained enigmatic so far. Herein, we report that NCgl2478 plays an important role in stress resistance. Deletion of the ncgl2478 gene increases the size of growth inhibition zones. The ncgl2478 expression is induced in the stress-responsive extra-cytoplasmic function-sigma (ECF-σ) factor SigH-dependent manner by stress. It receives electrons preferentially from the mycothiol (MSH)/mycothione reductase (Mtr)/NADPH pathway. Further, NCgl2478 reduces S-mycothiolated mixed disulfides and intramolecular disulfides via a monothiol-disulfide and a dithiol-disulfide exchange mechanism, respectively. NCgl2478 lacks oxidase activity; kinetic properties of its demycothiolation are different from those of Mrx1. Site-directed mutagenesis confirms Cys24 is the resolving Cys residue, while Cys21 is the nucleophilic cysteine that is oxidized to a sulfenic acid and then forms an intramolecular disulfide bond with Cys24 or a mixed disulfide with MSH under oxidative stress. In conclusion, our study presents the first evidence that NCgl2478 protects against various stresses by acting as an MSH-dependent thiol-disulfide reductase, belonging to a novel DsbA-Mrx1 cluster.
Keywords:
Corynebacterium glutamicum; Oxidative stress; SigH; Thiol–disulfide interchange protein (DsbA).