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.
Highlights
Corynebacterium glutamicum, a well-known various l-amino acid producer in industrial applications and a model organism in systems biology, unavoidably generates or encounters a series of unfavorable circumstances in the fermenting process (Bröer et al 1993)
A demonstration indicated that Rv2466c from M. tuberculosis and NCgl2339 from C. glutamicum, having high sequence similarity to DsbA, belong to a novel DsbA–mycoredoxin 1 (Mrx1) cluster (Rosado et al 2017)
Amino acid sequence comparison revealed NCgl2478 is only 29.8% identical to Rv2466c and 26.2% identical to NCgl2339 (Fig. 1a), we presumed it might be a member of the DsbA-Mrx1 cluster involved in stress response as it shared the C–P–F–C signature motif presumed to be an exclusive characteristic of the DsbA–Mrx1 cluster (Fig. 1b)
Summary
Corynebacterium glutamicum, a well-known various l-amino acid producer in industrial applications and a model organism in systems biology, unavoidably generates or encounters a series of unfavorable circumstances in the fermenting process (Bröer et al 1993). Mrx with the Cys–Pro–Tyr–Cys (C–P–Y–C) motif, an MSHdependent disulfide oxidoreductase with a glutaredoxin-like sequence and function, reduces mixed disulfides between MSH and CP of peroxidase with N-terminal cysteine of the active site via 1-Cys monothiol mechanism. Rosado et al believed NCgl2339 and Rv2466c were novel oxidoreductases, belonging to DsbA-like Mrx. Bioinformatics analysis reveals that C. glutamicum NCgl2478 preserves the C–P–F–C active-site sequence motif, similar to that of NCgl2339. The phenomenon indicated NCgl2478 may be a potentially new redox enzyme and protect C. glutamicum from oxidative stress. We selected ORF NCgl2478 and sought to explore the physiological and biochemical functions of NCgl2478 in C. glutamicum, paving the way for correctly classifying similar enzymes from other organisms
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