Corynebacterium glutamicum Methionine Sulfoxide Reductase A Uses both Mycoredoxin and Thioredoxin for Regeneration and Oxidative Stress Resistance

Abstract

A erobic metabolism and oxidative stress lead to the production and accumulation of reactive oxygen species (ROS). ROS induce oxidative damage to macromolecules, such as proteins, lipids, and DNA, ultimately inducing cell death and disease. In proteins, oxidation modification occurs mainly at the sulfur-containing amino acids, such as cysteine (Cys) and methionine (Met) (1). Oxidation of methionine leads to the formation of the R and S diastereomers of methionine sulfoxide (MetO) and results in either conformational change or loss of function of proteins (2). However, this oxidation can be reversed by the action of methionine sulfoxide reductase (Msr), a ubiquitous monomeric enzyme that reduces both free and protein-bound MetO by employing sulfenic acid chemistry (3). Two structurally unrelated classes of Msrs, MsrAs and MsrBs, reduce the S and R MetO diastereoisomers, respectively. MsrA has been implicated in physiological processes, such as protection of cells against oxidative damage and extension of the life span, and acts as a virulence factor in some bacterial pathogens (4-6).MsrAs can be classified into three groups, 3-Cys, 2-Cys, and 1-Cys MsrAs, according to the number of redox-active Cys residues. Three-Cys MsrAs, such as the Escherichia coli and Bos taurus enzymes, have three Cys residues involved in catalysis, one of which functions as a catalytic residue and the other two as resolving residues (7,8). Two-Cys MsrAs, such as the Saccharomyces cerevisiae and Streptococcus pneumoniae enzymes, possess a single resolving Cys in addition to the catalytic Cys (4, 9). The 3-Cys and 2-Cys MsrAs present similar catalytic mechanisms. First, the catalytic Cys residue attacks a sulfoxide moiety of the substrate and then forms a sulfenic acid intermediate, with a concomitant release of Met as a product. Second, the sulfenic acid of the catalytic Cys forms an intramolecular disulfide bond with a resolving Cys. For 2-Cys MsrAs, this disulfide bond is directly reduced by a reducing agent to regenerate the active form of the enzyme, such as thioredoxin (Trx), which is a natural reducing agent for Msrs. For 3-Cys MsrAs, which perform an extra thiol-disulfide exchange reaction, the first disulfide bond is further reduced by the second

Myo‐inositol‐1‐phosphate synthase (Ino‐1) functions as a protection mechanism in Corynebacterium glutamicum under oxidative stress

“…Since MSH is a key nonenzymatic antioxidant that protects the cells from ROS damage by directly scavenging free radicals and by serving as a cofactor for antioxidant enzymes, we were prompted to investigate whether Ino‐1 plays a role in removing ROS under stress conditions (Si, Xu, et al., ; Si, Zhang, et al., ). The ROS levels in vivo were detected using DCFH‐DA, a membrane permeable dye which passively diffuses into cells (Si et al., ).…”

“…Sensitivity assays for various stress conditions were investigated as previously described (Si, Xu, et al., ; Si, Zhang, et al., ). Overnight‐grown cultures of C. glutamicum (LB broth, 30°C) were diluted 100‐fold with LB medium and exposed to CHP (11 mM), MD (4 mM), H 2 O 2 (100 mM), CdCl 2 (300 μM), NiSO4 (6 mM), IAM (40 mM), MG (10 mM), and CDNB (70 mM) at 30°C with shaking for 30 min.…”

Functional comparison of methionine sulphoxide reductase A and B in <i>Corynebacterium glutamicum</i>

“…In a previous study, we have shown that MsrA plays an important role in hostile conditions, and not only can it reduce MetO to Met via the thioredoxin/thioredoxin reductase (Trx/TrxR) reducing system, but also the mycoredoxin 1/mycothione reductase/mycothiol (Mrx1/ Mtr/MSH) system (Si et al, 2015b). The purpose of the present study was to investigate the function of MsrB and compare it with MsrA.…”

“…We have earlier reported that the msrA deletion mutant showed a significant sensitivity in a hostile environment (Si et al, 2015b). To test whether MsrB has the same effect as MsrA does, the sensitivity of wild-type, DmsrA mutant, the complementary strain DmsrA (pXMJ19-msrA), DmsrB and the complementary strain DmsrB (pXMJ19-msrB) to H 2 O 2 (80 mM), cumene hydroperoxide (CHP, 8 mM) and CdCl 2 (0.3 mM) were tested (Fig.…”

“…During MsrA regeneration, the intramolecular disulfide bond is subject to attack by Trx and switches to a transient intermolecular disulfide bond to link MsrA with Trx (Kim et al, 2009a, b;Si et al, 2015b;Tarrago et al, 2009a). To clarify the mechanism for regenerating MsrB activity, Trx variants (TrxC32S and TrxC35S), Mrx1 variants (Mrx1C12S and Mrx1C15S) and their wild-type were incubated with MsrB, respectively, in the presence of MetO.…”