Characterization of Xi-class mycothiol S-transferase from Corynebacterium glutamicum and its protective effects in oxidative stress

Abstract

BackgroundOxidative stress caused by inevitable hostile conditions during fermentative process was the most serious threat to the survival of the well-known industrial microorganism Corynebacterium glutamicum. To survive, C. glutamicum developed several antioxidant defenses including millimolar concentrations of mycothiol (MSH) and protective enzymes. Glutathione (GSH) S-transferases (GSTs) with essentially defensive role in oxidative stress have been well defined in numerous microorganisms, while their physiological and biochemical functions remained elusive in C. glutamicum thus far.ResultsIn the present study, we described protein NCgl1216 belonging to a novel MSH S-transferase Xi class (MstX), considered as the equivalent of GST Xi class (GSTX). MstX had a characteristic conserved catalytic motif (Cys-Pro-Trp-Ala, C-P-W-A). MstX was active as thiol transferase, dehydroascorbate reductase, mycothiolyl-hydroquinone reductase and MSH peroxidase, while it showed null activity toward canonical GSTs substrate as 1-chloro-2,4-dinitrobenzene (CDNB) and GST Omega’s specific substance glutathionyl-acetophenones, indicating MstX had some biochemical characteristics related with mycoredoxin (Mrx). Site-directed mutagenesis showed that, among the two cysteine residues of the molecule, only the residue at position 67 was required for the activity. Moreover, the residues adjacent to the active Cys67 were also important for activity. These results indicated that the thiol transferase of MstX operated through a monothiol mechanism. In addition, we found MstX played important role in various stress resistance. The lack of C. glutamicum mstX gene resulted in significant growth inhibition and increased sensitivity under adverse stress condition. The mstX expression was induced by stress.ConclusionCorynebacterium glutamicum MstX might be critically involved in response to oxidative conditions, thereby giving new insight in how C. glutamicum survived oxidative stressful conditions.

Effect of the TetR family transcriptional regulator Sp1418 on the global metabolic network of Saccharopolyspora pogona

“…In summary, Sp1418 is an important global regulator in S. pogona that affects the sporulation, growth, and butenyl-spinosyn biosynthesis, which was likely due to the disturbed redox balance and abnormally expressed enzymes. During the fermentative process, oxidative stress is the most serious threat to the synthesis of cell survival [43], which has a prominent impact on the physiological and biochemical changes. In our study, the proteins CAT, RpoC and InfB involved in oxidative stress are speculated to be the main factor causing the alteration of the engineering strains, and their expression changes were regulated by Sp1418.…”

A novel mycothiol-dependent thiol–disulfide reductase in Corynebacterium glutamicum involving oxidative stress resistance

“…Hydroxyethyl disulfide (HED) was utilized to prepare and measure the mixed disulfide between MSH and 2-hydroxyethyl disulfide (HED) (HED-SSM) reduction activity (Si et al 2019 ). HED-SSM was formed by incubating 1000 mM HED with 50 mM MSH at 30 °C for 3 min.…”

“…The lacZY fusion reporter plasmid pK18 mobsacB-P ncgl2478 ::lacZY was transformed into the corresponding C. glutamicum RES 167 strain by electroporation and the chromosomal fusion reporter strains were selected on LB agar plates supplemented with kanamycin (Si et al 2019 ). The resulting strains were grown in LB medium to an optical density at 600 nm of 0.6–0.7 and then treated with different reagents of various concentrations at 30 °C for 30 min.…”

“…Isolation of the total RNA and transcript levels analysis was performed as described previously (Si et al 2019 ). To obtain standardized results, the relative abundance of the 16S rRNA gene was used as the internal standard.…”

“…C. glutamicum RES 167 strain by electroporation and the chromosomal fusion reporter strains were selected on LB agar plates supplemented with kanamycin (Si et al 2019).…”