Study On The Anti-oxidative Stress Mechanism Of Novel DsbA-type Branched Thiol Redox Proteins From Corynebacterium Glutamicu

Corynebacterium glutamicum is a non-pathogenic Gram-positive bacterium,widely used as an important industrial microorganism in the industrial production of amino acids such as L-glutamate and L-lysine,vitamins,organic acids,polymer precursors,aroma compounds and biofuels.In addition,C.glutamicum has been regarded as a model organism of Corynebacterium and Mycobacterium.However,in the process of industrial fermentation,unstable environmental changes usually cause oxidative stress to cells,that is,excessive production of reactive oxygen species（ROS）causes an imbalance of the intracellular redox state,resulting in the loss of cell function.In response to oxidative stress,organisms have evolved low-molecular-weight non-enzymatic defense systems,including mycothiol（MSH）,and antioxidant enzyme systems.Antioxidative enzymes such as superoxide dismutases（SOD）,mycoredoxin 1（Mrx1）,dithiol disulfide isomerase（DsbA）,and thioredoxin（Trx）,play important roles in removing ROS,enhancing the ability of strains to resist oxidative stress,and improving the survival rate of strains under stress.In 2017,a researcher discovered a novel DsbA-Mrx1 cluster through the study of Mycobacterium tuberculosis Rv2466c and C.glutamicum NCgl2339.At present,there are very few studies on the novel DsbA-Mrx1 cluster.Through bioinformatics analysis,we found that NCgl2478 in C.glutamicum may be a potential DsbA-Mrx1 enzyme,although it was annotated as dithiol-disulfide isomerase DsbA,it had the active site of Cys-Pro-Phe-Cys（C-P-F-C）,and this active site was considered to be the exclusive motif of the novel DsbA-Mrx1 cluster.Moreover,the biological function,biochemical characteristic,and expression regulation mechanism of NCgl2478 in C.glutamicum are still unknown.Therefore,we concentrate on NCgl2478 in this study,and the main results are as follows:（1）The resistance of ncgl2478 to oxidizing agents,alkylating agents,heavy metals,and antibiotics stress in C.glutamicum was detected with the wild type strain WT,the mutantΔncgl2478,and the complementary strainΔncgl2478⁺.And the results showed that theΔncgl2478 strain was highly sensitive to stress compared with the WT strain,indicating that ncgl2478 played an important role in stress resistance.（2）Oxidized NCgl2478 could be reduced by both the Trx/TrxR/NADPH pathway and the MSH/Mtr/NADPH pathway,but it preferred the MSH/Mtr/NADPH pathway as the electron donor pathway compared to the Trx/TrxR/NADPH pathway.In addition,comparing NCgl2478with Mrx1,it was found that the reduction rate of oxidizing Mrx1 by the MSH/Mtr/NADPH electron pathway was about 8 times faster than that of NCgl2478 by the MSH/Mtr/NADPH electron pathway.（3）Using HED-SSM（the mixed disulfide between HED and MSH）and insulin as substrates,the regeneration mechanism of oxidized NCgl2478 was investigated with NCgl2478WT,NCgl2478:C21S,NCgl2478:C24S.It was found that NCgl2478 reduced S-mycothiolated mixed disulfides and intramolecular disulfides via the monothiol-disulfide and the dithiol-disulfide exchange mechanism respectively.（4）NCgl2478 could not catalyze the oxidative refolding of RNase I,so it did not function as an oxidase.And NCgl2478 could not directly reduce hydrogen peroxide（H₂O₂）and cumene hydroperoxide（CHP）by steady-state kinetics.Therefore,NCgl2478 was neither an oxidase nor a peroxidase.（5）The expression of ncgl2478 was induced in the stress-responsive extra-cytoplasmic function-sigma（ECF-σ）factor Sig H-dependent manner by stress,and Sig H regulated the expression of ncgl2478 directly.（6）To further demonstrate that NCgl2478 belongs to the DsbA-Mrx1 cluster,the physiological and biochemical roles of the DsbA-Mrx1 type protein（NCgl2339）in C.glutamicum were investigated.Oxidized NCgl2339 could be reduced by both the MSH/Mtr/NADPH pathway and the Trx/TrxR/NADPH pathway,but it preferred the MSH/Mtr/NADPH as the electron donor pathway.NCgl2339 could play a role in demycothiolation.TheΔncgl2339 strain was more sensitive to oxidants and alkylating agents compared to the WT strain.All of these properties of NCgl2339 were highly similar to those of NCgl2478,further illustrating that NCgl2478 belonged to DsbA-Mrx1cluster.All in all,this study presents the first evidence that NCgl2478 protects against various stresses by acting as an MSH-dependent thiol-disulphide reductase,belonging to the novel DsbA-Mrx1 cluster.