Gene Cloning And Enzymatic Properties Of Glutathione S-Transferase From Monascus Purpureus

Monascus is a small,filamentous saprophytic fungus.Its metabolites such as monascus pigment and lovastatin have been widely used in food and medicine.Glutathione S-transferase（GST;EC2.2.1.18）is a kind of conserved supergene family enzyme line with multiple functions,which is widely involved in various physiological metabolic processes of organisms.In this study,the GST gene family of monascus was taken as the object,and bioinformatics analysis,gene cloning,enzymatic characteristics and expression patterns were studied.The main research results were as follows:1.A total of 17 GST genes were identified in the M.purpureus genome,which had typical GST protein domains by conserved domain analysis,belonging to Ure2p,e EF1Bγ,GTT1,Omega and other different subfamilies.Using M.purpureus h2019 as the experimental strain,17 GST members were cloned successfully,and bioinformatics analysis was performed on them,including analysis of physical and chemical properties（molecular weight,isoelectric point,hydrophobicity,etc.）,chromosome localization,evolutionary analysis,sequence analysis（sequence alignment,gene structure,conserved motifs,etc.）.Prediction of protein structure,and prediction of promoter cis-elements and protein interactions.The results showed that the physicochemical properties of different members of monascus GST gene family were different.The molecular weight of most members is about 25k Da,the instability coefficient of 52.3%members is less than 40,and the isoelectric point of 47.1%members is more than 7.Members belonging to e EF1Bγand Ure2p showed high sequence similarity and showed similar gene structure and Motif structure.The Ka/Ks of homologous gene pairs was less than 1,which may have experienced strong negative selection during evolution.Sequence alignment analysis showed that the sequences of different GST members were very different,but the 10 GST proteins obtained by homologous modeling method had similar 3D topologies and were all homologous dimers.The prediction of cis-element and protein interaction indicated that the GST genes from Monascus may be involved in a variety of abiotic stress response processes and is closely related to the growth,development,and physiological metabolism of Monascus.2.The expression pattern of GST genes from M.purpureus was analyzed by q RT-PCR.The results showed that the expression of GST genes was significantly different at different growth stages,and Mp GST7 began to express on the fifth day of monascus growth.The expression level of Mp GST1 was the highest on the second day,up to 21.56 folds of that of theβ-actin.Correlation analysis showed that Mp GST3,Mp GST14,Mp GST17 and other seven members had high correlation,which may play similar roles in different growth stages of monascus.In addition,the expression of key genes of monascus pigments,lovastatin and citrinomycin metabolites were also correlated with the expression of GST to varying degrees,suggesting that GST from Monascus may be involved in the synthesis and metabolism of important metabolites.The expression of 15 members was up regulated under 0.5m M hydrogen peroxide stress,indicating that GST from Monascus was involved in oxidative stress,and Mp GST3,Mp GST8 and Mp GST17 members may be one of the important strategies of M.purpureus to cope with high oxygen free radical stress.The expression of all members was down-regulated by HED induction,while some members were up-regulated by other substrates induction.Among them,7 genes were significantly upregulated during induction of CDNB,and the most noteworthy one was Mp GST1,whose expression could be upregulated by more than50 folds.Ure2p has high positive correlation with three genes,which is preliminarily predicted to have similar substrate spectra.Under different metal ion stress,all members showed different degrees of response.The expression of the most members was up-regulated under 2m M Zn²⁺treatment,and most members were down-regulated under Fe³⁺and Ba²⁺stress.The response to temperature was also different in high temperature environment.The 6 members were significantly up regulated in response to low temperature stress,and Mp GST17 and Mp GST11were highly expressed under low temperature and high temperature stress.3.The homology model of GST protein was used as the receptor,and the electron-phile substrates such as CDNB and NBC were used as ligands for molecular docking.The results showed that the binding energy and interaction force of different members on different substrates were different.The binding energy of all members on the first substrate GSH was higher than that on the second substrate,indicating that they had a low affinity for GSH.The binding energy of the second substrate HED was the highest,indicating that GST from M.purpureus had a low affinity for HED.The binding energy of the other second substrates was low,but some of the binding pockets did not fall on the C-terminal domain,which may lead to its inability to effectively catalyze the nucleophilic reaction of GSH with it.Eight soluble recombinant GST proteins were obtained by constructing prokaryotic expression vectors.The enzyme activity of recombinant Mp GST1 protein was detected only with CDNB as the substrate,but no activity was detected on other substrates.The Km values of purified recombinant Mp GST1 for GSH and CDNB were 0.78 m M and 0.41m M,respectively,which indicated that Mp GST1 had a higher affinity for CDNB than GSH.The optimum temperature of Mp GST1 was 20℃and the optimum p H was 8.The residual enzyme activity of MPGST1could reach 75.6%and 70.3%,respectively,when incubated at 40℃and p H 9 for 5h.Zn²⁺,Cu²⁺,Ni²⁺,Mn²⁺inhibited the activity of Mp GST1,while K⁺and Ca²⁺promoted the activity of Mp GST1.EDTA inhibited the activity of MPGst1 by 37.4%at 10m M,and SDS completely inactivated it.To further explore the catalytic type of Mp GST1,site-specific mutation showed that the R135A mutant was completely inactivated,and the enzyme activity of both N17A and V56A decreased to about 40%,indicating that Mp GST1 was an atypical catalytic type of GST with Asn17,Val 56,and Arg 135 as the catalytic active sites.In conclusion,17 GST genes were extracted from the M.purpureus genome and analyzed bioinformatically in this study.Studies on gene expression patterns and enzymatic properties showed that GST genes’function were differentiated.Combined with correlation results,GST genes played an important role in the growth,development,and metabolism of Monascus.