Characterization Of The Active Sites Of Protein Disulfide Isomerase From The Archaea Methanothermobacter Thermautotrophicus

Thermophilic autotrophic methanogens,micro-rod-like or filamentous,spore-free,nor athletic,the optimum growth temperature is 65 ?-70 ?,is a very strict anaerobic Of the thermophilic archaea.Protein disulfide isomerase(MtPDI)catalyzes the formation and isomerization of protein disulfide bonds,which have molecular chaperone activity and play a key role in the protection of host thermal damage.In order to study the key amino acid sites in the MtPDI activity domain,the active domain of Mt PDI(C74PAC77)was predicted by bioinformatics,and its effect on the enzyme activity of disulfide isomerase was analyzed by site-directed mutagenesis.The results are as follows:1.The reductase activities of MtPDI(C74S),MtPDI(C77S)and MtPDI(C74S / C77S)were significantly decreased in the Mt PDI activity domain,and the percentages were decreased by 32.2%,40.7% % And 51.2%,the effect of double mutant enzyme activity was greater than that of single point mutation.Molecular chaperone activity also decreased,single mutant enzyme activity than the double mutation site.However,mutations at C74 and C77 did not affect the chaperone activity of MtPDI.2.After heat treatment,the thermal damage protection of mutant MtPDI(C74S),MtPDI(C77S),MtPDI(C74S / C77S)to Escherichia coli was significantly lower than that of Mt PDI.but the growth of bacteria was basically the same under salt stress.In summary,the MtPDI activity domain C74,C77 site amino acid is the amino acid that plays a major role in reductase activity.And mutations in the C74 and C77 sites in the MtPDI activity domain affect MtPDI thermal damage protection.