Exploration Of Key Amino Acid Residues Of "Non-active Amino Acid Residues" In StDAPDH

There are only a small amount of D-amino acids in nature,but D-amino acids have very important applications in the pharmaceutical,food,and cosmetics industries.Meso-diaminopimelate dehydrogenase(meso-DAPDH)is a preferred enzyme by using one-step method,because of its high stereospecificity,it could be used for the synthesis of D-amino acids.In the previous study,we found that the meso-DAPDH family could be divided into two types: type I and type II.The meso-DAPDH from Corynebacterium glutamicum(CgDAPDH)belonged to type I.Type I meso-DAPDH showed a more specific oxidative deamination activity to meso-diaminopimelic acid(meso-DAP).While the meso-DAPDH from the Symbiobacterium thermophilum(StDAPDH))belonged to type II.Type II meso-DAPDH showed not only a more specific oxidative deamination activity to meso-diaminopimelic acid(meso-DAP),but also showed more obvious reversible amination activity than the others and had a broader spectrum of the substrate.By comparing the structures of CgDAPDH and StDAPDH,we found that they had very similar spatial folding structures.In this dissertation,we explored the key residues in the direction of reductive amination between StDAPDH and other meso-DAPDHs from the perspective of "non-active-site",found key aminating sites in the entire family,and analyzed its mechanism.Above all,based on the sequence and conservation analysis,we found that the two amino acid residues of StDAPDH,R35 and R71,were highly conserved within type II,while distinct from each other between the sub-types.Site mutagenesis studies identified R71 as a substrate preference-related residue of StDAPDH,which may serve as an indicator of the amination preference of type II,considering its high degree of conservation and that the R71 located next to the NADP(H)-binding site,but not at the Substrate binding site.Subsequently,site-directed mutagenesis of the R71 of StDAPDH was performed,and the functional role of type I meso-DAPDH CgDAPDH corresponding to the R71 of StDAPDH was studied.The results showed that the Arg of StDAPDH 71 was the best amino acid for its amination function,and the CgA69 R mutant showed higher catalytic efficiencies toward a series of 2-keto acids,ranging from 1.2-to 1.5-fold,after the mutation of the allele A69 of CgDAPDH to Arg.It was further proved that the 71 st amino acid of StDAPDH was the amination index of meso-DAPDH.Finally,through molecular docking and molecular dynamics simulations,it was found that in the enzyme-pyr complex system,the mutation at position 71 destroyed the cation-? interaction between R71 and Y205,caused the orientation rotation of pyruvate and the weak hydrogen-binding between the T70 and NADPH sites.The working mechanism of R71 related to amination and the effect on substrate selection were shown.These results deeping our understanding of the reductive amination ability of StDAPDH and provided new ideas for further understanding its catalytic mechanism.