Abstract:[Background] Compared with traditional genetic modification, molecular dynamics simulation of cyclodextrin glycosyltransferase can effectively improve the transformation efficiency and reduce blindness. [Objective] To explore the catalytic specificity mechanism of cyclodextrin glycosyltransferase, and to provide an efficient mutation method for obtaining cyclodextrin glycosyltransferase with higher specificity for producing γ-cyclodextrin. [Methods] Through molecular docking and molecular dynamics simulation, the docking simulation structures of three product types of CGTase and substrate were obtained, and verified by site-specific saturation mutation experiment. [Results] The results of molecular dynamics simulation showed that α- and β-CGTase and decarbose chains appeared closed in S1 region, while γ-CGTase and decarbose chains appeared more open in S1 region. There are seventeen corresponding sites in the three CGTase and ten sugar chain amino acids with hydrogen bonds at the same position, of which the amino acid types at fourteen sites are consistent. The corresponding α-CGTase sites of the three inconsistent amino acids were Y89, D234 and Y262, respectively. This study conducted site-directed mutagenesis and product specificity experiments on the Y262 locus. Y262L predicted by molecular dynamics was helpful to increase the specificity of γ-CD production, from 13.7% of the wild enzyme to 39.9%, and the percentage of γ-cyclodextrin products increased by three times. [Conclusion] The results of molecular dynamics simulation have positive significance for guiding the specificity mechanism of cyclodextrin glycosyltransferase.
