Abstract
A novel aspartokinase mutant M372I/T379W from Corynebacterium pekinense was constructed by using site-directed mutagenesis. The enzyme was then purified, characterized, and its molecular mechanism was comprehensively analyzed. Compared with wild-type AK, the catalytic activity of M372I/T379W AK was 16.51 fold higher and the optimum temperature increased from 28 to 35 °C. The thermostability of M372I/T379W AK was significantly improved. Microscale thermophoresis analysis indicated that M372I/T379W AK not only weakened the inhibitory effect of Lys, but also had stronger binding force with Asp. Molecular dynamics simulation showed that mutations M372I and T379W could regulate the activity of CpAK through affecting the flexibility of Asp and ATP binding pocket residues and the hydrogen bond between CpAK and Asp. In addition, mutations could affect the relative position of protein domains. The width of the Asp binding pocket entrance gate Arg169-Ala60 of M372I/T379W AK was greater than that in wild-type AK and the CpAK switched from T-state to R-state, which promoted the binding of the enzyme to Asp and improving the catalytic efficiency of this enzyme. These results explain the molecular mechanism of M372I/T379W AK, which will greatly facilitate the rational design of more aspartokinase mutants, with have potential applications in aspartic acid metabolism.