Isolation and mechanism analysis of a catalytic efficiency improved L-aspartate -decarboxylase toward 3-methylaspartic acid

L-Aspartate beta-decarboxylase from Acinetobacter radioresistens (ArASD) has been modified to convert 3-methylaspartic acid into 2-aminobutyric acid, which activated a novel process for biosynthesis of 2-aminobutyric acid. However, the process is limited by the low activity of the ArASD. Here, the activity of ArASD was significantly improved by modification based on sequence alignment and structural analysis. The 38th residue of ArASD is speculated to be the key residue for regulating the conformation of the internal aldimine, and site-directed mutagenesis on R38 residue was carried out. A variant, K18A/R38K/V287I, with 2.2 times higher specific activity was isolated. Molecular dynamics simulation indicated that the torsion angle of the imine bond of the variant decreased, which was beneficial to the protonation of the internal aldimine and the increase in the initial energy of the enzyme. Therefore, the energy barrier of the transition state was reduced, resulting in improved catalytic activity toward 3-methylaspartic acid. These results provide a reference and a new point of view for enzyme modification by increasing the energy of the initial state.