Redesigning transamination and decarboxylation characteristics of L-aspartate aminotransferase by site directed mutation of non-active site

Aspartate aminotransferase (L-AspAT) is a highly substrate-specific biocatalyst for chiral amino acid splitting and unnatural amino acid synthesis, with both transamination and decarboxylation functions, but it is frequently interfered with each other in the catalytic process. The characterized mutant strains for the isolation of transamination and decarboxylation were obtained by bioinformatics analysis and site-directed mutagenesis in this research. The results showed that the mutants T296D and C180I enhanced decarboxylation by hydrogen bonding to α-ketoglutarate. In contrast, the mutants W130S and W130P increased the transamination activities to 157 % and 144 % of the WT respectively, which improved the binding efficiency through the expansion of the substrate binding pocket of the mutations. In addition, the catalytic efficiency of W130P for o-chlorobenzoylformate was also increased to 226 % of the WT, and the size of the binding pocket with o-chlorobenzoylformate was increased from 9.2 Å to 11.5 Å, a big change improved the probability of the substrate's entry. These results provided an extremely important idea and theoretical basis for changing the preference of bifunctional enzymes by mutating.