Abstract
In microorganisms and plants, aspartate kinase (AK) is the initial committed enzyme of the biosynthesis of the aspartate acid family amino acids and is inhibited by end products. In the paper, we mutated the key allosteric regulatory site A380 around the binding site of the Lys inhibitor in Corynebacterium pekinense AK (CpAK). A single-mutant A380C was obtained with 12.35-fold higher enzyme activity through high-throughput screening. On this basis, T379 as another key allosteric regulatory site was further modified, and the double-mutant T379N/A380C with 22.79-fold higher enzyme activity was obtained. Molecular dynamics (MD) simulations were used to investigate the mechanism of allosteric inhibition by Lys. The results indicated that the binding of Lys with CpAK resulted in conformational changes and a larger distance between the phosphorus atom of ATP and the oxygen atom of Asp, which was detrimental for the catalytic reaction. However, the mutation of allosteric sites opens the "switch" of allosteric regulation and can prevent the conformational transformation. Some key residues such as G168, R203, and D193 play an important role in maintaining the substrate binding with CpAK and further enhance the enzyme activity.