Mechanism of the feedback-inhibition resistance in aspartate kinase of : from experiment to MD simulations

• Published in: RSC advances 2020-12, Vol.11 (1), p.3-38
• Main Authors: Liu, Xiaoting, Han, Caijing, Fang, Li, Fan, Zhanqing, Wang, Yanan, Gao, Xin, Shi, Junhua, Min, Weihong
• Format: Article
• ISSN: 2046-2069
• DOI: 10.1039/d0ra09153g

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 ( Cp AK). 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 Cp AK 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 Cp AK and further enhance the enzyme activity. Corynebacterium pekinense AK was successfully modified and two mutants A380C and T379N/A380C with high enzyme activity were constructed. The mechanism of their feedback-inhibition resistance was thoroughly studied from experiment to MD simulations.