关键词: 天冬氨酸激酶；定点突变；酶活力；分子动力学模拟；酶学性质

Abstract: The purpose of this study is to improve the catalytic activity of aspartate kinase (AK), the first key rate-limiting enzyme in aspartic acid metabolism pathway by site-directed random mutagenesis; reduce or remove the synergistic feedback inhibition of metabolites on it; and analyze its spatial structure using Discovery Studio (DS) software and the underlying mechanism through molecular dynamics simulation (MDS). First, the key residue sites around ATP were chosen for further construction of mutants based on the mutant obtained in our previous study, T379N/A380C/G171I. Then, through high-throughput screening, the mutant T379N/A380C/G171I/S227D with significantly increased AK activity was selected from the mutants. Kinetic analysis showed that the Vmax value of T379N/A380C/G171I/S227D was 242.05 U/(mg·min), which was 1.28 and 80.41 times higher than that of T379N/A380C/G171I (187.88 U/(mg·min)) and the wild-type (WT) strain (3.01 U/(mg·min)), respectively. The Km value decreased to 1.35 mmol/L, and the substrate affinity was increased. Through DS software and MDS analysis, it was found that the system became more stable after the mutation, the hydrogen bond occupancy rate with ATP was increased, and the substrate binding stability was enhanced, thereby favoring the catalytic reaction. The enzymatic properties showed that the optimal reaction temperature of T379N/A380C/G171I/S227D was 30 ℃, 5 and 2 ℃ higher than the wild-type strain and T379N/A380C/G171I, respectively; the optimal pH was 8.5 compared to 9.0 for T379N/A380C/G171I; the half-life was 3.9 h, which was 0.8 h longer than T379N/A380C/G171I. In addition, in the presence of different concentrations of the inhibitors Lys, Thr and Met as well as different combinations of Lys + Thr, Lys + Met, Thr + Met and Lys + Thr + Met, the mutant strain was activated by up to 143.35% rather than inhibited.

Key words: aspartate kinase; site-directed mutagenesis; enzyme activity; molecular dynamics simulation; enzymatic properties