Abstract
In this study, a novel monomer aspartokinase (AK) from Corynebacterium pekinense was identified, and its monomer model was constructed. Site 380 was identified by homologous sequencing and monomer model comparison as the key site which was conserved and located around the binding site of the inhibitor Lys. Furthermore, the mutant A380I with enzyme activity 11.32-fold higher than wild type AK (WT-AK), was obtained by site-directed mutagenesis and high throughput screening. In the mutant A380I, the optimal temperature was raised from 26 °C (WT-AK) to 28 °C, the optimal pH remained unchanged at 8.0, and the half-life was prolonged from 4.5 h (WT-AK) to 6.0 h, indicating enhanced thermal stability. The inhibition of A380I was weakened at various inhibitor concentrations and even activated at certain inhibitor concentrations (10 mM of Lys, 5 mM or 10 mM of Lys + Thr, 10 mM of Lys + Met, 5 mM of Lys + Thr + Met). Molecular dynamics simulation results indicated that the occupancy rate of hydrogen bond between A380I and ATP was enhanced, the effect of Lys (inhibitor) on the protein was weakened, and the angle between Ser281-Tyre358 and Asp359-Gly427 was increased after mutation, leading to an open conformation (R-state) that favored the binding of substrate.