The mutant T379L of novel aspartokinase from Corynebacterium pekinense: A combined experimental and molecular dynamics simulation study

Aspartokinase (AK) is known to be inhibited by the synergistic feedback of threonine and lysine in the relevant biosynthesis pathway in Corynebacterium, thereby hindering downstream metabolite accumulation. In this study, site-directed mutagenesis was performed to modify AK, and the mutant's characteristics were found to be different, laying the foundation for the downstream metabolite accumulation. Position 379 was identified as the key inhibitor-binding amino acid residue via homologous sequencing and comparison with the 3AAW conformation (Protein Data Bank). The mutant strain T379L, with 9.16-fold higher enzymatic activity, was obtained via site-directed mutagenesis and high-throughput screening. An analysis of the properties of the mutant enzyme revealed that the optimal temperature increased from 26 to 35 degrees C, the optimal pH stayed at 8.0, and the enzyme stability decreased from 4.5 to 3.32 h. The inhibition of T379L was weaker at various inhibitor concentrations; however, the inhibition turned into activation at low concentrations of inhibitors: Lys, Lys + Thr, or Lys + Met. Molecular dynamics simulations revealed that, with an increase in the enzymatic activity of the mutant T379L, the affinity between AK and Asp increased, and the catalytic residues (S192 and D193) shifted toward Asp.