This study aimed to improve the catalytic activity of aspartate kinase (AK), the first key rate-limiting enzyme in the aspartic acid metabolism pathway, by site-directed saturation mutagenesis, and to weaken the synergistic feedback inhibition of metabolites and analyze its mechanism using molecular dynamics simulation (MD). The key residual sites around the inhibitor lysine (Lys) were selected to construct the mutant strains. The mutant A380M with significantly increased enzyme activity was obtained through enzyme activity screening. Kinetic analysis showed that the Vmax value increased to 15.73 U/mg, which was 4.8 times higher than that of wild-type AK (WT AK) (3.28 U/mg). The Kn value decreased to 0.61 mM, which was significantly lower than that of the wild type (4.77 mM), indicating that the substrate affinity increased. The enzyme properties analysis showed that the optimum temperature of the mutant A380M increased from 26 °C to 35 °C, the optimum pH remained unchanged. The stability was determined at optimum temperature (35 °C) and optimum pH 8.0, and it decreased from 4.8 h to 2.7 h. The feedback inhibition was weakened, showing a significant activation with the highest relative enzyme activity of 123.29% (Water was used instead of inhibitor as blank control group, and the highest enzyme activity was defined as 100%). Molecular dynamics simulations showed that the distance between ATP and Asp was shortened after mutation. The binding force and interaction between AK and ATP and substrate Asp were enhanced. The distance between catalytic residues D193 and S192 and substrate Asp was shortened.
Keywords: Aspartate kinase; Enzymatic properties; Feedback inhibition; Molecular dynamics simulation; Site-directed saturation mutagenesis.
© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.