Improved catalytic activity of a novel aspartate kinase by site-directed saturation mutagenesis

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 V-max 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 K-n 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 degrees C to 35 degrees C, the optimum pH remained unchanged. The stability was determined at optimum temperature (35 degrees 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.

Automated summary

This study aimed to improve the catalytic activity of aspartate kinase (AK), the first key enzyme in the aspartic acid metabolism pathway, and weaken the feedback inhibition of metabolites. The mutant A380M with significantly increased enzyme activity showed enhanced substrate affinity. Molecular dynamics simulations revealed changes in the binding force and interaction between AK and its substrates.