LAUSR

Metalloprotease PT121Y114S, an effective catalyst for Z-aspartame synthesis under the substrate (Z-Asp:l-Phe-OMe) molar ratio of 1:5, was obtained previously. Herein, a computational strategy combining molecular dynamics simulation of the enzyme-substrate complex with binding free energy (ΔG) calculations was established to guide the further engineering of PT121Y114S. One His224 residue proximal to the PT121Y114S active site was selected on the basis of the difference in ΔG decomposition of PT121Y114S toward l-Phe-NH2 and l-Phe-OMe. Site-saturation mutagenesis of His224 resulted in the mutants H224D, H224N, and H224S, which showed great improvement in Z-aspartame synthesis under an economical substrate molar ratio approaching 1:1. Furthermore, the kinetic constants of PT121Y114S and its mutants revealed that the affinity of mutants toward the l-Phe-OMe was significantly higher than that of PT121Y114S. Molecular dynamic simulation revealed that the enhanced synthetic activity may be attributed to the mutation stabilizing the transient state of the enzyme-l-Phe-OMe complex.