Enhancement of Z-aspartame synthesis by rational engineering of metalloprotease

Abstract Metalloprotease PT121 Y114S , 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 (Δ G ) calculations was established to guide the further engineering of PT121 Y114S . One His224 residue proximal to the PT121 Y114S active site was selected on the basis of the difference in Δ G decomposition of PT121 Y114S toward l -Phe-NH 2 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 PT121 Y114S and its mutants revealed that the affinity of mutants toward the l- Phe-OMe was significantly higher than that of PT121 Y114S . 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.