Improvement in the catalytic performance of a phenylpyruvate reductase from Lactobacillus plantarum by site-directed and saturation mutagenesis based on the computer-aided design.

ABSTRACT: To enhance the specific activity and catalytic efficiency (k _cat/K _m) of an NADH-dependent LpPPR, its directed modification was performed based on the computer-aided design using molecular docking simulation and multiple sequence alignment. Firstly, five single-site variants of an LpPPR-encoding gene (lpppr) were amplified and expressed in E. coli BL21 (DE3). The asymmetric reduction of 20 mM phenylpyruvic acid (PPA) was carried out using 50 mg/mL E. coli/lpppr ^R53Q or /lpppr ^A79V whole wet cells at 37 °C for 20 min, giving d-phenyllactic acid (PLA) with 41.1 or 44.3% yield, being 1.17- or 1.26-fold that by E. coli/lpppr. Secondly, double-site variants were obtained by saturation mutagenesis of Ala79 in LpPPR^R53Q. Among all tested E. coli transformants, E. coli/lpppr ^R53Q/A79V exhibited the highest d-PLA yield of 85.3%. The specific activity and k _cat/K _m of the purified LpPPR^R53Q/A79V increased to 67.5 U/mg and 169.8 mM^-1 s^-1, which were 3.0- and 13.2-fold those of LpPPR, respectively. Finally, the catalytic mechanism analysis of LpPPR^R53Q/A79V by molecular docking simulation indicated that the replacement of Arg53 in LpPPR with Gln expanded its substrate-binding pocket, while that Ala79 with Val formed an additional π-sigma interaction with phenyl group of PPA.

Supplementary material

The online version of this article (10.1007/s13205-020-02633-3) contains supplementary material, which is available to authorized users.

SUBMITTER: Zhang D 

PROVIDER: S-EPMC7806690 | biostudies-literature |

REPOSITORIES: biostudies-literature