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

UNLABELLED

To enhance the specific activity and catalytic efficiency ( / ) of an NADH-dependent PPR, 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 PPR-encoding gene () were amplified and expressed in BL21 (DE3). The asymmetric reduction of 20 mM phenylpyruvic acid (PPA) was carried out using 50 mg/mL / or / 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 /. Secondly, double-site variants were obtained by saturation mutagenesis of Ala79 in PPR. Among all tested transformants, / exhibited the highest d-PLA yield of 85.3%. The specific activity and / of the purified PPR increased to 67.5 U/mg and 169.8 mM s, which were 3.0- and 13.2-fold those of PPR, respectively. Finally, the catalytic mechanism analysis of PPR by molecular docking simulation indicated that the replacement of Arg53 in PPR 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.