Structure-guided steric hindrance engineering of Bacillus badius phenylalanine dehydrogenase for efficient l-homophenylalanine synthesis

Background Direct reductive amination of prochiral 2-oxo-4-phenylbutyric acid (2-OPBA) catalyzed by phenylalanine dehydrogenase (PheDH) is highly attractive in the synthesis of the pharmaceutical chiral building block l-homophenylalanine (l-HPA) given that its sole expense is ammonia and that water is the only byproduct. Current issues in this field include a poor catalytic efficiency and a low substrate loading. Results In this study, we report a structure-guided steric hindrance engineering of PheDH from Bacillus badius to create an enhanced biocatalyst for efficient l-HPA synthesis. Mutagenesis libraries based on molecular docking, double-proximity filtering, and a degenerate codon significantly increased catalytic efficiency. Seven superior mutants were acquired, and the optimal triple-site mutant, V309G/L306V/V144G, showed a 12.7-fold higher k(cat) value, and accordingly a 12.9-fold higher k(cat)/K-m value, than that of the wild type. A paired reaction system comprising V309G/L306V/V144G and glucose dehydrogenase converted 1.08 M 2-OPBA to l-HPA in 210 min, and the specific space-time conversion was 30.9 mmol g(-1) L-1 h(-1). The substrate loading and specific space-time conversion are the highest values to date. Docking simulation revealed increases in substrate-binding volume and additional degrees of freedom of the substrate 2-OPBA in the pocket. Tunnel analysis suggested the formation of new enzyme tunnels and the expansion of existing ones. Conclusions Overall, the results show that the mutant V309G/L306V/V144G has the potential for the industrial synthesis of l-HPA. The modified steric hindrance engineering approach can be a valuable addition to the current enzyme engineering toolbox.

Automated summary

This study successfully enhanced the efficiency of l-HPA synthesis by engineering PheDH through a structure-guided steric hindrance approach, resulting in the identification of seven superior mutants. The optimal triple-site mutant, V309G/L306V/V144G, showed significantly higher catalytic activity compared to the wild type, achieving the highest substrate conversion rates to date. Docking simulations revealed changes in substrate-binding volume and enzyme tunnels, indicating the potential industrial applications of this mutant for l-HPA synthesis.