T211K substitution in Pseudomonas putida phenylserine aldolase improves catalytic efficiency towards l-threo-4-nitrophenylserine with reversed stereoselectivity

Phenylserine aldolase from Pseudomonas putida (PSALD) catalyzes the conversion of glycine and 4-nitrobenzaldehyde to 4-nitrophenylserine with unsatisfactory enantioselectivity at the Cβ position. In this report, mutations adjacent to the substrate-binding pocket of PSALD were introduced using site-directed mutagenesis to improve product stereoselectivity. The activity of the variants towards native substrates l-threonine and l-allo-threonine decreased by 2- to 10-fold in the retro-aldol reaction when compared with that of wild-type (WT) PASLD. In particular, the T211K mutant biosynthesizes l-erythro-4-nitrophenylserine from glycine and 4-nitrobenzaldehyde as substrates with improved catalytic efficiency and reversed product stereoselectivity. This mutant synthesized l-erythro-4-nitrophenylserine with diastereoselectivity (de) of 84% and a conversion of 57%, whereas WT PSALD yielded a de of 5% and a conversion of 14%. In order to study the binding force between proteins and ligands, thermodynamic parameters were calculated by fluorescence spectroscopy. The determined thermodynamic parameters (ΔH = −76.52 kJ·mol–1, ΔS = −0.16 kJ·mol–1·K–1 and ΔG = −28.48 kJ·mol–1) indicate that the T211K mutation alters hydrogen bonds between PSALD and 4-nitrophenylserine. Molecular dynamics simulations elucidated the formation of a hydrogen bond between the amino group of T211K and the nitro group of 4-nitrophenylserine, which enhances the interaction between the mutant and product. This agent modifies the stereo-preference of threonine aldolases for the preparation of desired chiral β-hydroxy-α-amino acids by protein engineering.