Expansion of the Substrate Specificity of Porcine Kidney D-Amino Acid Oxidase for S-Stereoselective Oxidation of 4-Cl-Benzhydrylamine.

Abstract

Discovery and development of enzymes for the synthesis of chiral amines have been a hot topic for basic and applied aspects of biocatalysts. Based on our X‐ray crystallographic analyses of porcine kidney D‐amino acid oxidase (pkDAO) and its variants, we rationally designed a new variant that catalyzed the oxidation of (S)‐4‐Cl‐benzhydrylamine (CBHA) from pkDAO and obtained it by functional high‐throughput screening with colorimetric assay. The variant I230A/R283G was constructed from the variant R283G which had completely lost the activity for D‐amino acids, further gaining new activity toward (S)‐chiral amines with the bulky substituents. The variant enzyme (I230A/R283G) was characterized to have a catalytic efficiency of 1.85 s−1 for (S)‐CBHA, while that for (R)‐1‐phenylethylamine was diminished 10‐fold as compared with the Y228L/R283G variant. The variant was efficiently used for the synthesis of (R)‐CBHA in 96 % ee from racemic CBHA by the deracemization reaction in the presence of reducing agent such as NaBH4 in water. Furthermore, X‐ray crystallographic analysis of the new variant complexed with (S)‐CBHA, together with modelling study clearly showed the basis of understanding the structure‐activity relationship of pkDAO.