Redesigning an (R)-Selective Transaminase for the Efficient Synthesis of PharmaceuticalN-Heterocyclic Amines

Transaminases show potential for the industrial synthesis of important pharmaceutical ingredients. However, these naturally occurring enzymes show poor activity toward bulky N-heterocyclic compounds. To produce a catalyst with enhanced catalytic efficiency, this study redesigned an (R)-selective transaminase from Rhodobacter sp. 140A (RbTA). Key residues for substrate binding were identified by molecular docking and molecular dynamics simulations. A “simplified amino acid alphabet,” consisting of amino acids of different sizes (Phe, Asn, Val, and Ala), was then used to fine-tune the substrate-binding pocket by producing a small but smart variant library. Residue Y125 was found to be critical for substrate binding, and variant RbTAM1(Y125A), exhibiting a remarkable activity enhancement, was obtained. Through combined mutation, the most active variant, RbTAM2(Y125A/I6A/L7A/L158V), was constructed, exhibiting 1064-fold greater catalytic efficiency (kcat/Km) toward substrate N-Boc-3-piperidone (7a) than the wild-type enzyme. This variant also exhibited significantly improved activity (4–110-fold) toward a series of cyclic and bulky heterocyclic ketones. Structure-guided analysis of variant Y125A and molecular simulations revealed that the introduction of residue A125 enlarged the substrate-binding pocket volume and enabled additional hydrophobic interactions with the substrate, facilitating binding in a more favorable conformation for catalysis. The activity of variant RbTAM2 was verified in the gram-scale synthesis of chiral N-heterocyclic amine (R)-1-Boc-3-piperidinamine (7b), achieving 99% conversion and a space-time yield of 222 g L–1 d–1.