Engineering an ( R )‐Selective Transaminase for High‐Substrate‐Loading Asymmetric Amination of a Sterically Demanding N ‐Heterocyclic Ketone

ABSTRACT The biocatalytic asymmetric amination of bulky N ‐heterocyclic ketones is often limited by low enzyme activity and poor performance at high substrate loading. Herein, we developed an efficient ( R )‐selective transaminase for the synthesis of ( R )‐1‐Boc‐3‐aminopiperidine through an integrated strategy combining phylogeny‐guided enzyme mining, structure‐guided engineering, and mechanistic analysis. Screening of transaminases identified Mb ATA as the “best” parent enzyme, affording the target amine from N ‐Boc‐3‐piperidone (1B3OP) in specific activities of 0.269 U·g −1 and > 99% ee . Subsequent engineering yielded variant M3 (H61L/Q57R/P158Q), which exhibited a 45‐fold increase in activity over the wild type (WT) while retaining strict enantioselectivity (> 99% ee ). M3 achieved completely aminated 500 mM substrate loading within 4 h, 1000 mM in an extended duration of 24 h. Preparative biotransformation afforded 8.5 g and 15.6 g of product in 85% and 78% isolated yields, respectively. Structural and mechanistic analyses showed that the beneficial mutations enlarged and remodeled the substrate‐binding pocket, strengthened favorable substrate interactions, shortened the reactive cofactor–substrate distance, and promoted productive binding conformations. These results establish M3 as a practical biocatalyst for high‐substrate‐loading synthesis of chiral piperidine amines.