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

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 MbATA 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.