Semirational Protein Engineering of a Decarboxylative Aldolase for the Regiodivergent and Stereodivergent Synthesis of Cyclic Imino Acids.

Aldolases are powerful C-C bond-forming enzymes for asymmetric organic synthesis because of their supreme stereoselectivity, compatibility with diverse electrophiles and nucleophiles, and promising scalability. Stereodivergent engineering of aldolases to tune the selectivity for the synthesis of stereoisomers of chiral molecules is highly desirable but has rarely been reported. Herein we report the semirational engineering of the decarboxylative aldolase UstD with a focused rational iterative site-specific mutagenesis (FRISM) strategy to perform a C-C bond-forming reaction with dione electrophiles. The variant obtained from a small mutant library showed divergent regioselectivity and diastereoselectivity compared to the wild-type enzyme, resulting in the production of 30 cyclic imino acids with stereocenters at the α and γ positions. Molecular dynamics simulation and kinetic data revealed the basis of selectivity.