Semirational Protein Engineering of a Decarboxylative Aldolase for 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, 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. This study documented the semirational engineering of the decarboxylative aldolase UstD with the 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 to the wild-type enzyme, which resulted in the production of thirty cyclic imino acids with stereocenters at the α and γ positions. Molecular dynamics simulation and kinetic data revealed the basis of selectivity.