Directed Evolution and Unusual Protonation Mechanism of Pyridoxal Radical C-C Coupling Enzymes for the Enantiodivergent Photobiocatalytic Synthesis of Noncanonical Amino Acids.

Visible light-driven pyridoxal radical biocatalysis has emerged as a new strategy for the stereoselective synthesis of valuable noncanonical amino acids in a protecting-group-free fashion. In our previously developed dehydroxylative C-C coupling using engineered PLP-dependent tryptophan synthases, an enzyme-controlled unusual α-stereochemistry reversal and pH-controlled enantiopreference were observed. Herein, through high-throughput photobiocatalysis, we evolved a set of stereochemically complementary PLP radical enzymes, allowing the synthesis of both l- and d-amino acids with enhanced enantiocontrol across a broad pH window. These newly engineered l- and d-amino acid synthases permitted the use of a broad range of organoboron substrates, including boronates, trifluoroborates, and boronic acids, with excellent efficiency. Mechanistic studies unveiled unexpected PLP racemase activity with our earlier PLP enzyme variants. This promiscuous racemase activity was abolished in our evolved amino acid synthases, shedding light on the origin of enhanced enantiocontrol. Further mechanistic investigations suggest a switch of proton donor to account for the stereoinvertive formation of d-amino acids, highlighting an unusual stereoinversion mechanism that is rare in conventional two-electron PLP enzymology.