Directed Evolution of Threonine Aldolases for the Photobiocatalytic Synthesis of β-Branched-α-Tetrasubstituted and Other Noncanonical Amino Acids

threonine aldolases, we transformed a traditionally two-electron pyridoxal phosphate (PLP)-dependent enzyme into a highly active and stereoselective biocatalyst for single-electron C-C bond formation, enabling enantioconvergent conversion of a broad range of racemic organoboron substrates. Mechanistic studies using radical clock probes and electron paramagnetic resonance (EPR) spectroscopy uncovered an unexpected role of free PLP in oxidative radical generation under photochemical conditions, highlighting the unique photochemical properties of this natural cofactor. Molecular dynamics (MD) simulations further elucidated the origin of enantio- and diastereoselectivity in the key radical addition step between the enzymatic quinonoid and the carbon-centered radical. Collectively, these results underscore the power of pyridoxal radical biocatalysis to access a broad spectrum of valuable noncanonical amino acid products via intermolecular, enzyme-controlled asymmetric radical chemistry─a transformation that remains elusive to both state-of-the-art small-molecule catalysis and native enzymology.