amine dehydrogenase

Amine dehydrogenases (AmDHs) catalyze the reductive amination of ketones with inorganic ammonia as the amino donor, offering an attractive method for the synthesis of chiral amines. Because the wild-type AmDHs show low activity and limited substrate scope, most synthetic applications involve the engineered AmDHs from L-amino acid dehydrogenases. However, they continue the stereoselectivity of L-amino acid dehydrogenases. In this study, using 4-phenylbutan-2-one (1a) as the model substrate, a (R)-enantiospecific AmDH (M0) derived from L-amino acid dehydrogenase was converted to (S)-enantioselective enzymes (M2-M7) through several rounds of semi-rational iterative mutations. Among the variants, M7 achieved 98% conversion and 95% ee (S) for 1a, and its catalytic efficiency (kcat/Km) is 20 times that of M0. Variant M6-1 exhibits excellent stereoselectivity (>99% ee) towards para-substituted aralkylamines. The substrate spectra of these variants involve 49 aryl alkyl ketones. Molecular docking and dynamics simulations provided insights into the role of mutations in substrate recognition and stereoselective control, revealing that the mutations resulted in movement of the benzene ring and methyl group of 1c, that facilitated the Re-face of iminium intermediate toward the coenzyme, thus achieving the enantioselectivity inversion. This study is the first to create a highly active engineered AmDH using L-amino acid dehydrogenases as the template for the direct synthesis of (S)-amines and (R)-β-amino alcohols.