Iterative Alanine Scanning Mutagenesis Confers Aromatic Ketone Specificity and Activity of L‐Amine Dehydrogenases

Abstract Direct reductive amination of prochiral ketones catalyzed by amine dehydrogenases is attractive in the synthesis of active pharmaceutical ingredients. Here, we report the protein engineering of L‐ Bacillus cereus amine dehydrogenase to allow reactivity on synthetically useful aromatic ketone substrates using an iterative, multiple‐site alanine scanning mutagenesis approach. Mutagenesis libraries based on molecular docking, iterative alanine scanning, and double‐proximity filter approach significantly expand the scope of active pharmaceutical ingredients relevant building blocks. The eventual quintuple mutant (A115G/T136A/L42A/V296A/V293A) showed reactivity toward aromatic ketones 12 a (5‐phenyl‐pentan‐2‐one) and 13 a (6‐phenyl‐hexan‐2‐one), which have not been reported to serve as targets of reductive amination by currently available amine dehydrogenases. Docking simulation and tunnel analysis provided valuable insights into the source of the acquired specificity and activity.