Generation of Oxidoreductases with Dual Alcohol Dehydrogenase and Amine Dehydrogenase Activity

The l-lysine-e-dehydrogenase (LysEDH) from Geobacillus stearothermophilus naturally catalyzes the oxidative deamination of the e-amino group of l-lysine. We previously engineered this enzyme to create amine dehydrogenase (AmDH) variants that possess a new hydrophobic cavity in their active site such that aromatic ketones can bind and be converted into a-chiral amines with excellent enantioselectivity. We also recently observed that LysEDH was capable of reducing aromatic aldehydes into primary alcohols. Herein, we harnessed the promiscuous alcohol dehydrogenase (ADH) activity of LysEDH to create new variants that exhibited enhanced catalytic activity for the reduction of substituted benzaldehydes and arylaliphatic aldehydes to primary alcohols. Notably, these novel engineered dehydrogenases also catalyzed the reductive amination of a variety of aldehydes and ketones with excellent enantioselectivity, thus exhibiting a dual AmDH/ADH activity. We envisioned that the catalytic bi-functionality of these enzymes could be applied for the direct conversion of alcohols into amines. As a proof-of-principle, we performed an unprecedented one-pot hydrogen-borrowing cascade to convert benzyl alcohol to benzylamine using a single enzyme. Conducting the same biocatalytic cascade in the presence of cofactor recycling enzymes (i.e., NADH-oxidase and formate dehydrogenase) increased the reaction yields. In summary, this work provides the first examples of enzymes showing alcohol aminase activity.

Automated summary

This study showcases the creation of new variants utilizing the ADH activity of LysEDH, resulting in enhanced catalytic activity for the reduction of substituted benzaldehydes and arylaliphatic aldehydes. Additionally, the engineered dehydrogenases exhibited dual AmDH/ADH activity for the reductive amination of aldehydes and ketones, showing potential for direct conversion of alcohols into amines.