Dynamically Coupled Network-Guided Engineering of Amine Dehydrogenase With Cofactor Recycling for Efficient...

(R)-3-Aminobutanol is a valuable chiral amino alcohol widely used in the pharmaceutical industry. The amine dehydrogenase (AmDH)-catalyzed synthesis with inorganic ammonia as the amine donor represents one of the most promising routes for its production. However, this approach is limited by the low catalytic efficiency of AmDHs toward the unnatural substrate 4-hydroxy-2-butanone, as well as the high cost associated with coenzyme requirements. To address these issues, a structure-guided engineering strategy integrating loop remodeling with dynamically coupled network analysis was employed to modify an AmDH derived from leucine dehydrogenase (GKGB-AmDH). A pentamutant was obtained with a 7.8-fold increase in catalytic efficiency (k_cat/K_m) and a 6.8-fold increase in specific activity. Molecular dynamics simulations were performed to elucidate the molecular mechanism underlying the improved catalytic performance. A dual^-enzyme co-expression system for GKGB-AmDH-M5 and formate dehydrogenase (FDH) was established for in situ NADH regeneration. As a result, the catalytic efficiency toward 4-hydroxy-2-butanone was enhanced, and the conversion reached 83.8% at a substrate concentration of 300 mM, with an enantiomeric excess (e.e.) of > 99.99%. These results demonstrate the feasibility of this engineering strategy and provide a theoretical basis for the efficient and green biomanufacturing of chiral amino alcohols.