Unlocking Catalytic Diversity of a Formate Dehydrogenase: Formamide Activity for NADPH Regeneration and Amine Supply for Asymmetric Reductive Amination

Abstract

The formate dehydrogenase (FDH) from Candida boidinii is a well-studied and applied enzyme for NADH regeneration in various reactions. As many oxidoreductases require NADPH, FDH mutants were created with shifted cofactor specificity toward NADP+. However, less effort was made to elucidate the substrate specificity for the hydride donors. Here, we report the FDH-catalyzed cleavage of formamide (F) and derivatives thereof into CO2 and amines, while regenerating the cofactors NADH and NADPH. Wild-type FDH and the NADP+-accepting variant FDH C23S/D195Q/Y196R/Q197N (FDH M5) showed both activity with 10% (v/v) F, N-methylformamide (MF), and N,N-dimethylformamide of 80, 67, and 4.5 mU/mg, and 4.9, 4.7, and 0.5 mU/mg, respectively. In silico docking and molecular dynamics simulation gave insights into substrate binding, indicating an altered binding conformation. NADP+-accepting variants were utilized in a cascade set up for the reductive amination of cyclohexanone by means of reductive aminase from Aspergillus oryzae with MF as hydride and amine donor, thereby reaching conversion rates of 72% in a whole cell approach. This work broadens the applicability of FDHs in biocatalysis.