Engineering a Formate Dehydrogenase for NADPH Regeneration

Chembiochem. 2023 Oct 17;24(20):e202300390. doi: 10.1002/cbic.202300390. Epub 2023 Aug 28.

Abstract

Nicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide phosphate (NADPH) constitute major hydrogen donors for oxidative/reductive bio-transformations. NAD(P)H regeneration systems coupled with formate dehydrogenases (FDHs) represent a dreamful method. However, most of the native FDHs are NAD+ -dependent and suffer from insufficient reactivity compared to other enzymatic tools, such as glucose dehydrogenase. An efficient and competitive NADP+ -utilizing FDH necessitates the availability and robustness of NADPH regeneration systems. Herein, we report the engineering of a new FDH from Candida dubliniensis (CdFDH), which showed no strict NAD+ preference by a structure-guided rational/semi-rational design. A combinatorial mutant CdFDH-M4 (D197Q/Y198R/Q199N/A372S/K371T/▵Q375/K167R/H16L/K159R) exhibited 75-fold intensification of catalytic efficiency (kcat /Km ). Moreover, CdFDH-M4 has been successfully employed in diverse asymmetric oxidative/reductive processes with cofactor total turnover numbers (TTNs) ranging from 135 to 986, making it potentially useful for NADPH-required biocatalytic transformations.

Keywords: cofactor preference; cofactor regeneration; formate dehydrogenase; protein engineering.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Formate Dehydrogenases* / genetics
  • Formate Dehydrogenases* / metabolism
  • NAD* / metabolism
  • NADP / metabolism
  • Oxidation-Reduction
  • Protein Engineering / methods

Substances

  • NADP
  • NAD
  • Formate Dehydrogenases