Engineering of Formate Dehydrogenase: Synergistic Effect of Mutations Affecting Cofactor Specificity and Chemical Stability

Appl Microbiol Biotechnol. 2013 Mar;97(6):2473-81. doi: 10.1007/s00253-012-4142-9. Epub 2012 May 17.

Abstract

Formate dehydrogenases (FDHs) are frequently used for the regeneration of cofactors in biotransformations employing NAD(P)H-dependent oxidoreductases. Major drawbacks of most native FDHs are their strong preference for NAD(+) and their low operational stability in the presence of reactive organic compounds such as α-haloketones. In this study, the FDH from Mycobacterium vaccae N10 (MycFDH) was engineered in order to obtain an enzyme that is not only capable of regenerating NADPH but also stable toward the α-haloketone ethyl 4-chloroacetoacetate (ECAA). To change the cofactor specificity, amino acids in the conserved NAD(+) binding motif were mutated. Among these mutants, MycFDH A198G/D221Q had the highest catalytic efficiency (k cat/K m) with NADP(+). The additional replacement of two cysteines (C145S/C255V) not only conferred a high resistance to ECAA but also enhanced the catalytic efficiency 6-fold. The resulting quadruple mutant MycFDH C145S/A198G/D221Q/C255V had a specific activity of 4.00 ± 0.13 U mg(-1) and a K m, NADP(+) of 0.147 ± 0.020 mM at 30 °C, pH 7. The A198G replacement had a major impact on the kinetic constants of the enzyme. The corresponding triple mutant, MycFDH C145S/D221Q/C255V, showed the highest specific activity reported to date for a NADP(+)-accepting FDH (v max, 10.25 ± 1.63 U mg(-1)). However, the half-saturation constant for NADP(+) (K m, NADP(+) , 0.92 ± 0.10 mM) was about one order of magnitude higher than the one of the quadruple mutant. Depending on the reaction setup, both novel MycFDH variants could be useful for the production of the chiral synthon ethyl (S)-4-chloro-3-hydroxybutyrate [(S)-ECHB] by asymmetric reduction of ECAA with NADPH-dependent ketoreductases.

MeSH terms

  • Amino Acid Substitution
  • Binding Sites
  • Coenzymes / metabolism*
  • Enzyme Stability
  • Formate Dehydrogenases / chemistry
  • Formate Dehydrogenases / genetics*
  • Formate Dehydrogenases / metabolism*
  • Kinetics
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / metabolism
  • Mutation, Missense*
  • Mycobacterium / enzymology*
  • Mycobacterium / genetics
  • NADP / metabolism
  • Protein Conformation
  • Protein Engineering*

Substances

  • Coenzymes
  • Mutant Proteins
  • NADP
  • Formate Dehydrogenases