High-resolution Structures of Formate Dehydrogenase From Candida Boidinii

Protein Sci. 2007 Jun;16(6):1146-56. doi: 10.1110/ps.062741707.

Abstract

The understanding of the mechanism of enzymatic recovery of NADH is of biological and of considerable biotechnological interest, since the essential, but expensive, cofactor NADH is exhausted in asymmetric hydrogenation processes, but can be recovered by NAD(+)-dependent formate dehydrogenase (FDH). Most accepted for this purpose is the FDH from the yeast Candida boidinii (CbFDH), which, having relatively low thermostability and specific activity, has been targeted by enzyme engineering for several years. Optimization by mutagenesis studies was performed based on physiological studies and structure modeling. However, X-ray structural information has been required in order to clarify the enzymatic mechanism and to enhance the effectiveness and operational stability of enzymatic cofactor regenerators in biocatalytic enantiomer synthesis as well as to explain the observed biochemical differences between yeast and bacterial FDH. We designed two single-point mutants in CbFDH using an adapted surface engineering approach, and this allowed crystals suitable for high-resolution X-ray structural studies to be obtained. The mutations improved the crystallizability of the protein and also the catalytic properties and the stability of the enzyme. With these crystal structures, we explain the observed differences from both sources, and form the basis for further rational mutagenesis studies.

MeSH terms

  • Candida / enzymology*
  • Candida / genetics
  • Crystallography, X-Ray
  • Dimerization
  • Enzyme Stability
  • Formate Dehydrogenases / chemistry
  • Formate Dehydrogenases / genetics
  • Formate Dehydrogenases / metabolism*
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • NAD / chemistry
  • NAD / metabolism
  • Protein Binding
  • Protein Engineering / methods
  • Protein Folding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Structure-Activity Relationship

Substances

  • Recombinant Proteins
  • NAD
  • Formate Dehydrogenases

Associated data

  • PDB/2FSS
  • PDB/2J6I