Engineering of coenzyme specificity of formate dehydrogenase from Saccharomyces cerevisiae

Biochem J. 2002 Nov 1;367(Pt 3):841-7. doi: 10.1042/BJ20020379.

Abstract

A eukaryotic formate dehydrogenase (EC 1.2.1.2, FDH) with its substrate specificity changed from NAD(+) to NADP(+) has been constructed by introducing two single-point mutations, Asp(196)-->Ala (D196A) and Tyr(197)-->Arg (Y197R). The mutagenesis was based on the results of homology modelling of a NAD(+)-specific FDH from Saccharomyces cerevisiae (SceFDH) using the Pseudomonas sp.101 FDH (PseFDH) crystal structure as a template. The resulting model structure suggested that Asp(196) and Tyr(197) mediate the absolute coenzyme specificity of SceFDH for NAD(+).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Coenzymes / metabolism*
  • Computer Simulation
  • DNA Primers
  • Formate Dehydrogenases / chemistry
  • Formate Dehydrogenases / genetics
  • Formate Dehydrogenases / metabolism*
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis
  • Protein Engineering
  • Saccharomyces cerevisiae / enzymology*
  • Sequence Homology, Amino Acid
  • Substrate Specificity

Substances

  • Coenzymes
  • DNA Primers
  • Formate Dehydrogenases