A eukaryotic formate dehydrogenase (EC 22.214.171.124, FDH) with its substrate specificity changed from NAD+ to NADP+ has been constructed by introducing two single-point mutations, Asp196→Ala (D196A) and Tyr197→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 Asp196 and Tyr197 mediate the absolute coenzyme specificity of SceFDH for NAD+.
- cofactor preference
- Pseudomonas sp.101
- substrate binding
- three-dimensional structure modelling
Abbreviations used: D196A, Asp196→Ala; D195S, Asp195→Ser; FDH, formate dehydrogenase; CmeFDH, Candida methylica FDH; PseFDH, Pseudomonas sp.101 FDH; SceFDH, Saccharomyces cerevisiae FDH; Y197R, Tyr197→Arg.
- The Biochemical Society, London ©2002