Engineering of coenzyme specificity of formate dehydrogenase from Saccharomyces cerevisiae

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(+).