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(+).
Serov
Alexander E
AE
Department of Chemical Enzymology, Faculty of Chemistry, M.V. Lomonosov Moscow State University, Leninskie Gory, 119992 Moscow, Russian Federation.
Popova
Anna S
AS
Fedorchuk
Vladimir V
VV
Tishkov
Vladimir I
VI
D000595:Amino Acid Sequence; D001483:Base Sequence; D003067:Coenzymes / Q000378:metabolism*; D003198:Computer Simulation; D017931:DNA Primers; D005560:Formate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015202:Protein Engineering; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D003067:Coenzymes; D017931:DNA Primers; D005560:Formate Dehydrogenases