title: Identification of cofactor discrimination sites in NAD-isocitrate dehydrogenase from Pyrococcus furiosus.
abstract: The role of Asp-328 and Ile-329 as a cofactor discrimination site of the NAD-dependent isocitrate dehydrognase (NAD-IDH) from Pyrococcus furiosus has been verified by replacing these residues with Lys and Tyr, respectively, which are the corresponding residues in NADP-IDH from Escherichia coli. The Asp-328-Lys mutant showed dual coenzyme specificity, whereas introduction of the double mutation, Asp-328-Lys/Ile-329-Tyr shifted the cofactor preference from NAD to NADP. NADP-dependent P. furiosus IDH retained thermostability and thermoactivity compared with NAD-IDH.
authors.affiliation: Department of Microbiology, University of Bergen, PO Box 7800, Jahnebakken 5, 5020 Bergen, Norway. ida.steen@im.uib.no
authors.forename: Ida Helene
authors.initials: IH
authors.lastname: Steen
authors.forename: Torleiv
authors.initials: T
authors.lastname: Lien
authors.forename: Marit Steine
authors.initials: MS
authors.lastname: Madsen
authors.forename: Nils-Kåre
authors.initials: NK
authors.lastname: Birkeland
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002152:Calorimetry, Differential Scanning; D003067:Coenzymes / Q000378:metabolism*; D004795:Enzyme Stability; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D003067:Coenzymes; D009243:NAD; D009249:NADP; D007521:Isocitrate Dehydrogenase