Identification of cofactor discrimination sites in NAD-isocitrate dehydrogenase from Pyrococcus furiosus |
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Authors: | Steen Ida Helene Lien Torleiv Madsen Marit Steine Birkeland Nils-Kåre |
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Institution: | Department of Microbiology, University of Bergen, PO Box 7800, Jahnebakken 5, 5020 Bergen, Norway. ida.steen@im.uib.no |
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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. |
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