Steen Ida Helene, Lien Torleiv, Madsen Marit Steine, Birkeland Nils-Kåre
Department of Microbiology, University of Bergen, PO Box 7800, Jahnebakken 5, 5020 Bergen, Norway.
Arch Microbiol. 2002 Oct;178(4):297-300. doi: 10.1007/s00203-002-0439-x. Epub 2002 Jun 28.
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.
通过分别将嗜热栖热菌NAD依赖型异柠檬酸脱氢酶（NAD-IDH）中的Asp-328和Ile-329替换为大肠杆菌NADP-IDH中的相应残基Lys和Tyr，验证了它们作为该酶辅因子识别位点的作用。Asp-328-Lys突变体表现出双辅酶特异性，而引入双突变Asp-328-Lys/Ile-329-Tyr则使辅因子偏好从NAD转变为NADP。与NAD-IDH相比，NADP依赖的嗜热栖热菌IDH保留了热稳定性和热活性。
