Imada Katsumi, Tamura Takashi, Takenaka Ryo, Kobayashi Issei, Namba Keiichi, Inagaki Kenji
Department of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan.
Proteins. 2008 Jan 1;70(1):63-71. doi: 10.1002/prot.21486.
The crystal structure of Acidithiobacillus thiooxidans isocitrate dehydrogenase complexed with NAD+ and citrate has been solved to a resolution of 1.9 A. The protein fold of this NAD+-dependent enzyme shares a high similarity with those of NADP+-dependent bacterial ICDHs. The NAD+ and the citrate are clearly identified in the active site cleft with a well-defined electron density. Asp-357 is the direct cofactor-specificity determinant that interacts with 2'-OH and 3'-OH of the adenosine ribose. The adenosine ribose takes a C2'-endo puckering conformation as previously reported for an NAD+-specific isopropylmalate dehydrogenase. The nicotinamide moiety of NAD+ has the amide NH2 group oriented in cis conformation with respect to the C4 carbon of the nicotinamide ring, slanted toward the bound citrate molecule with a dihedral angle of -21 degrees . The semi-empirical molecular orbital calculation suggests that the pro-R hydrogen atom at C4 of NADH would bear the largest negative charge when the amide NH2 group is in such conformation, suggesting that the amide group has a catalytically significant role in stabilizing the transition state as NADH is being formed during the hydride transfer catalysis.
嗜酸氧化硫硫杆菌异柠檬酸脱氢酶与NAD⁺和柠檬酸盐复合物的晶体结构已解析至1.9 Å的分辨率。这种依赖NAD⁺的酶的蛋白质折叠与依赖NADP⁺的细菌异柠檬酸脱氢酶具有高度相似性。在活性位点裂隙中，NAD⁺和柠檬酸盐通过明确的电子密度清晰可辨。Asp-357是与腺苷核糖的2'-OH和3'-OH相互作用的直接辅因子特异性决定因素。腺苷核糖采取C2'-内型褶皱构象，如先前报道的NAD⁺特异性异丙基苹果酸脱氢酶那样。NAD⁺的烟酰胺部分的酰胺NH₂基团相对于烟酰胺环的C4碳呈顺式构象，以-21度的二面角朝向结合的柠檬酸盐分子倾斜。半经验分子轨道计算表明，当酰胺NH₂基团处于这种构象时，NADH的C4处的前R氢原子将带有最大负电荷，这表明酰胺基团在氢化物转移催化过程中形成NADH时稳定过渡态方面具有催化显著作用。
