Structure and fanctional analysis of NAD^+-dependent isocitrate dehydrogenase from the chemolithotroph Aci dithiobacillus thiooxidans

1)异柠檬酸脱氢酶（Isocitrate Dehydrogenase，ICDH）催化D-异柠檬酸氧化脱羧生成2-酮戊二酸和CO_2，NAD^+或NADP^+是TCA循环中的辅因子。来自氧化硫硫杆菌的ICDH是NAD^+依赖性的，尽管大多数细菌含有NADP^+依赖性的ICDH。ICDH和3-异丙基苹果酸脱氢酶（IPMDH）通常显示NAD^+依赖性，属于一类蛋白质家族，脱羧脱氢酶，其缺乏通常存在于大多数脱氢酶中的典型βαβ核苷酸结合折叠。虽然这些酶在三维结构上具有高度的同源性和相似性，但底物结合位点的氨基酸序列不同。这些酶也提供了一个有吸引力的模型系统，研究辅酶和底物的识别。因此，我们试图利用pkk-ICDH转化大肠杆菌JM 109，建立一个产酶量（ICDH）的生产系统。和 ...更多信息 通过离心、热休克、DEAE-Toyoprotein和Sephacryl S-200 HR纯化ICDH。纯化的结果是，我们获得了57.8倍的纯化酶。对At-ICDH的热稳定性、pH稳定性等进行了分析。2）At-ICDH的晶体学和量子酶化学细菌ICDH通常需要NADP作为辅因子，但At-ICDH对NAD具有特异性。获得了ICDH-NAD配合物的双锥晶体，空间群为P43212，晶胞参数为a=B=125.99，c=268.35。用锇衍生物晶体的SAD法解析了结构，并将其分辨率提高到1.9 μ m。虽然在PDB中发现的大多数酶结合烟酰胺辅因子具有与烟酰胺环的C4碳反式的酰胺NH 2基团，但我们确定在At-ICDH中氮与C4碳顺式倾斜23°朝向底物。利用MNDO哈密顿量计算了烟酰胺环上氢原子的电势随酰胺转动的沿着。酰胺NH_2基团呈顺式构象，二面角为23°时，C_4原子上的一个氢原子带最大负电荷。由酰胺NH 2诱导的负电荷似乎表明在氢化物转移催化过程中稳定过渡态的催化意义。少

1)Functional analysis of Isocitrate Dehydrogenase from Acidithiobacillus thiooxidansIsocitrate dehydrogenase(ICDH) catalyzes the oxidative decarboxylation of D-isocitrate to 2-oxoglutarate and CO_2 with NAD^+ or NADP^+ as cofactor in the TCA cycle. ICDH from Acidithiobacillus thiooxidans is NAD^+-dependent, although most bacteria contain NADP^+-dependent ICDH. ICDH and 3-isopropylmalate dehydrogenase(IPMDH) which generally shows NAD^+-dependency belong to a class of protein family, decarboxylating dehydrogenases, that lack a typical βαβ nucleotide-binding fold which is commonly present in most dehydrogenases. Although these enzymes show high sequence homology and similarity in their 3-D structure, amino acid sequence of substrate-binding site is different. These enzymes also provide an attractive model system to study the coenzyme and substrate recognition. So we tried to build a product system which produces amount of enzyme (ICDH) by use of E.coli JM109 transformed with pkk-ICDH. And … More we purified ICDH by centrifugation, heat-shock, DEAE-Toyopearl and Sephacryl S-200HR. As a result of purification we obtained purified enzyme by 57.8-fold. We also analyzed the enzyme with thermostability, pH stability etc of At-ICDH. In result, ICDH from Acidithiobacillus thiooxidans is superior to that of ICDH from yeast.2)Crystallography and Quantum Enzyme Chemistry of At-ICDHBacterial ICDHs normally require NADP as the cofactor, but At-ICDH is specific to NAD. We obtained bi-pyramidal crystals of ICDH-NAD complex with a space group P43212 and the unit cell of a=b=125.99Å, c=268.35Å. The structure was solved by SAD method using Os-derivative crystals and refined to 1.9Å resolution. Although most of enzyme-bound nicotinamide cofactors found in PDB have the amide NH2 group in trans to the C4 carbon of nicotinamide ring, we determined in At-ICDH the nitrogen was in cis to C4 carbon slanting by 23° toward substrate. Electro-potentials of hydrogen atoms on nicotinamide ring were computed along with the amide rotation using MNDO Hamiltonian. A hydrogen atom on C4 atom had the greatest negative charge when amide NH2 group was in the cis conformation with dihedral angle of 23°. Negative charge induced by the amide NH2 appeared to suggest a catalytic significance in stabilizing the transition state during the hydride transfer catalysis. Less