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

1)异柠檬酸脱氢酶（ICDH）在TCA循环中以NAD^+或NADP^+作为辅助因子催化d -异柠檬酸盐氧化脱羧生成2-氧葡萄糖酸盐和CO_2的功能分析。来自硫氧化硫硫杆菌的ICDH依赖于NADP^+，尽管大多数细菌含有NADP^+依赖的ICDH。ICDH和3-异丙基苹果酸脱氢酶（IPMDH）同属于一类脱氢酶蛋白家族，缺乏在大多数脱氢酶中普遍存在的典型βαβ核苷酸结合折叠。虽然这些酶在三维结构上具有较高的序列同源性和相似性，但底物结合位点的氨基酸序列不同。这些酶也为研究辅酶和底物识别提供了一个有吸引力的模型系统。因此，我们试图利用转化pkk-ICDH的大肠杆菌JM109构建一个产酶量（ICDH）的产物体系。采用离心分离法、热休克法、DEAE-Toyopearl法和Sephacryl S-200HR法纯化ICDH。经纯化，得到纯度为57.8倍的纯化酶。并对酶的热稳定性、pH稳定性等进行了分析。结果表明，酸性硫氧化硫杆菌的ICDH优于酵母的ICDH。细菌的icdh通常需要NADP作为辅助因子，但At-ICDH对NAD具有特异性。我们得到了空间群为P43212的ICDH-NAD配合物双锥体晶体，其胞格为a=b=125.99Å， c=268.35Å。利用os -导数晶体的SAD方法对结构进行求解，并将其细化到1.9Å分辨率。虽然在PDB中发现的大多数酶结合的烟酰胺辅助因子的酰胺NH2基团在烟酰胺环的C4碳上反式，但我们在At-ICDH中确定氮在C4碳上顺式，向底物倾斜23°。利用MNDO哈密顿量计算了烟酰胺环上氢原子的电势。酰胺NH2基团呈23°二面角顺式构象时，C4原子上的氢原子负电荷最大。酰胺NH2诱导的负电荷似乎对氢化物转移催化过程中过渡态的稳定具有催化意义。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