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

来自化能营养菌 Aci 二硫杆菌氧化硫的 NAD^ 依赖性异柠檬酸脱氢酶的结构和功能分析

基本信息

批准号：
14580648
负责人：
INAGAKI Kenji
金额：
$ 2.24万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

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

1)氧化硫硫杆菌异柠檬酸脱氢酶的功能分析。在TCA循环中，异柠檬酸脱氢酶催化D-异柠檬酸氧化脱羧制2-羟基戊二酸和CO_2，辅酶NAD~+或NADP~+。尽管大多数细菌都含有依赖于NADP^+的ICDH，但氧化硫硫杆菌的ICDH是NAD^+依赖性的。ICDH和3-异丙基苹果酸脱氢酶属于蛋白质家族中的一类，它缺乏大多数脱氢酶中常见的βαβ核苷酸结合折叠，通常表现为NAD^+依赖性。虽然这些酶在三维结构上具有很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很有吸引力的模型系统。因此，我们尝试用pKK-ICDH转化E.ColiJM109，建立一种产酶系统。和…进一步采用离心、热休克、DEAE-TOYOPEL和Sephacryl S-200HR等方法纯化ICDH。纯化后得到的酶纯度为57.8倍。并对该酶的热稳定性、pH稳定性等进行了分析。2)at-ICDHBICDHs的结晶学和量子酶化学通常需要NADP作为辅因子，但at-ICDh对NAD是特异的。得到了空间群为P43212，晶胞参数为a=b=125.99Å，c=268.35Å的双锥晶体。以Os-衍生物晶体为原料，用SAD法对其结构进行了解析，并对其进行了细化至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都含有反式到烟酰胺环的C4碳的酰胺NH2基团，但我们在AT-ICDH中确定氮在顺式到C4碳的位置上向底物倾斜了23°。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变化。在二面角为23°的顺式构象中，C4原子上的一个氢原子的负电荷最大。在氢化物转移催化过程中，酰胺NH2产生的负电荷对稳定过渡态具有催化意义。较少