Crystal structure of porcine mitochondrial NADP+-dependent isocitrate dehydrogenase complexed with Mn2+ and isocitrate -: Insights into the enzyme mechanism
Crystal structure of porcine mitochondrial NADP+-dependent isocitrate dehydrogenase complexed with Mn2+ and isocitrate -: Insights into the enzyme mechanism
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DOI：
10.1074/jbc.m207306200
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发表时间：
2002-11-08
影响因子：
4.8
通讯作者：
Bahnson, BJ
中科院分区：
文献类型：
--
作者：
Ceccarelli, C;Grodsky, NB;Bahnson, BJ
猪心脏线粒体NADP（+） - 依赖性异位酸脱氢酶（IDH）与MN2+复合和异氯酸酯溶液溶液以分辨率为1.85 Angstrom。该酶在大肠杆菌中表达，纯化为具有麦芽糖结合蛋白的融合蛋白，并用凝血酶裂解以产生均匀的酶。该结构是通过多波长异常衍射的形式来确定的，以硒甲氨酸为异常的散射剂的形式进行硒取代。猪NADP（+）-IDH酶与从大肠杆菌和枯草芽孢杆菌的先前溶液结构进行比较，分别与哺乳动物酶具有16％和17％的身份。猪酶的蛋白质折叠与细菌IDH结构相似，每个单体折叠成两个结构域。然而，在连接核心二级结构的区域中，细菌和哺乳动物形式的IDH形式存在很大差异。基于猪，大肠杆菌和枯萎病IDH之间的序列和结构对齐，已经为哺乳动物酶确定了推定的磷酸化位点。包括绑定的MN2+ - 异晶酸酯复合物在内的活动位点是高度有序的，因此在机械上有用。共识IDH机制预测，在其NADP（+） - 依赖性氧化之前，将异氯酸盐的MN2+结合羟基羟基脱去。当前的晶体结构具有活性的位点水，可以很好地接受质子，并最终将质子转移到额外的结合水中。
The crystal structure of porcine heart mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH) complexed with Mn2+ and isocitrate was solved to a resolution of 1.85 Angstrom. The enzyme was expressed in Escherichia coli, purified as a fusion protein with maltose binding protein, and cleaved with thrombin to yield homogeneous enzyme. The structure was determined by multi-wavelength anomalous diffraction phasing using selenium substitution in the form of selenomethionine as the anomalous scatterer. The porcine NADP(+)-IDH enzyme is structurally compared with the previously solved structures of IDH from E. coli and Bacillus subtilis that share 16 and 17% identity, respectively, with the mammalian enzyme. The porcine enzyme has a protein fold similar to the bacterial IDH structures with each monomer folding into two domains. However, considerable differences exist between the bacterial and mammalian forms of IDH in regions connecting core secondary structure. Based on the alignment of sequence and structure among the porcine, E. coli, and R subtilis IDH, a putative phosphorylation site has been identified for the mammalian enzyme. The active site, including the bound Mn2+-isocitrate complex, is highly ordered and, therefore, mechanistically informative. The consensus IDH mechanism predicts that the Mn2+-bound hydroxyl of isocitrate is deprotonated prior to its NADP(+)-dependent oxidation. The present crystal structure has an active site water that is well positioned to accept the proton and ultimately transfer the proton to solvent through an additional bound water.