Modis Y, Filppula S A, Novikov D K, Norledge B, Hiltunen J K, Wierenga R K
European Molecular Biology Laboratory, Heidelberg, Germany.
Structure. 1998 Aug 15;6(8):957-70. doi: 10.1016/s0969-2126(98)00098-7.
The degradation of unsaturated fatty acids is vital to all living organisms. Certain unsaturated fatty acids must be catabolized via a pathway auxiliary to the main beta-oxidation pathway. Dienoyl-coenzyme A (dienoyl-CoA) isomerase catalyzes one step of this auxiliary pathway, the isomerization of 3-trans,5-cis-dienoyl-CoA to 2-trans,4-trans-dienoyl-CoA, and is imported into both mitochondria and peroxisomes. Dienoyl-CoA isomerase belongs to a family of CoA-binding proteins that share the enoyl-CoA hydratase/isomerase sequence motif.
The crystal structure of rat dienoyl-CoA isomerase has been determined at 1.5 A resolution. The fold closely resembles that of enoyl-CoA hydratase and 4-chlorobenzoyl-CoA dehalogenase. Dienoyl-CoA isomerase forms hexamers made up of two trimers. The structure contains a well ordered peroxisomal targeting signal type-1 which is mostly buried in the inter-trimer space. The active-site pocket is deeply buried and entirely hydrophobic, with the exception of the acidic residues Asp176, Glu196 and Asp204. Site-directed mutagenesis of Asp204 revealed that this residue is essential for catalysis. In a molecular modeling simulation, a molecule of 3-trans,5-cis-octadienoyl-CoA was docked into the active site.
The structural data, supported by the mutagenesis data, suggest a reaction mechanism where Glu196 acts as a proton acceptor and Asp204 acts as a proton donor. Asp176 is paired with Glu196 and is important for optimizing the catalytic proton transfer properties of Glu196. In the predicted mode of substrate binding, an oxyanion hole stabilizes the transition state by binding the thioester oxygen. The presence of a buried peroxisomal targeting signal suggests that dienoyl-CoA isomerase is prevented from reaching its hexameric structure in the cytosol.
不饱和脂肪酸的降解对所有生物都至关重要。某些不饱和脂肪酸必须通过一条辅助于主要β-氧化途径的途径进行分解代谢。二烯酰辅酶A（dienoyl-CoA）异构酶催化该辅助途径的一步反应，即将3-反式，5-顺式-二烯酰辅酶A异构化为2-反式，4-反式-二烯酰辅酶A，并且该酶可被导入线粒体和过氧化物酶体。二烯酰辅酶A异构酶属于一类共享烯酰辅酶A水合酶/异构酶序列基序的辅酶A结合蛋白家族。
已确定大鼠二烯酰辅酶A异构酶的晶体结构，分辨率为1.5埃。其折叠结构与烯酰辅酶A水合酶和4-氯苯甲酰辅酶A脱卤酶的折叠结构非常相似。二烯酰辅酶A异构酶形成由两个三聚体组成的六聚体。该结构包含一个排列有序的1型过氧化物酶体靶向信号，该信号大部分埋藏在三聚体间的空间中。活性位点口袋深埋且完全疏水，除了酸性残基天冬氨酸176、谷氨酸196和天冬氨酸204。对天冬氨酸204进行定点诱变表明该残基对催化作用至关重要。在分子模拟中，一个3-反式，5-顺式-辛二烯酰辅酶A分子对接至活性位点。
结构数据在诱变数据的支持下，提示了一种反应机制，即谷氨酸196作为质子受体，天冬氨酸204作为质子供体。天冬氨酸176与谷氨酸196配对，对优化谷氨酸196的催化质子转移特性很重要。在预测的底物结合模式中，一个氧负离子洞通过结合硫酯氧来稳定过渡态。埋藏的过氧化物酶体靶向信号的存在表明二烯酰辅酶A异构酶在细胞质中无法形成其六聚体结构。
