Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids
Crystal Structures of Xanthomonas campestris OleA Reveal Features That Promote Head-to-Head Condensation of Two Long-Chain Fatty Acids
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DOI：
10.1021/bi300386m
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发表时间：
2012-05-22
期刊：
影响因子：
2.9
通讯作者：
Wilmot, Carrie M.
中科院分区：
文献类型：
--
作者：
Goblirsch, Brandon R.;Frias, Janice A.;Wilmot, Carrie M.
OleA是一种硫解酶超家族酶，可催化两种长链脂肪酰基辅酶A(CoA)底物的缩合反应。该酶是一个更大的基因簇的一部分，该基因簇负责产生长链烯烃产品，这是一种潜在的生物燃料前体。在硫解酶超家族中，催化是通过乒乓球机制实现的。第一底物与活性部位半胱氨酸形成共价中间体，半胱氨酸随后与第二底物反应。对于Olea，这种共轭是通过非脱羧基的Claisen缩合进行的。野油菜黄单胞菌的油已经结晶并确定了其结构，以及抑制剂结合结构和氙衍生物结构，以提高我们在酶周转背景下对底物定位的理解。OleA是第一个表征硫解酶超家族成员的成员，它有两个需要同时结合的长链烷基底物，因此唯一需要一个额外的烷基结合通道。脂肪酸生物合成抑制物蓝蓝蛋白具有已知的Olea底物范围内的烷基链长，结合单个氙气结合位点的位置，导致了这一新的烷基结合通道的推定。在油同系物3-羟基-3-甲基戊二酰辅酶A(HMG-CoA)合成酶和脂肪酸生物合成酶FabH之间的结构覆盖允许分配剩下的两个通道：一个用于含有硫酯的泛氨酸臂，另一个用于底物的烷基。一个短的β-发夹区域只在一种晶体形式中排序，这可能表明与底物结合相关的开放和关闭状态。Cys143是超家族中保守的催化型半胱氨酸，也是蓝精蛋白的烷基化位点。烷基化结构表明，谷氨酸残基(Glu117β)可能通过作为催化碱促进Claisen缩合。出乎意料的是，Glu117β来自生理二聚体的另一种单体。
OleA is a thiolase superfamily enzyme that has been shown to catalyze the condensation of two long-chain fatty acylcoenzyme A (CoA) substrates. The enzyme is part of a larger gene cluster responsible for generating long-chain olefin products, a potential biofuel precursor. In thiolase superfamily enzymes, catalysis is achieved via a ping-pong mechanism. The first substrate forms a covalent intermediate with an active site cysteine that is followed by reaction with the second substrate. For OleA, this conjugation proceeds by a nondecarboxylative Claisen condensation. The OleA from Xanthomonas campestris has been crystallized and its structure determined, along with inhibitor-bound and xenon-derivatized structures, to improve our understanding of substrate positioning in the context of enzyme turnover. OleA is the first characterized thiolase superfamily member that has two long-chain alkyl substrates that need to be bound simultaneously and therefore uniquely requires an additional alkyl binding channel. The location of the fatty acid biosynthesis inhibitor, cerulenin, that possesses an alkyl chain length in the range of known OleA substrates, in conjunction with a single xenon binding site, leads to the putative assignment of this novel alkyl binding channel. Structural overlays between the OleA homologues, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase and the fatty acid biosynthesis enzyme FabH, allow assignment of the two remaining channels: one for the thioester-containing pantetheinate arm and the second for the alkyl group of one substrate. A short beta-hairpin region is ordered in only one of the crystal forms, and that may suggest open and closed states relevant for substrate binding. Cys143 is the conserved catalytic cysteine within the superfamily, and the site of alkylation by cerulenin. The alkylated structure suggests that a glutamic acid residue (Glu117 beta) likely promotes Claisen condensation by acting as the catalytic base. Unexpectedly, Glu117 beta comes from the other monomer of the physiological dimer.