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首页> 外文期刊>The FEBS journal >Fatty acid synthesis - Role of active site histidines and lysine in Cys-His-His-type beta-ketoacyl-acyl carrier protein synthases
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Fatty acid synthesis - Role of active site histidines and lysine in Cys-His-His-type beta-ketoacyl-acyl carrier protein synthases

机译：脂肪酸合成-活性位点组氨酸和赖氨酸在Cys-His-His型β-酮酰基-酰基载体蛋白合成酶中的作用

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摘要

beta-Ketoacyl-acyl carrier protein (ACP) synthase enzymes join short carbon units to construct fatty acyl chains by a three-step Claisen condensation reaction. The reaction starts with a trans thioesterification of the acyl primer substrate from ACP to the enzyme. Subsequently, the donor substrate malonyl-ACP is decarboxylated to form a carbanion intermediate, which in the third step attacks C1 of the primer substrate giving rise to an elongated acyl chain. A subgroup of beta-ketoacyl-ACP synthases, including mitochondrial beta-ketoacyl-ACP synthase, bacterial plus plastid beta-ketoacyl-ACP synthases I and II, and a domain of human fatty acid synthase, have a Cys-His-His triad and also a completely conserved Lys in the active site. To examine the role of these residues in catalysis, H298Q, H298E and six K328 mutants of Escherichia coli beta-ketoacyl-ACP synthase I were constructed and their ability to carry out the trans thioesterification, decarboxylation and/or condensation steps of the reaction was ascertained. The crystal structures of wild-type and eight mutant enzymes with and/or without bound substrate were determined. The H298E enzyme shows residual decarboxylase activity in the pH range 6-8, whereas the H298Q enzyme appears to be completely decarboxylation deficient, showing that H298 serves as a catalytic base in the decarboxylation step. Lys328 has a dual role in catalysis: its charge influences acyl transfer to the active site Cys, and the steric restraint imposed on H333 is of critical importance for decarboxylation activity. This restraint makes H333 an obligate hydrogen bond donor at N-epsilon, directed only towards the active site and malonyl-ACP binding area in the fatty acid complex.
机译：β-酮酰基-酰基载体蛋白（ACP）合酶通过三个步骤的Claisen缩合反应加入短碳单元以构建脂肪酰基链。反应开始于酰基引物底物从ACP到酶的反硫基酯化反应。随后，将供体底物丙二酰基-ACP脱羧形成碳负离子中间体，在第三步中，该碳负离子中间体侵蚀底漆底物的C1并产生细长的酰基链。 β-酮酰基-ACP合酶的一个亚组，包括线粒体β-酮酰基-ACP合酶，细菌加上质体β-酮酰基-ACP合酶I和II，以及人脂肪酸合酶的结构域，具有Cys-His-His三联体和在活动位点也是完全保守的赖氨酸。为了检查这些残基在催化中的作用，构建了大肠杆菌β-酮酰基-ACP合酶I的H298Q，H298E和六个K328突变体，并确定了它们进行反应的反硫代酯化，脱羧和/或缩合步骤的能力。确定了具有和/或没有结合的底物的野生型和八种突变酶的晶体结构。 H298E酶在6-8的pH范围内显示残留的脱羧酶活性，而H298Q酶似乎完全脱羧，这表明H298在脱羧步骤中起催化碱的作用。 Lys328在催化中具有双重作用：其电荷影响酰基转移至活性位点Cys，并且对H333施加的空间限制对于脱羧活性至关重要。这种限制使H333在N-ε上成为专性的氢键供体，仅指向脂肪酸复合物中的活性位点和丙二酰-ACP结合区域。

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