Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: Two catalytic activities in one active site
Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: Two catalytic activities in one active site
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DOI：
10.1016/s0969-2126(96)00030-5
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发表时间：
1996-03-15
期刊：
影响因子：
5.7
通讯作者：
Smith, JL
中科院分区：
文献类型：
--
作者：
Leesong, M;Henderson, BS;Smith, JL
背景：大肠埃希氏菌β-羟基癸酰硫醇酯脱水酶(脱水酶)是不饱和脂肪酸生物合成所必需的，它将饱和脂肪酸途径中的10碳中间体分流到不饱和脂肪酸途径中。脱水酶催化酰基载体蛋白(ACP)10-碳硫醇酯的脱水反应和双键异构化反应。这项工作的目的是阐明这两个酶反应发生在一个不寻常的双功能活性部位的机制，并了解该酶对含有10-碳脂肪酰链的底物的专一性。结果：确定了游离脱水酶和其经典的基于机理的钝化剂3-十月烯基-N-乙酰半胱胺修饰的晶体结构。脱水酶是一种对称的二聚体，具有不同寻常的α+β‘热狗折叠。两个独立的活性中心都位于酶的两个亚基之间，是一个与普通溶剂完全隔离的隧道状口袋。一个亚基的组氨酸侧链和另一个亚基的天冬氨酸侧链是活性中心中唯一潜在的活性蛋白质基团，结论：组氨酸和天冬氨酸共同催化脱水和异构化反应的双碱基机制与活性中心的结构一致。蛋白质折叠的独特拓扑结构和活性部位组分的鉴定揭示了另一种酶FabZ的预测价值，该酶可能是参与脂肪酰链延长的非特异性脱水酶。还发现活性部位入口处周围有一个带正电的区域，可以与带负电的ACP相互作用。
Background: Escherichia coli beta-hydroxydecanoyl thiol ester dehydrase (dehydrase) is essential to the biosynthesis of unsaturated fatty acids, by shunting a 10-carbon intermediate from the saturated fatty acid pathway into the unsaturated fatty acid pathway. Dehydrase catalyzes reactions of dehydration and of double-bond isomerization on 10-carbon thiol esters of acyl carrier protein (ACP). The aim of this work is to elucidate mechanisms for the two enzymatic reactions, which occur in an unusual bifunctional active site, and to understand the specificity of the enzyme for substrates with 10-carbon fatty acyl chains.Results: Crystal structures at 2.0 Angstrom resolution for free dehydrase and for the enzyme modified by its classic, mechanism-based inactivator, 3-decynoyl-N-acetylcysteamine, have been determined. Dehydrase is a symmetric dimer with an unusual alpha + beta 'hot dog' fold. Each of the two independent active sites is located between the two subunits of the enzyme, and is a tunnel-shaped pocket completely isolated from the general solvent. Side chains of histidine from one subunit and aspartic acid from the other are the only potentially reactive protein groups in the active site,Conclusions: A two-base mechanism by which the histidine and aspartic acid together catalyze dehydration and isomerization reactions is consistent with the active-site structure. The unique topology of the protein fold and the identification of the active-site components reveal features of predictive value for another enzyme, FabZ, which may to be the non-specific dehydratase involved in elongation of fatty acyl chains. A positively charged area surrounding the entrance to the active site, which could interact with the negatively charged ACP, was also found.