The 1.8 Å crystal structure and active-site architecture of β-ketoacyl-acyl carrier protein synthase III (FabH) from Escherichia coli
The 1.8 Å crystal structure and active-site architecture of β-ketoacyl-acyl carrier protein synthase III (FabH) from Escherichia coli
复制标题
DOI：
10.1016/s0969-2126(00)00094-0
复制
发表时间：
2000-02-15
期刊：
影响因子：
5.7
通讯作者：
Rock, CO
中科院分区：
文献类型：
--
作者：
Davies, C;Heath, RJ;Rock, CO
背景：β-酮酰基-酰基载体蛋白合酶 III (FabH) 启动细菌和植物中发现的 II 型脂肪酸合酶系统的延伸。 FabH 是 II 型系统中普遍存在的成分，并且位于控制脂肪酸产生的途径中的理想位置。 FabH 结构的阐明对于理解其反馈抑制调节及其与药物的相互作用具有重要意义。虽然两种相关缩合酶的结构已知，但活性位点残基的作用尚未经过实验测试。结果：利用12位点硒多波长反常色散实验确定了FabH的1.8埃晶体结构。活性位点（Cys112、His244 和 Asn274）由两个 a 螺旋汇聚形成，并通过狭窄的疏水通道进入。包含两个紧密结合的水分子的氢键网络固定了 His244 和 Asn274 的位置，这对于脱羧和缩合反应至关重要。令人惊讶的是，His244-->Ala 突变并不影响转酰基反应，表明 His244 对 Cys112 的亲核性影响很小。结论：组氨酸和天冬酰胺活性位点残基都是缩合反应中的脱羧步骤。 α-螺旋偶极效应增强了活性位点半胱氨酸的亲核性，并且氧阴离子空穴促进了四面体过渡态的形成。
Background: beta-Ketoacyl-acyl carrier protein synthase III (FabH) initiates elongation in type II fatty acid synthase systems found in bacteria and plants. FabH is a ubiquitous component of the type II system and is positioned ideally in the pathway to control the production of fatty acids. The elucidation of the structure of FabH is important for the understanding of its regulation by feedback inhibition and its interaction with drugs. Although the structures of two related condensing enzymes are known, the roles of the active-site residues have not been experimentally tested.Results: The 1.8 Angstrom crystal structure of FabH was determined using a 12-site selenium multiwavelength anomalous dispersion experiment. The active site (Cys112, His244 and Asn274) is formed by the convergence of two a helices and is accessed via a narrow hydrophobic tunnel. Hydrogen-bonding networks that include two tightly bound water molecules fix the positions of His244 and Asn274, which are critical for the decarboxylation and condensation reactions, Surprisingly, the His244-->Ala mutation does not affect the transacylation reaction suggesting that His244 has only a minor influence on the nucleophilicity of Cys112.Conclusions: The histidine and asparagine active-site residues are both required for the decarboxylation step in the condensation reaction. The nucleophilicity of the active-site cysteine is enhanced by the alpha-helix dipole effect, and an oxyanion hole promotes the formation of the tetrahedral transition state.