Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: Versatility from a unique substrate channel
Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: Versatility from a unique substrate channel
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DOI：
10.1073/pnas.011399198
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发表时间：
2001-12-18
影响因子：
11.1
通讯作者：
Stroud, RM
中科院分区：
文献类型：
--
作者：
Tsai, SC;Miercke, LJW;Stroud, RM
作为模块化聚酮化合物合酶 (PKS) 结构域的首次结构阐明，通过多重同晶置换和多波长反常色散的组合解决了来自 6-脱氧赤酮内酯 B 合酶 (DEBS) 的大环形成硫酯酶 (TE) 结构域的晶体结构，并将 R 因子精化至 24.1% 至 2.8 埃分辨率。其整体三级结构属于α/β-水解酶家族，具有该家族中前所未有的两个不寻常特征：疏水性富含亮氨酸的晚餐界面和穿过整个蛋白质的底物通道。活性位点三联体由 Asp-169、His-259 和 Ser-142 组成，位于底物通道的中间，表明底物通过蛋白质。建模表明，活性位点可以独特地容纳和定向 6-脱氧赤酮内酯 B 前体，同时保护活性位点免受外部水的影响，并通过大内酯形成催化环化。官能团的几何形状和组织解释了观察到的该 TE 的底物特异性，并为工程大环生物合成提供了策略。上游酰基载体蛋白（ACP6）与TE的同源模型对接表明，TE二聚体的2倍轴也可能是决定整个DEBS中结构域排列的对称轴。序列保守性表明来自模块化聚酮合酶的所有 TE 具有相似的折叠、二聚体 2 折叠轴和底物通道几何形状。
As the first structural elucidation of a modular polyketide synthase (PKS) domain, the crystal structure of the macrocycle-forming thioesterase (TE) domain from the 6-deoxyerythronolide B synthase (DEBS) was solved by a combination of multiple isomorphous replacement and multiwavelength anomalous dispersion and refined to an R factor of 24.1% to 2.8-Angstrom resolution. Its overall tertiary architecture belongs to the alpha/beta -hydrolase family, with two unusual features unprecedented in this family: a hydrophobic leucine-rich dinner interface and a substrate channel that passes through the entire protein. The active site triad, comprised of Asp-169, His-259, and Ser-142, is located in the middle of the substrate channel, suggesting the passage of the substrate through the protein. Modeling indicates that the active site can accommodate and orient the 6-deoxyerythronolide B precursor uniquely, while at the same time shielding the active site from external water and catalyzing cyclization by macrolactone formation. The geometry and organization of functional groups explain the observed substrate specificity of this TE and offer strategies for engineering macrocycle biosynthesis. Docking of a homology model of the upstream acyl carrier protein (ACP6) against the TE suggests that the 2-fold axis of the TE dimer may also be the axis of symmetry that determines the arrangement of domains in the entire DEBS. Sequence conservation suggests that all TEs from modular polyketide synthases have a similar fold, dimer 2-fold axis, and substrate channel geometry.