Theoretical Studies on the Mechanism of Thioesterase-Catalyzed Macrocyclization in Erythromycin Biosynthesis
Theoretical Studies on the Mechanism of Thioesterase-Catalyzed Macrocyclization in Erythromycin Biosynthesis
复制标题
红霉素生物合成中硫酯酶催化大环化作用机制的理论研究
DOI：
10.1021/acscatal.6b01154
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发表时间：
2016-07-01
期刊：
影响因子：
12.9
通讯作者：
Zhao, Yi-Lei
中科院分区：
文献类型：
--
作者：
Chen, Xiong-Ping;Shi, Ting;Zhao, Yi-Lei
由模块化聚酮合酶（PKS）生物合成的大环聚酮已成功开发成为许多治疗领域的一代又一代药物。大量的实验和理论研究致力于阐明PKS的生物合成机制，特别是硫酯酶（TE）介导的大环化，它控制着PKS生物合成的最后一步，并决定了最终产物的化学结构。为了更好地了解大环化过程（即，释放步骤），我们对6-脱氧腺苷酸B合酶（DEBS）TE与两种底物（一种是大环产物，另一种是线性水解产物）的复合物进行了MD模拟、QM和QM/MM计算。我们的研究显示了TE酶和底物之间的诱导匹配相互识别：在大环化的情况下，在酶和底物1之间形成关键的氢键网络，并且疏水袋适当地容纳盖区域中的底物，其中一个关键的预反应状态（1（IV））的能量势垒为11.6千卡/摩尔被捕获的势能面计算。伴随着预反应状态的去质子化，亲核攻击发生与计算的势垒为9.9千卡/摩尔，并导致带电四面体中间体。在中间体分解之后，最终的大环产物以相对低的屏障释放。然而，在水解的情况下，这样的预反应状态的环化没有观察到类似的分子模拟。这些计算结果与以前关于TE介导的反应的生物化学和结构研究一致。我们的研究表明，酶的底物特异性源于通过DEBS TE和底物之间的预反应状态的相互分子识别，这表明TE大环化和PKS产物释放的预反应和作用机制。
Macrocyclic polyketides, biosynthesized by modular polyketide synthases (PKSs), have been developed successfully into generation-by-generation pharmaceuticals for numerous therapeutic areas. A great effort has been made experimentally and theoretically to elucidate the biosynthesis mechanisms, in particular for thioesterase (TE)-mediated macrocyclization, which controls the final step in the PKS biosynthesis and determines chemical structures of the final products. To obtain a better insight into the macrocyclization process (i.e., releasing step), we carried out MD simulations, QM and QM/MM calculations on complexes of 6-deoxyerythronolide B synthase (DEBS) TE and two substrates, one toward a macrocyclic product and another toward a linearly hydrolytic product. Our investigation showed the induced-fit mutual recognition between the TE enzyme and substrates: in the case of macrocydization, a critical hydrogen-bonding network is formed between the enzyme and substrate 1, and a hydrophobic pocket appropriately accommodates the substrate in the lid region, in which a pivotal prereaction state (1(IV)) with an energy barrier of 11.6 kcal/mol was captured on the potential energy surface calculation. Accompanied with the deprotonation of the prereaction state, the nucleophilic attack occurs with a calculated barrier of 9.9 kcal/mol and leads to the charged tetrahedral intermediate. Following the decomposition of the intermediate, the final macrocyclic product releases with a relatively low barrier. However, in the case of hydrolysis, such a prereaction state for cyclization was not observed in similar molecular simulations. These calculations are consistent with the previous biochemical and structural studies about the TE-mediated reactions. Our study indicated that the enzyme substrate specificity stems from mutual molecular recognition via a prereaction state between DEBS TE and substrates, suggesting a prereaction-and-action mechanism in the TE macrocyclization and release of PKS product.