Mechanisms of molecular recognition in the pikromycin polyketide synthase
Mechanisms of molecular recognition in the pikromycin polyketide synthase
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DOI：
10.1016/s1074-5521(00)00039-9
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发表时间：
2000-12-01
影响因子：
--
通讯作者：
Reynolds, KA
中科院分区：
文献类型：
--
作者：
Chen, S;Xue, YQ;Reynolds, KA
背景：模块化聚酮合酶（PKS）产生多种具有医学意义的化合物。就委内瑞拉链霉菌的匹克霉素 PKS 而言，四个独立的多肽 (PikAl-PikAIV) 总共包含一个装载结构域和六个延伸模块，生成 14 元环大环内酯 narbonolide。多肽 PikAIV 含有硫酯酶 (TE) 结构域，负责催化甲基丙二酰辅酶 A 的最后延伸步骤，以及随后从 PKS 中释放最终聚酮化合物链延伸中间体。在某些生长条件下，该多肽从另一个翻译起始位点合成，产生 PikAIV N 端截短形式，仅包含一半的酮合酶 (KS6) 结构域。截短形式的 PikAIV 无法催化最终的延伸步骤，但能够从 PikAIII (ACP(5)) 上的前一个模块中裂解聚酮化合物链，产生 12 元环产物 10-脱氧甲内酯。 结果：表达含有酰基载体蛋白 (ACP) 和丙二酰辅酶 A 特异性酰基转移酶的杂合 PikAIV 多肽的委内瑞拉葡萄球菌突变体雷帕霉素 PKS 的 (AT) 结构域无法催化 12 或 14 元环大环内酯产物的生产。然而，含有天然 KS6 和 TE 结构域的杂种 PikAIV 的基于质粒的表达，在委内瑞拉链球菌 AX912 突变体中恢复了 narbonolide 和 10-deoxymethynolide 的产生，该突变体产生了 TE 缺失形式的 PikAIV。在混合 PikAIV 中使用替代 KS 结构域或删除 KS6 结构域会导致两种产品的损失。在 AX912 突变体中，PikAIV TE 结构域作为单独多肽的基于质粒的表达导致 10-脱氧甲基内酯恢复超过 50%，但在表达带有非天然 AT 结构域的杂合 PikAIV 的突变体中则不然。表达含有天然 ATG 结构域和不同 ACP 结构域的杂合 PikAIV 多肽的突变体有效地产生聚酮化合物产物，但与用天然 PikAIV 观察到的相比，具有显着更高的 10-脱氧甲内酯/纳波内酯比率。 结论：KS6 模块的二聚化允许使用含有不同 AT 和 ACP 结构域的 PikAIV 多肽在体内形成 PKS 异二聚体。在此类异二聚体中，负责形成narbonolide产物的TE结构域和ATE结构域位于不同的多肽链上。 PikAIV 的 ATG 结构域在促进 PikAIII 中后续模块的 TE 催化链终止（10-脱氧甲内酯形成）方面发挥着重要作用。匹克霉素 PKS 可以耐受多种形式（活性和非活性）PikAIV 的存在，并且 PikAIV 延伸效率的降低可能导致 10-脱氧甲内酯水平升高。这些结果为模块化 PKS 中的功能性分子相互作用和域间识别提供了新的见解。
Background: Modular polyketide synthases (PKSs) produce a wide range of medically significant compounds. In the case of the pikromycin PKS of Streptomyces venezuelae, four separate polypeptides (PikAl-PikAIV), comprising a total of one loading domain and six extension modules, generate the 14-membered ring macrolactone narbonolide. The polypeptide PikAIV contains a thioesterase (TE) domain and is responsible for catalyzing both the last elongation step with methylmalonyl CoA, and subsequent release of the final polyketide chain elongation intermediate from the PKS. Under certain growth conditions this polypeptide is synthesized from an alternative translational start site, giving rise to an N-terminal truncated form of PikAIV, containing only half of the ketosynthase (KS6) domain. The truncated form of PikAIV is unable to catalyze the final elongation step, but is able to cleave a polyketide chain from the preceding module on PikAIII (ACP(5)), giving rise to the 12-membered ring product 10-deoxymethynolide.Results: S. venezuelae mutants expressing hybrid PikAIV polypeptides containing acyl carrier protein (ACP) and malonyl CoA specific acyltransferase (AT) domains from the rapamycin PKS were unable to catalyze production of 12-or 14-membered ring macrolactone products. Plasmid-based expression of a hybrid PikAIV containing the native KS6 and TE domains, however, restored production of both narbonolide and 10-deoxymethynolide in the S. venezuelae AX912 mutant that generates a TE-deleted form of PikAIV. Use of alternative KS domains or deletion of the KS6 domain within the hybrid PikAIV resulted in loss of both products. Plasmid-based expression of a TE domain of PikAIV as a separate polypeptide in the AX912 mutant resulted in greater than 50% restoration of 10-deoxymethynolide, but not in mutants expressing a hybrid PikAIV bearing an unnatural AT domain. Mutants expressing hybrid PikAIV polypeptides containing the natural ATG domains and different ACP domains efficiently produced polyketide products, but with a significantly higher 10-deoxymethynolide/ narbonolide ratio than observed with native PikAIV.Conclusions: Dimerization of KS6 modules allows in vivo formation of a PKS heterodimer using PikAIV polypeptides containing different AT and ACP domains. In such heterodimers, the TE domain and the ATE domain responsible for formation of the narbonolide product are located on different polypeptide chains. The ATG domain of PikAIV plays an important role in facilitating TE-catalyzed chain termination (10-deoxymethynolide formation) at the proceeding module in PikAIII. The pikromycin PKS can tolerate the presence of multiple forms (active and inactive) of PikAIV, and decreased efficiency of elongation by PikAIV can result in increased levels of 10-deoxymethynolide. These results provide new insight into functional molecular interactions and interdomain recognition in modular PKSs.