Notwithstanding the one-module-one-elongation-cycle paradigm of assembly line polyketide synthases (PKSs), some PKSs harbor modules that iteratively elongate their substrates through a defined number of cycles. While some insights into module iteration, also referred to as stuttering, have been derived through in vivo and in vitro analysis of a few PKS modules, a general understanding of the mechanistic principles underlying module iteration remains elusive. This report serves as the first interrogation of a stuttering module from a trans-AT subfamily PKS that is also naturally split across two polypeptides. Previous work has shown that Module 5 of the NOCAP (nocardiosis associated polyketide) synthase iterates precisely three times in the biosynthesis of its polyketide product, resulting in an all trans-configured triene moiety in the polyketide product. Here we describe the intrinsic catalytic properties of this NOCAP synthase module. Through complementary experiments in vitro and in E. coli, the split-and-stuttering module was shown to catalyze up to five elongation cycles, although its dehydratase domain ceased to function after three cycles. Unexpectedly, the central olefinic group of this truncated product had a cis configuration. Our findings set the stage for further in-depth analysis of a structurally and functionally unusual PKS module with contextual biosynthetic plasticity.

中文翻译：

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组装线聚酮化合物合酶的“分裂和口吃”模块分析

尽管流水线聚酮化合物合酶（PKS）的单模块一伸长循环范例，但一些PKS包含通过一定次数的循环迭代将其底物拉长的模块。尽管通过对几个PKS模块的体内和体外分析得出了一些有关模块迭代的见解，也称为口吃，但对模块迭代基础的机械原理的一般理解仍然难以捉摸。该报告是对反式-AT亚家族PKS口吃模块的首次询问，该家族也自然分裂成两个多肽。先前的工作表明，NOCAP（与心脏病有关的聚酮化合物）合酶的第5单元在其聚酮化合物产品的生物合成中精确地重复了3次，在聚酮化合物产物中产生了所有反式构型的三烯部分。在这里，我们描述此NOCAP合酶模块的内在催化性能。通过体外和在大肠杆菌中的补充实验，显示了分裂和口吃模块最多可催化五个延长周期，尽管其脱水酶结构域在三个周期后便不再起作用。出乎意料的是，该截短产物的中心烯基具有顺式构型。我们的发现为进一步深入分析具有上下文生物合成可塑性的结构和功能异常的PKS模块奠定了基础。尽管其脱水酶结构域在三个循环后停止起作用。出乎意料的是，该截短产物的中心烯基具有顺式构型。我们的发现为进一步深入分析具有上下文生物合成可塑性的结构和功能异常的PKS模块奠定了基础。尽管其脱水酶结构域在三个循环后停止起作用。出乎意料的是，该截短产物的中心烯基具有顺式构型。我们的发现为进一步深入分析具有上下文生物合成可塑性的结构和功能异常的PKS模块奠定了基础。