Acyltransferases (ATs) of modular polyketide synthases catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. Subsequently, AT domains have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, critically limiting the efficiency and scope of AT engineering. Here, molecular dynamics simulations were used to prioritize ~50 mutations in the active site of EryAT6 from erythromycin biosynthesis. Following detailed in vitro studies, 13 mutations across 10 residues were identified to significantly impact extender unit selectivity, including nine residues that were previously unassociated with AT specificity. Unique insights gained from the MD studies and the novel EryAT6 mutations led to identification of two previously unexplored structural motifs within the AT active site. Remarkably, exchanging both motifs in EryAT6 with those from ATs with unusual extender specificities provided chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy is expected to advance our ability to engineer PKSs towards designer polyketides.

中文翻译：

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模块化聚酮化合物合酶酰基转移酶中底物选择性基元的计算指导交换

模块化聚酮化合物合酶的酰基转移酶（ATs）催化将丙二酰辅酶A增量剂安装到聚酮化合物支架中。随后，AT结构域已被广泛用于将各种扩展剂选择性地引入聚酮化合物中。然而，尚未建立负责底物选择的AT残留物的完整清单，从而严重限制了AT工程的效率和范围。在这里，分子动力学模拟被用来区分红霉素生物合成中EryAT6活性位点的〜50个突变。以下详细体外研究发现，在10个残基上有13个突变可显着影响扩展单元的选择性，其中包括9个以前与AT特异性无关的残基。从MD研究和新颖的EryAT6突变中获得的独特见解导致在AT活性位点内鉴定出两个先前未探索的结构基序。值得注意的是，将EryAT6中的两个基序与具有异常扩展剂特异性的AT的基序进行交换，为嵌合PKS模块提供了扩展的和反向的底物特异性。我们对AT底物选择性和这种基元交换策略的应用有了更深入的了解，有望进一步提高我们将PKS设计为聚酮化合物的能力。