Modular polyketide synthases (PKSs) are molecular-scale assembly lines comprising multiple gigantic polypeptide subunits. Faithful ordering of the subunits is mediated by extreme C- and N-terminal regions called docking domains (DDs). Decrypting how specificity is achieved by these elements is important both for understanding PKS function and modifying it to generate useful polyketide analogues for biological evaluation. Here we report the biophysical and structural characterisation of all six PKS/PKS interfaces in the unusual, chimaeric cis-AT/trans-AT PKS pathway responsible for biosynthesis of the antibiotic enacyloxin IIa in Burkholderia ambifaria. Taken together with previous work, our data reveal that specificity is achieved in the enacyloxin PKS by deploying at least three functionally orthogonal classes of DDs. We also demonstrate for the first time that cis-AT PKS subunits incorporate DDs with intrinsically disordered character, reinforcing the utility of such regions for achieving both medium affinity and high specificity at PKS intersubunit junctions. Overall, this work substantially increases the number of orthogonal DDs available for creating novel, highly-specific interfaces within cis- and trans-AT PKSs and their hybrids. It also reveals unexpected sequence/structure relationships in PKS DDs, identifying major current limitations to both accurately predicting and categorising these useful protein–protein interaction motifs.

模块化聚酮合酶 (PKS) 是包含多个巨大多肽亚基的分子级装配线。亚基的忠实排序由称为对接域 (DD) 的极端 C 和 N 端区域介导。解密这些元素如何实现特异性对于理解 PKS 功能和修改它以生成用于生物学评估的有用聚酮化合物类似物都很重要。在这里，我们报告了不寻常的嵌合顺式-AT/反式-AT PKS 途径中所有六个PKS/PKS 界面的生物物理和结构表征，该途径负责Burkholderia ambifaria 中抗生素enacyloxin IIa 的生物合成. 结合以前的工作，我们的数据表明，通过部署至少三个功能正交类别的 DD，在 enacyloxin PKS 中实现了特异性。我们还首次证明cis- AT PKS 亚基包含具有内在无序特征的 DD，增强了这些区域在 PKS 亚基间连接处实现中等亲和力和高特异性的效用。总的来说，这项工作大大增加了正交 DD 的数量，可用于在顺式和反式中创建新颖的、高度特异性的界面-AT PKS 及其混合动力车。它还揭示了 PKS DD 中意想不到的序列/结构关系，确定了当前在准确预测和分类这些有用的蛋白质-蛋白质相互作用基序方面的主要局限性。