Modular polyketide synthases (PKSs) are biosynthetic assembly lines that construct structurally diverse natural products with wide-ranging applications in medicine and agriculture. Various mechanisms contribute to structural diversification during PKS-mediated chain assembly, including dehydratase (DH) domain-mediated elimination of water from R and S-configured 3-hydroxy-thioesters to introduce E- and Z-configured carbon–carbon double bonds, respectively. Here we report the discovery of a DH domain variant that catalyzes the sequential elimination of two molecules of water from a (3R, 5S)-3,5-dihydroxy thioester during polyketide chain assembly, introducing a conjugated E,Z-diene into various modular PKS products. We show that the reaction proceeds via a (2E, 5S)-2-enoyl-5-hydroxy-thioester intermediate and involves an additional universally conserved histidine residue that is absent from the active site of most conventional DH domains. These findings expand the diverse range of chemistries mediated by DH-like domains in modular PKSs, highlighting the catalytic versatility of the double hotdog fold.

模块化聚酮合酶 (PKS) 是生物合成装配线，可构建结构多样的天然产物，在医学和农业中具有广泛的应用。各种机制有助于 PKS 介导的链组装过程中的结构多样化，包括脱水酶 (DH) 结构域介导的从R和S构型的 3-羟基硫酯中去除水以分别引入E和Z构型的碳-碳双键. 在这里，我们报告了 DH 结构域变体的发现，该变体在聚酮化合物链组装过程中催化从 (3 R , 5 S )-3,5-二羟基硫酯中连续消除两个水分子，引入共轭E、Z-二烯转化为各种模块化 PKS 产品。我们表明反应通过 (2 E , 5 S )-2-enoyl-5-hydroxy-thioester 中间体进行，并涉及一个额外的普遍保守的组氨酸残基，该残基在大多数常规 DH 结构域的活性位点中不存在。这些发现扩大了模块化 PKS 中 DH 样结构域介导的化学反应的多样性，突出了双热狗折叠的催化多功能性。