Cycloaddition reactions are among the most widely used reactions in chemical synthesis. Nature achieves these cyclization reactions with a variety of enzymes, including Diels–Alderases that catalyse concerted 4 + 2 cycloadditions, but biosynthetic enzymes with 2 + 2 cyclase activity have yet to be discovered. Here we report that PloI4, a β-barrel-fold protein homologous to the exo-selective 4 + 2 cyclase that functions in the biosynthesis of pyrroindomycins, catalyses competitive 2 + 2 and 4 + 2 cycloaddition reactions. PloI4 is believed to catalyse an endo-4 + 2 cycloaddition in the biosynthesis of pyrrolosporin A; however, when the substrate precursor of pyrroindomycins was treated with PloI4, an exo-2 + 2 adduct was produced in addition to the exo- and endo-4 + 2 adducts. Biochemical characterizations, computational analyses, (co)crystal structures and mutagenesis outcomes have allowed the catalytic versatility of PloI4 to be rationalized. Mechanistic studies involved the directed engineering of PloI4 to variants that produced the exo-4 + 2, endo-4 + 2 or exo-2 + 2 product preferentially. This work illustrates an enzymatic thermal 2 + 2 cycloaddition and provides evidence of a process through which an enzyme evolves along with its substrate for specialization and activity improvement.

环加成反应是化学合成中应用最广泛的反应之一。大自然通过多种酶实现这些环化反应，包括催化协同 4 + 2 环加成的 Diels-Alderases，但尚未发现具有 2 + 2 环化酶活性的生物合成酶。在这里，我们报告 PloI4，一种与在吡咯吲哚霉素生物合成中起作用的外切选择性 4 + 2 环化酶同源的 β-桶折叠蛋白，催化竞争性 2 + 2 和 4 + 2 环加成反应。PloI4 被认为在吡咯孢菌素 A 的生物合成中催化内切-4 + 2 环加成；然而，当吡咯吲哚霉素的底物前体用 PloI4 处理时，除了exo - 和endo -4 + 2 加合物。生化特征、计算分析、（共）晶体结构和诱变结果使 PloI4 的催化多功能性合理化。机制研究涉及将 PloI4 定向工程化为优先产生exo -4 + 2、endo -4 + 2 或exo -2 + 2 产物的变体。这项工作说明了酶促热 2 + 2 环加成，并提供了酶与其底物一起进化以实现专业化和活性改进的过程的证据。