Complex polyketides of bacterial origin are biosynthesised by giant assembly-line like megaenzymes of the type 1 modular polyketide synthase (PKS) class. The trans-AT family of modular PKSs, whose biosynthetic frameworks diverge significantly from those of the archetypal cis-AT type systems represent a new paradigm in natural product enzymology. One of the most distinctive enzymatic features common to trans-AT PKSs is their ability to introduce methyl groups at positions β to the thiol ester in the growing polyketide chain. This activity is achieved through the action of a five protein HCS cassette, comprising a ketosynthase, a 3-hydroxy-3-methylglutaryl-CoA synthase, a dehydratase, a decarboxylase and a dedicated acyl carrier protein. Here we report a molecular level description, achieved using a combination of X-ray crystallography, in vitro enzyme assays and site-directed mutagenesis, of the bacillaene synthase dehydratase/decarboxylase enzyme couple PksH/PksI, responsible for the final two steps in β-methyl branch installation in this trans-AT PKS. Our work provides detailed mechanistic insight into this biosynthetic peculiarity and establishes a molecular framework for HCS cassette enzyme exploitation and manipulation, which has future potential value in guiding efforts in the targeted synthesis of functionally optimised ‘non-natural’ natural products.

细菌来源的复杂聚酮化合物由巨型装配线生物合成，如 1 型模块化聚酮化合物合酶 (PKS) 类的巨酶。模块化PKS的反式-AT 家族，其生物合成框架与原型顺式-AT 型系统的生物合成框架显着不同，代表了天然产物酶学的新范式。反式常见的最独特的酶学特征之一-AT PKS 是它们能够在 β 位将甲基引入正在生长的聚酮链中的硫羟酸酯。该活性是通过五蛋白 HCS 盒的作用实现的，包括酮合酶、3-羟基-3-甲基戊二酰辅酶 A 合酶、脱水酶、脱羧酶和专用酰基载体蛋白。在这里，我们报告了分子水平的描述，结合 X 射线晶体学、体外酶测定和定点诱变，对芽孢杆菌合酶脱水酶/脱羧酶偶 PksH/PksI 进行了描述，负责 β-的最后两个步骤。在这个反式中安装甲基分支- 在 PKS。我们的工作为这种生物合成特性提供了详细的机械洞察力，并为 HCS 盒酶的开发和操作建立了一个分子框架，这在指导功能优化的“非天然”天然产物的靶向合成方面具有未来的潜在价值。