Structure and mechanism of a dehydratase/decarboxylase enzyme couple involved in polyketide β-methyl branch incorporation

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 signifcantly 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 fve 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 sitedirected mutagenesis, of the bacillaene synthase dehydratase/decarboxylase enzyme couple PksH/
PksI, responsible for the fnal 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 eforts in the targeted synthesis of functionally optimised ‘non-natural’ natural products.