Structure and mechanism of a dehydratase/decarboxylase enzyme couple involved in polyketide ß-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 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 ß 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 ß-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.

Synthesis of Diarylheptanoid Scaffolds Inspired by Calyxins I and J.

γ,δ-Unsaturated alcohols are prepared efficiently in two steps from o-hydroxycinnamaldehyde. The TMSOTf-mediated reaction of the γ,δ-unsaturated alcohols with aldehydes creates two oxygen heterocycles and three new stereocenters in a single pot. The approach is versatile, and by varying the boronic acid, Grignard reagent, and aldehyde, different substituents may be introduced, while use of a chiral base in the conjugate addition gives enantioenriched products.