The mechanism of activation of the polyketide toxin mycolactone. (360G-Wellcome-079241_Z_06_Z)

The genes for the biosynthesis of the lipophilic polyketide toxin mycolactone, which plays a decisive role in creating the lesions that are characteristic of the emerging disease known as Buruli ulcer, are known to be clustered on an unusual plasmid housed in Mycobacterium ulcerans. This strain is very difficult to work with, requires specialised P3 facilities, taking months to grow, and there are no reliable systems developed for its genetic manipulation. In this project we aim to uncover the identity of the key enzyme that catalyses the activation of the polyketide core of mycolactone by the specific attachment of a polyketide sidechain. Recombinant candidate enzymes will be cloned, expressed and purified, assayed using purified core and chemically synthesised acyl donors, and the formation of mycolactones monitored by LC-MS. Once identified, the joinase will be studied to reveal its mechanism of action and substrate specificity. In principle, purified joinase can be used to synt hesise a library of variants of mycolactone differing in the side-chain. It is already known that natural cytotoxic mycolactones differ in their side-chain, and in parallel we will also determine the structure of several novel natural mycolactones from extracts of clinical isolates of M. ulcerans.