Probing enzyme reaction cascades in polyketide biosynthesis at atomic resolution by NMR

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Many antibiotics and other potentially therapeutic products are produced by type I polyketide synthases. These systems work like assembly lines in order to build up molecules, such that by manipulating enzymes within the cascade, the end product can be altered. By advancing our understanding of these enzymes, we can even begin to look at using them for the design of novel molecules.One system of particular interest for this project is the bongkrekic acid system, named after the toxin it produces. Bongkrekic acid works by inhibiting the adenine nucleotide translocase (ANT), blocking oxidative phosphorylation. Interestingly, compounds which selectively target the ANT have been put forward as antiapoptotic agents that may be used in chemotherapy. Bongkrekic acid therefore represents an excellent starting structure for this rational development, a process which this work will provide key information for. It will highlight how synthesis and flux is controlled by the enzymes, and therefore how this may be manipulated to produce analogues. Key questions in the project surround how the enzymes control which beta branching events include a final decarboxylation and how synthesis is terminated without the presence of a terminal thioesterase enzyme. Novel NMR assays and supplementary ESI-MS will be used that allow monitoring of intermediates in the enzyme cascade. This allows probing of bongkrekic acid synthesis at atomic resolution, without the need for these intermediates to be individually isolated. Where appropriate, structural techniques for analysis of protein interactions may be used, such as x-ray crystallography and cryo-EM. This project falls within the EPSRC Chemical Biology and Biological Chemistry research area.