Polyketide synthases (PKSs) are a family of structurally and mechanistically related multi-enzyme assemblies that catalyze the biosynthesis of numerous structurally complex and medicinally important natural products. Their overall catalytic cycles are comprised of a large number (typically greater than 25) of enzyme-catalyzed reactions through which simple building block molecules (usually CoA thioesters) are incrementally elaborated into structurally intricate products. Bacterial aromatic PKSs are one sub-class of PKSs. They are composed of 3-10 distinct subunits, which together synthesize a polyfunctional aromatic product. Over the past five years, the modularity of these multi-enzyme systems has been exploited by us and others for the engineered biosynthesis of numerous unnatural natural products. However, our understanding of the structural and mechanistic principles by which these remarkable enzymes assemble and catalyze multi-step transformations involving highly reactive intermediates is rudimentary at best. Towards this end, the specific goals for this proposal are: 1) Purification and reconstitution of the actinorhodin (act) and tetracenomycin (tcm) minimal PKSs; 2) Overexpression, purification, and reconstitution of representative auxiliary PKS components such as the act ketoreductase, act aromatase/cyclase (ARO/CYC), griseusin (gris) /ARO/CYC, tmc ARO/CYC, and the second ring cyclase from the act PKS; 3) Physico-chemical studies on minimal and extended PKS complexes, including kinetic analysis, detection of functionally relevant protein- protein interactions, and measurement of the subunit stoichiometries; 4) In vitro mutant complementation studies to probe the pathway for chain transfer between active sites of the act PKS; 5) Probing the molecular recognition features of aromatic PKSs using candidate unnatural substrates, including CoA and N-acetylcysteamine thioesters of primer and extender units, acyl carrier protein (ACP) and acyl-ACP analogs, as well as simplified mimics of the natural substrates of various auxiliary enzymes; 6) Probing the structural basis for limited interchangeability between the subunits of the act and tcm minimal PKS using mutagenesis as well as protein chemical approaches; 7) Dissecting chain length specificity of the minimal PKS using mutagenesis; 8) Dissecting the molecular recognition features of ARO/CYC subunits as well as their ability to influence the properties of other PKS subunits using mutagenesis and protein chemical approaches; 9) Development of sensitive assays capable of extracting short-chain intermediates from minimal aromatic PKSs; 10) (If time permits) Use of the above information for the synthesis of novel polyketides; and 11) (If time permits) Preliminary evaluation of NMR and X-ray crystallography for structural studies on these purified proteins.
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