Bacterial aromatic polyketide synthases (PKSs) have already shown promise in producing new modified polyaromatic compounds that mimic the cores of many anti-tumor agents such as daunorubicin. However, the enzymatic mechanisms of these multi-protein complexes remain obscure. What kinds of starter units do these PKSs tolerate and what do these primers tell us about the mechanism of these proteins? A clear understanding of the tolerances of natural and synthetic substrates, the structural determinants of the substrate that dictate polyketide chain length, and an expanded repertoire of available starter units would provide a model to guide future engineering efforts toward larger libraries of potential therapeutics. In order to test the structural determinants of the polyketide chain starter unit that determine its processability and ultimate chain length, the proposed research uses primarily the purified actinorhodin (act) PKS as a representative aromatic PKS system: 1) to explore the ability of the PKS to accept synthetic starter units with different steric parameters to test the steric tolerances of the system; 2) to test the ability of the PKS to accept synthetic starter units that closely mimic the natural starter units to see if the chain length is determined by the number of ketones or the spacing of ketones in the developing chain; 3) to probe the ability of the PKS to accept synthetic starter units that are classical and non-classical bioisosteres of the natural chain; 4) to probe the ability of the PKS to process various N- acetylcysteamine derived starter units; and 5) to clone another, highly similar aromatic PKS complex from Streptomycetes that naturally use a butyl or larger starter unit to compare and contrast to the act system, as well as to explore the possibility of greater synthetic utility.
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