Natural aromatic polyketides such as the antibiotic tetracycline and the anticancer agent daunorubicin represent an important class of pharmaceuticals that, together with their semi-synthetic derivatives, command a vital role in human health. A basic understanding of aromatic polyketide assembly catalyzed by iterative type II polyketide synthases (PKSs) at the biochemical and structural levels will undoubtedly increase our appreciation for these important biosynthetic processes and will aid in the rational engineering of new chemical entities. While the past decade has witnessed substantial growth in our basic knowledge on how aromatic polyketides are naturally synthesized, a number of fundamental gaps still persist today. We thus propose in this competitive renewal application to further our biosynthetic studies on the polyketide antibiotic enterocin, which has emerged as an important vehicle to address the early stages in aromatic polyketide assembly involving starter unit selection, timing of the ketoreduction reaction, and cyclization potential as well as post-PKS modification reactions. Their simple gene and protein architecture makes them amendable for study using a variety of sophisticated approaches including heterologous biosynthesis, in vitro and in vivo biochemical analysis, directed and random approaches towards enzyme engineering, and atomic resolution protein x-ray crystallography.
The specific aims for this proposal are thus: (1) to biochemically analyze the proposed acyl carrier protein-independent enterocin PKS priming mechanism with the goal of engineering aromatic polyketide libraries; (2) to biochemically characterize the flavoprotein EncM, which catalyzes an unprecedented series of biosynthetic reactions involving oxidative Favorskii rearrangement, aldol condensation and heterocycle-forming reactions; and (3) to structurally characterize several enterocin biosynthetic enzymes in association with our structural biology collaborators. The outcomes of this research plan will illuminate new biochemical reactions in natural product biosynthesis and will provide new enzymes for the combinatorial biosynthesis of novel chemistry with the potential of leading to new drugs.
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