Biosynthesis of the Antibiotic Polyketide Enterocin

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DESCRIPTION (provided by applicant): 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 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, 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 of this proposal are thus: (1) to biochemically characterize the enterocin PKS priming and extension reactions using a recently developed in vitro process together with high-resolution protein mass spectrometry; (2) to biochemically characterize the flavoprotein EncM, which catalyzes an unprecedented series of biosynthetic reactions involving oxidative favorskii-like rearrangement, aldol condensation and heterocycle-forming reactions; and (3) to mechanistically and structurally characterize phenylalanine ammonia-lyase, a rare prokaryotic enzyme first discovered in the enterocin biosynthetic pathway and now in pre-clinical trials to treat the childhood disorder phenylketonuria. The outcome of this research plan will illuminate new biochemical reactions in natural product biosynthesis and will provide the opportunity to develop novel biocatalysts in bioorganic chemistry as well as therapeutic enzymes in the case of PAL.