The importance of polyketides to human health and welfare is recognized both by major pharmaceuticals and important environmental carcinogens and mammalian toxins, as well as phytotoxins that impose heavy costs on agriculture and endanger the food supply. In three major Aims, we propose to undertake fundamental biochemical and structural studies of representatives from two major families of iterative polyketide synthases, which exemplify some of the most sophisticated catalytic systems known and pose many unanswered questions about how the coordinated function of their individual catalytic domains is achieved. Unequivocal determination of the programmed product of the enediyne highly-reducing (HR)-PKSs will be extended to investigation of how this simple, shared intermediate is converted to the enediyne and anthraquinone ?halves? of dynemicin A and other enediyne architectures. With advances in antibody technology, conjugates of enediyne natural products are coming again as valuable anti-cancer therapies. The biosynthesis of these structurally intriguing DNA-cleaving molecules remains one of the principal unsolved problems in natural product biosynthesis. Application of precise CRISPR/Cas9 gene deletions in the dynemicin A pathway has brought exciting experimental advances to isolate and characterize the first post-PKS intermediates in any enediyne biosynthetic pathway. Additional mutational studies will be carried out, the structures of other possible intermediates will be elucidated and a strategy of paired CRISPR mutations will be developed to finally crack how these fascinating structures are made. Having first detected and functionally characterized ?starter-unit acyl transferase? (SAT) domains and ?product template? (PT) domains in NR-PKSs, we are now poised to build from static structures of individual domains, stepwise to tridomains and tetradomains to, finally, full-length structures. In this Aim collaboration with the laboratory of Timm Maier (Biozentrum, Univ. of Basel) will couple biochemical studies, ACP?client crosslinking, x-ray crystallography and cryo-electron microscopy to achieve the next level of understanding to visualize how these separate components integrate their actions into fully functional molecular machines. Cercospora sp. cause immense damage to a range of vital food crops through the production of cercosporin, a diabolically efficient photosensitizer of reactive oxygen species and pathogenic to plants (and animals). The mostly unexplored biosynthesis of this perylenequinone will be undertaken and previous biogenetic proposals will be corrected. The roles of a laccase/fasciclin-family protein and other newly discovered biosynthetic proteins encoded in an expanded biosynthetic gene cluster will be studied in collaboration with scientists at the USDA with the added goal to find ?green? ways to combat toxin production by this pathogen. !

Public Health Relevance

Polyketide natural products occupy a central place in human health and welfare as both medicines and environmental toxins that endanger human health and our food supply. Advances using CRISPR/Cas9 deletions of dynemicin A biosynthetic genes have led to a breakthrough in understanding the formation of the broader class of enediynes and the potential of their antibody?drug conjugates in cancer therapy. Recent success obtaining the x-ray structure of an iterative, non-reducing polyketide synthase tridomain, and analysis of it crosslinked to its cognate acyl-carrier protein by cryo-electron microscopy have opened the way to higher order structures and unprecedented insights into not only whole-protein structure but also extensive conformational changes that accompany the catalytic cycle.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Synthetic and Biological Chemistry B Study Section (SBCB)
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Reinlib, Leslie J
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Johns Hopkins University
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