A great majority of compounds important for the treatment and study of human disease have their origin in natural products. These compounds are frequently modified with carbohydrate appendages that are critical for their biological activities and, in many cases, modulate their medicinal properties. It therefore comes as no surprise that these carbohydrates demonstrate an incredible range of structural variability, despite their origin in only a handful of precursors from primary metabolism. By exploiting the biosynthetic machinery of these unusual sugars it is possible to enhance or vary the physiological characteristics of the parent molecules and apply the principles learned to new systems. However, in order to fully realize the potential of such an approach, the biosynthetic pathways of these sugars must be characterized and the underlying chemistry thoroughly understood at the mechanistic level. In this spirit, our previous efforts funded by this grant have yielded many notable and important contributions to the goal of providing a scientific foundation for pharmaceutical research and potential drug development. As a result of these studies, we have identified three key areas that warrant further investigation in the next grant period. Accordingly, this proposal outlines experiments targeting the biosynthetic pathways and enzymes for the production of methylthiolincosamide, desosamine, tobramycin, oxetanocin, and the carbohydrate appendages of gentamicin. The specific objectives include: (1) study of the fundamental principles by which organic radicals are controlled to effect deamination, dehydrogenation and dehydration reactions catalyzed by the radical SAM enzymes DesII and AprD4, (2) pioneering mechanistic investigations into the B12-dependent radical SAM enzymes responsible for C- methylation reaction catalyzed by GenK and the ribose-to-oxetane ring-contraction catalyzed by OxsB, and (3) elucidation of the biosynthetic mechanism of sulfur incorporation into methylthiolincosamide. The enzymes OxsB and GenK are of particular interest because they are members of an emerging class of cobalamin- dependent radical SAM enzymes about which virtually nothing is presently known. These research directions are identified on the basis of their novelty, implications for the field of mechanistic enzymology, and potential utility in biomedical research at the basic and translational levels. We believe this wor will continue to address standing questions in biological chemistry and open new avenues of discovery in secondary metabolism and pharmaceutical research.
Carbohydrates with chemically complex and unusual structures play a central role in drug design, because they are often the critical component for the antimicrobial, antiviral, or anticancer properties of natural products. The carbohydrate systems proposed herein for study are specifically selected not only for their antibacterial (desosamine, tobramycin, gentamicin, lincomycin A) and antiviral (oxetanocin) properties, but also because of the unknown chemistry involved in their biosynthesis. These studies will open new directions in drug discovery and provide the scientific foundation for their realization.
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