Reversal Of Substrate Specificity Of Cmp N-Glycosidase To Dcmp And Crystallographic Studies On Bacimethrin And Azinomycin B Biosynthesis Enzymes

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The concept that structure defines function is fundamental to understanding biology and this theme is repeated again and again at the molecular, cellular, and organism levels. Remarkable advances in X-ray crystallography allow scientists to probe the structure of enzymes at atomic resolution. Protein crystal structures allow a greater understanding of the chemical and biological functions of these cellular machines. Enzymes from prokaryotes that are involved in the biosynthesis and degradation of various metabolites often present novel chemistry and curious functionalities. The enzymes classified in the nucleoside 2!-deoxyribosyltransferase superfamily have conserved catalytic residues and share a mechanism for the Nglycosidic bond cleavage of nucleosides, but demonstrate varied substrate specificity and biochemical functions. The structures of two members of this superfamily, Streptomyces rimofaciens MilB and Clostridium botulinum BcmB, provide novel information about how these enzymes confer specificity at their substrate 2!-position. The biosynthetic pathways of bacterial natural products also frequently present enzymes with unique functions and specificities. To better understand the biology of the thiamin antimetabolite bacimethrin, the C. botulinum genes responsible for its biosynthesis were identified and several enzymes implicated in its production were structurally characterized. Here, the structure of the bacimethrin kinase, BcmD is analyzed to better understand its specificity and relationship to other ribokinases. Another enzyme clustered with the bacimethrin genes, Cb-thiaminase I, was thought to serve as a bacimethrin-detoxifying enzyme. While this was not supported, structural solution and supporting kinetic characterization provides a new mechanistic understanding of the thiaminase I class of enzymes. Finally, the biosynthetic genes for the Streptomyces sahachiroi antitumor antibiotic, azinomycin B, were recently identified and reveal a complex network of enzymes involved in its assembly. Biochemical studies suggest that production of the azinomycin B naphthoate moiety requires a thioesterase enzyme in addition to a polyketide synthase megaenzyme. Structural characterization of the thioesterase SsAziG co-crystallized with a product analog supports this finding and provides a basis for its proposed mechanism.
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structural biology; crystallography; bacimethrin; azinomycin B; N-glycosidase; thiaminase; thioesterase
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Ealick, Steven Edward
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Cerione, Richard A
Lin, Hening
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Chemistry and Chemical Biology
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Ph. D., Chemistry and Chemical Biology
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Doctor of Philosophy
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Government Document
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dissertation or thesis
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