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D-Desosamine, a 3-(dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin, neomethymycin, pikromycin, and narbomycin produced by Streptomyces venezuelae, plays an essential role in conferring biological activities to its parent aglycones. The proteins encoded by the desI and desII genes in the methymycin/pikromycin biosynthetic gene cluster have been proposed to catalyze C-4 deoxygenation in D-desosamine biosynthesis. DesI is a pyridoxal 5'-phosphate-dependent C4-aminotransferase and catalyzes a transamination reaction converting thymidine diphosphate (TDP)-4-keto-6-deoxy-D-glucose to TDP-4-amino-4,6-dideoxy-D-glucose. DesII, which contains a [4Fe-4S] cluster binding motif, CXXXCXXC, has been identified as a member of the radical S-adenosylmethionine (SAM) enzyme superfamily by sequence analysis. To study the catalytic function of DesII, the desII gene was heterologously overexpressed in Escherichia coli and the DesII protein was purified to near homogeneity. Biochemical studies clearly established that the substrate for DesII is TDP-4-amino-4,6-dideoxy-D-glucose, and DesI and DesII function independently to carry out C-4 deoxygenation. DesII requires a [4Fe-4S]¹⁺ center and Sadenosylmethionine for activity. Accordingly, the originally proposed mechanism for C-4 deoxygenation in which DesI and DesII function together was revised. Two possible mechanisms have subsequently been proposed for the DesII reaction. The DesII catalysis is likely initiated by the formation of a 5'-deoxyadenosyl radical followed by the C-3 hydrogen atom abstraction. In the first possible route, the key step is a radical-induced deamination followed by the readdition of ammonia to the resulting cation radical intermediate, which is effectively a 1,2-amino shift, to form an aminol radical. Alternatively, the reaction may involve deprotonation of the 3-hydroxyl group to yield a ketyl radical anion to facilitate the [beta]-elimination of the ammonia group. Interestingly, DesII is flexible towards TDP-D-quinovose and TDP-3-amino-3,6-dideoxy-D-glucose. A possible biological reducing system, flavodoxin, flavodoxin reductase, and NADPH, for the reduction of the [4Fe-4S]²⁺ cluster, was also identified. Deuterium incorporation into SAM using C-3 deuterium-labeled substrate provides solid evidence for C-3 hydrogen atom abstraction by the 5'-deoxyadenosyl radical in the proposed mechanism. TDP-3-fluoro-3,6-dideoxy-D-glucose serves as a competitive inhibitor for DesII, which is in favor of deprotonation of the C-3 hydroxyl group being involved in DesII catalysis. / text

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Typescript. / Thesis (Ph. D.)--University of Hawaii, 1964. / Bibliography: leaves [107]-109. / ix, 109, [1] l mounted graphs, tables

xiii, 107 leaves : ill. ; 28 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1973) from the Dept. of Biochemistry, University of Adelaide

xii, 152 leaves : ill. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1972