Genetic and biochemical studies of the biosynthesis and attachment of D-desosamine, the deoxy sugar component of macrolide antibiotics produced by Streptomyces venezuelae

Borisova, Svetlana Alekseyevna, 1976-
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Macrolide antibiotics are clinically important drugs widely used to treat the infections caused by gram-positive bacteria. They consist of a macrolactone aglycone unit and one or more deoxy sugar component(s). The presence of an at least one deoxy sugar moiety in the structure of macrolides is essential for their antimicrobial activity. Modifications of the deoxy sugar substituent hold promise as a valuable approach towards generating new macrolide antibiotics with improved biological properties. The development of new antimicrobial drugs is essential to combat the growing problem of the pathogen resistance to the existing antibiotics. This thesis describes a part of our ongoing effort to investigate the mechanistic details of the biosynthetic pathway to D-desosamine, an amino deoxy sugar component of macrolide antibiotics methymycin, neomethymycin, pikromycin, and narbomycin produced by Streptomyces venezuelae. D- Desosamine also exists in many other clinically important macrolides, e.g. erythromycin, clarithromycin, and oleandomycin. In particular, the gene knockout technique was used to explore the functions of the desI, desII, and desVIII genes of the D-desosamine biosynthetic gene cluster. The analysis of the macrolides produced by the resulting S. venezuelae mutants has led to the revision of the originally proposed pathway to desosamine. A new mechanism for the C-4 deoxygenation step was also envisioned. Four new macrolides were isolated in these studies, which established the relaxed substrate specificity of the glycosyl- transferase DesVII involved in the coupling of TDP-sugar derivatives to the aglycone in the pathway. To further explore the potential of DesVII to couple various sugar and aglycone substrates, desVII was expressed in E. coli and the recombinant DesVII protein was used to study the in vitro glycosyltransferase activity. Our results demonstrate that DesVII requires an additional protein component, DesVIII, to perform the catalysis, and the activity is optimal at pH 9. These conditions, unusual for the known glycosyl transfer reactions, may prove to be the general requirements for other macrolide glycosyltransferases. The preliminary study of the substrate specificity of the DesVII/DesVIII catalytic pair was conducted in vitro and showed the potential for their application to glycosylation in combinatorial biosynthesis.