Molecular genetics and enzymology of secondary metabolite biosynthesis. I, Isolation of natural product biosynthesis gene clusters from symbiotic marine organisms. II, Enzymology of blasticidin S biosynthesis
| Autor: | Grochowski, Laura L. |
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| Rok vydání: | 2004 |
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| Druh dokumentu: | Thesis/Dissertation |
| Popis: | Molecular genetic and enzymological techniques have been employed to study secondary metabolite biosynthesis. These investigations have focused on two projects: the cloning and heterologous expression of biosynthetic gene clusters from unculturable marine organisms and the characterization of individual enzymes involved in the biosynthesis of the antifungal agent blasticidin S. The marine environment is proving to be a valuable source of biologically active compounds, but problems associated with sustainable harvest, laboratory culture, and organic synthesis make obtaining sufficient quantities of compounds for drug development both difficult and expensive. A method has been developed for the isolation of biosynthetic gene clusters from complex marine microbe/invertebrate associations. Using this method a mixed polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene cluster has been cloned from the marine sponge Jaspis splendens. The cloned gene cluster was found to code for a PKS with three extension modules and an NRPS with three extension modules. In addition, several open reading frames (ORFs) were identified that may be involved in the biosynthesis of the PKS starter molecule. Partial characterization of catalytic domains from the NRPS was also completed. The second project centers on the characterization of enzymes involved in blasticidin S (BS) biosynthesis. Two ORFs were identified in the BS gene cluster encoding gene products predicted to be involved in the early steps of BS biosynthesis. The blsG gene product has sequence similarity to lysine 2,3-aminomutase and is believed to be involved in the formation of the β-arginine moiety of BS. A series of heterologous expression studies were undertaken to determine the function of B1sG. The product of blsM exhibits sequence homology with several nucleosidetransferases. blsM was cloned from the BS gene cluster, heterologously expressed in E. coli, and shown to catalyze the formation of cytosine using cytidine 5'- monophosphate as the preferred substrate. Point mutations were introduced in blsM to generate three B1sM mutant enzymes: S92D, E98A, and E98D. All three mutants lost cytidine 5'-monophosphate hydrolysis activity. Surprisingly, the B1sM S92D mutant exhibits cytidine deaminase activity when incubated with cytidine or deoxycytidine, resulting in the formation of uridine and deoxyuridine, respectively. Graduation date: 2005 |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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