Studies on the Biosynthesis of Nucleocidin and Related Nucleoside Analogues
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Authors
Pasternak, Ola
Date
Type
thesis
Language
eng
Keyword
biosynthesis , nucleocidin , glycosyltransferase , natural products , fluoronucleoside
Alternative Title
Abstract
Natural products (NPs), also known as secondary metabolites, are compounds produced by an organism that are non-essential to its survival. These compounds are commonly biologically active and thus, are useful in the medical and pharmaceutical industries. Many pharmaceuticals, including antibiotics, are halogenated. Notably, about a quarter of the top-selling pharmaceuticals are fluorine-containing compounds. Fluorine is a small element which does not sterically hinder drug-receptor interactions and forms a strong bond with carbon. In stark contrast to the vast number of fluorinated compounds in industrial and pharmaceutical use, there are only six known naturally occurring fluorinated NPs, and one known enzyme capable of fluorination.
This work is focused on elucidating the biosynthesis of nucleocidin, a nucleoside which contains rare 4’-fluorine and 5’-O-sulfamoyl substituents. The biosynthetic pathway to formation of these two groups remains unknown. Nucleocidin was first discovered in cultures of S. calvus in 1956. Production by this organism was extremely low, thus the first goal of this project was to enhance production. Through a 16S rRNA genome mining approach, improved producers, with an approximately 30-fold increase in nucleocidin production, were identified. The increase in production helped to facilitate gene disruption experiments and identify genes which are needed for fluorination and sulfamate formation. NucG, nucI and nucJ, encoding two sulfatases and a radical SAM enzyme, respectively, and are essential for sulfamate formation, but have no effect on fluorination. Preliminary work towards the characterization of NucG and NucI revealed that NucG can function as a phosphodiesterase, while NucI can function as a sulfatase, phosphatase and phosphodiesterase. The exact catalytic role of NucG and NucI in nucleocidin biosynthesis is unknown. NucK, nucO and nucN, encoding two sulfotransferases and an amidinotransferase, respectively, are required both for sulfamation and fluorination. Disruption of nucP, encoding a DNA topoisomerase, produced a new unknown fluoronucleoside, while disruption of orf208, encoding a lycopene cyclase, had no effect on the fluoronucleoside profile. Additionally, two new acetylated nucleocidin derivatives were identified in culture extracts of wild type S. virens and S. aureorectus.
Key genes within the nucleocidin cluster were used to guide genome mining for the discovery of novel glycosyl and sulfamate containing nucleosides, including 3’-O-β-glucosyltubercidin. The substrate scope and kinetic parameters were determined on the glycosyltransferase, AvpGT, responsible for glucosylating tubercidin. Following the logic that new nucleocidin derivatives may be discovered through searching for clusters containing nucleocidin homologs, preliminary work towards in vivo rational design of an amino acid nucleocidin analogue was performed by introducing genes from the biosynthetic gene cluster encoding the related 5’-O-sulfamated nucleoside, ascamycin. Work towards characterization of this natural product and the biosynthetic enzymes is presented here.
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ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Attribution-NonCommercial 3.0 United States