<p> The extensive;: use of antibiotics in the clinic, veterinary medicine, and
agricultures has imposed an immense selective pressure for the emergence of antibiotic
resistant bacteria In order to maintain the upper hand against pathogenic bacteria, we
must constantly seek new antimicrobials. Most antibiotics used in the clinic were
discovered as natural products or are derivatives thereof. Therefore, we must seek means
of increasing the chemical diversity of natural products in our quest for new antibiotics.
Herein, we investigate methyltransferases as agents to increase chemical diversity. More
specificity, we have performed biochemical studies on a tetracycline and a glycopeptide
methyltransferase. </p> <p> In our studies of the putative tetracycline N-methyltransferase OxyT, we determined the conditions required to overexpress the protein in an E. coli host.
Subsequently, using purified protein we examined substrate specificity using
commercially available compounds. However, we were unable to detect methylation of
the compounds tested and therefore we made an effort to secure a biologically relevant
substrate by insertionally inactivating the oxyT gene in S. rimosus but were unsuccessful. </p> <p> In our studies of the glycopeptide N-methyltransferase MtfA, we examined the
biochemical activity of this enzyme on the glycopeptide desulfo-A47934. We purified
desulfo-A47934 as a fermentation product of S. toyocaensis Ξ”staL and determined an
extinction co-efficient of 4200 Lmol^-1cm^-1. Furthermore, based on a crystal structure of
MtfA we biochemically characterized the enzyme and its four mutants Y32F, E144A, H228A, and R230A to study residues involved in substrate binding and catalysis. We demonstrated that these mutations did not alter quaternary protein structure but did lead to a significant decrease in enzyme activity as compared to the wild-type enzyme. </p> / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/21601 |
| Date | 07 1900 |
| Creators | Zakeri, Bijan |
| Contributors | Wright, Gerard, Chemical Biology |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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