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|Title:||Methyltransferases as Agents of Chemical Diversity in Natural Products|
|Keywords:||Methyltransferases;Chemical Diversity;Natural Products|
|Abstract:||<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 ΔstaL 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>|
|Appears in Collections:||Digitized Open Access Dissertations and Theses|
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