Antibiotic resistance is a worldwide problem and has led to the emergence of the so-called 'superbugs' which are resistant to most of the clinically used antibiotics, and thus there is an urgent need for new antibiotics with new structures and/or unexploited modes of action. Pacidamycins, secondary metabolites produced by Streptomyces coeruleorubidus, possess a unique scaffold. These compounds are active against the superbug Pseudomonas aeruginosa through inhibiting translocase I; this represents a clinically-unexploited mode of action. This thesis describes the research carried out to elucidate and harness the biosynthetic flexibility of pacidamycin for generation of new analogues and to probe the biogenesis of its unique structural features. The system's inherent flexibility at both the C and N termini of the peptide was addressed and led to the generation of halogenated pacidamycins which were assessed for their antibiotic activity. The biosynthetic precursor for the nonproteinogenic amino acid m-tyrosine was identified as phenylalanine through feeding of synthesised [I, 2, 3, 4, 5_2H]_ phenylalanine to the pacidamycin producer. Biosynthesis of the pacidamycin nucleoside motif was studied through feeding of labeled precursors, mutant generation, metabolite screening of the mutant strains and chemical complementation with proposed biosynthetic intermediates that were synthesised for this purpose. Genes involved in the generation of the nucleoside were cloned and heterologously expressed, and the purified proteins were shown to be capable of the in vitro synthesis of the nucleoside motif. The nucleoside motif was shown to be derived from uridine by the catalysis of three enzymes Pac l l , Pac5 and Pac13 .
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:577685 |
| Date | January 2012 |
| Creators | Ragab, Amany Elsayed |
| Publisher | University of East Anglia |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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