| Summary: | There is an ever increasing need for the development of new antibiotics to fight the emergence of antibacterial resistant strains of pathogens. Developing antimicrobials with ‘novel scaffolds’ and modes of action is an effective way to combat pathogens that are resistant to compounds currently in clinical use. The pacidamycins are a member of the uridyl peptide class of antibiotics that are produced by the soil dwelling bacterium Streptomyces coeruleorubidus. They show specific activity against the pathogen Pseudomonas aeruginosa, using a currently unexploited mode of action against a cell wall biosynthetic enzyme target. This thesis reports the investigation into the biosynthesis of pacidamycin, more specifically, into the function of the hypothetical protein genes present in the pacidamycin gene cluster and the biosynthesis of the non-proteinogenic amino acid, (2S, 3S)-diaminobutyric acid (DABA), which is at the core of the pacidamycin structure and other related antimicrobials. A multidisciplinary approach has been taken in this investigation, utilising biophysical, biochemical and genetic approaches. Protein crystallographic studies have deduced the structure of Pac17, postulated to be a lyase involved in DABA biosynthesis along with structural determination of the protein bound to the proposed substrate aspartate. Site directed mutagenesis of a number of the Pac17 active site amino acids also showed their essentiality for aspartate binding. In vitro biochemical approaches to study the enzymatic activity of the DABA biosynthetic proteins were inconclusive, with no activity observed. Genetic disruptions of the genes under investigation revealed the function of pac13 as a dehydratase, responsible for dehydrating the furan ring of the uridyl nucleoside present in the pacidamycin structure. Further to this, these studies established the essentiality of the DABA biosynthetic genes pac19 and pac20 for pacidamycin production in the native producer.
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