Investigating the biosynthesis of the streptomycete antibiotic pacidamycin

Tromans, Daniel (2013) Investigating the biosynthesis of the streptomycete antibiotic pacidamycin. Doctoral thesis, University of East Anglia.

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Abstract

Abstract
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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Chemistry
Depositing User: Mia Reeves
Date Deposited: 11 Mar 2014 10:30
Last Modified: 11 Mar 2014 13:00
URI: https://ueaeprints.uea.ac.uk/id/eprint/48041
DOI:

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