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Title: Studies on the chromosomal beta-lactamase of Pseudomonas aeruginosa
Author: Toomer, Carol Ann
ISNI:       0000 0001 3535 1415
Awarding Body: University of Aston in Birmingham
Current Institution: Aston University
Date of Award: 1992
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The chromosomal ~-lactamase of Pseudomonas aeruginosa SAlconst (a derepressed laboratory strain) was isolated and purified. Two peaks of activity were observed on gel permeation chromatography (one major peak mol. wt. 45 kD and one minor peak of 54 kD). Preparations from 12 clinical derepressed strains showed identical results. Chromosomal ~-lactamase production in both normal and derepressed P aeruginosa strains was induced both by iron restricted growth conditions and by penicillin G. The majority of the enzyme (80-90%) was found in the periplasm and cytoplasm but a significant amount (2-20%) was associated with the outer membrane (OM). The growth conditions did not affect the distribution of the enzyme between subcellular fractions although higher activity was found in the cells grown under iron limitation and/ or in the presence of ~-lactams. The penicillanate sulphone inhibitor, tazobactam, displayed irreversible kinetics whilst cloxacillin, cefotaxime, ampicillin and penicillin G were all competitive inhibitors of the enzyme. Similar results were obtained for the Enterobacter cloacae P99 [)-lactamase, but tazobactam displayed a non-classical kinetic pattern for the Staphylococcus aureus PC 1 ~-lactamase. The residues involved in f3-lactam hydrolysis by the P aeruginosa SAlconst enzyme were detennined by affinity labelling with tazobactam. A tryptic digestion fragment of the inhibited enzyme contained the amino acids D, T, S, E, P, G, A, C, V, M, I, Y, F, H, K, R. This suggests the involvement of the conserved SVSK, DAE and KTG motifs found in all penicillin sensitive proteins. A model of the 3-D structure of the active site of the P aeruginosa SAlconst chromosomal f3-!actamase was constructed from the published amino acid sequence of P aeruginosa chromosomal ~-lactamase and the a-carbon coordinates of the S. aureus PCI ~-lactamase by homology modelling and energy minimisation. The crystal structure of tazobactam was determined and energy minimised. Computer graphics docking identified Ser 72 as a possible residue involved in a secondary attack on the C5 position of tazobactam after initial ~-lactam hydrolysis by serine 70. The enhanced activity of tazobactam over sulbactam might be explained by the triazole substituent which might participate in favourable hydrogen bonding between N3 and active site residues. Keywords: Pseudomonas aeruginosa SAyonst, chromosomal ~-lactamase, tazobactam, molecular modelling, growth conditions, [)-lactam antibiotics, f3-lactamase inhibitors.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Pharmacy