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Title: Novel chloramphenicol acetyltransferase variants
Author: Murray, Iain Ashmore
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1986
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The nucleotide sequences of genes, cat, encoding chloramphenicol acetyltransferases in Shigella flexneri and Streptomyces acrimycini were determined following subcloning of the determinants in bacteriophage M13. The predicted open reading frames, 213 and 219 amino acids respectively, demonstrate 30% or greater homology with one another and with the published sequences of other variants. Homology is most evident within the region flanking an essential histidyl residue (His-195) which is believed to function as a general base during catalysis. The 5' and 3' non-coding DNA of the Enterobacterial gene (cat) contains sequences equivalent to the E. coli consensus transcriptional initiation and termination motifs. Oligonucleotide site-directed mutagenesis of cat was utilised to test the hypothesis that the His-195 imidazole interacts, via a hydrogen-bond, with the carboxylate anion of an acidic amino acid - a process defined as tautomeric stabilisation. Five highly conserved aspartyl and glutamyl residues were replaced by asparagine or glutamine and the catalytic competence and substrate affinities of the mutant enzymes were determined. The K values for each substrate, chloramphenicol and acetyl-CoA, were essentially unchanged in the mutants but in one (Asp-199 replaced by Asn) the turnover coefficient, k, was diminished over 700-fold relative to wild type indicating that Asp-199 has an important, though non-essential, role in catalysis and is therefore a strong candidate for the tautomeric stabilisation of His-195. Inactivation of both the Asn-199 mutant and wild type enzyme by the affinity reagent 3-bromoacetyl chloramphenicol results in specific alkylation of the 3-nitrogen of the His-195 imidazole. This suggests that Asp-199 modulates the catalytic competence of His-195 by increasing the basicity of N-3 rather than by ensuring productive orientation of the imidazole. Plasmid and Ml 3 vectors were constructed to facilitate future studies of chloramphenicol acetyltransferase employing site-directed mutagenesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available