Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.305682
Title: Molecular studies of the kefC glutathione gated potassium efflux system of Escherichia coli
Author: Munro, Andrew W.
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 1991
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Abstract:
The kefC gene of Escherichia coli encodes a potassium efflux system regulated by glutathione and glutathione metabolites. The gene has been cloned and sequenced and appears to encode a protein of 620 amino acids and mass 79,010 Dal. Exonuclease III mediated deletions from both ends of the plasmid-borne gene provided targeted breakpoints for sequencing of the gene (Munro et al (1991)). Comparisons of the primary sequence of kefC with those of other enzymes revealed homologies with two other cation-transporting proteins and two glutathione-utilisingenzymes. One of these, glyoxalase I from P.putida, catalyses the first step in the degradation of the toxic metabolite methylglyoxal - acting on its glutathione conjugate (hemithiolacetal) (Murata et al (1989)). Methylglyoxal was also found to stimulate KefC mediated potassium efflux - and represents the first recognised endogenous activator of the KefC system. The kinetics of KefC mediated potassium efflux are rapid and similar to those of ion channels rather than ion transporter proteins (Gennis (1989)). Plasmids carrying an intact wild-type kefC gene suppress the potassium retention defect associated with E.coli strain TK121 (kefC121). However, the kinetics of ESG activated potassium efflux are significantly altered in the transformants - suggesting that KefC may have an oligomeric structure. Preliminary cross-linking studies give further indication that this could be the case (G.Y.Ritchie, personal communication). Oligometric organisation would be consistent with the structure of an ion channel. Hydrophobicity profiling of KefC reveals that the N-terminal two-thirds of the protein are sufficiently hydrophobic to form transmembrane helices while the C-terminal one third is likely to be located in the cytoplasm. Since the central one third of the protein shows homology with glutathione-utilising enzymes which possess hydrophobic cytoplasmic glutathione binding `pockets', the model favoured is one in which the N-terminal third of KefC forms the potassium translocating transmembrane channel and the C-terminal two thirds are cytoplasmically located and involved in the glutathione gating of potassium efflux. It is possible that KefC represents the first recognised chemically regulated ion channel of prokaryotes, although a potassium-proton antiporter protein cannot yet be ruled out. Growth and growth-rate analyses on TK121 transformants with plasmids carrying partial and intact functional kefC genes were performed. The region of the kefC121 mutation was refined to approximately 0.5kb. Regions of the kefC gene were amplified by PCR, including the mutated region in TK121. Direct sequencing of PCR products should reveal the nature of the kefC missense mutation in TK121. A similar approach could be used to identify altered residues in a number of kefC mutant strains with potassium retention defects similar to that of TK121.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.305682  DOI: Not available
Keywords: Genetics Molecular biology Cytology Genetics Microbiology Biochemistry
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