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I A Murray

Publications and source records attributed to I A Murray.

26 records · Page 2Linked to original sources
PubMed1990

Evidence for transition-state stabilization by serine-148 in the catalytic mechanism of chloramphenicol acetyltransferase.

The function of conserved Ser-148 of chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Modeling studies (P. C. E. Moody and A. G. W. Leslie, unpublished results) suggested that the hydroxyl group of Ser-148 could be involved in transition-state stabilization via a hydrogen bond to the oxyanion of the putative tetrahedral intermediate. Replacement of serine by alanine results in a mutant enzyme (Ala-148 CAT) with kcat reduced 53-fold and only minor changes in Km values for chloramphenicol and acetyl-CoA. The Ser-148----Gly substitution gives rise to a mutant enzyme (Gly-148 CAT) with kcat reduced only 10-fold. A water molecule may partially replace the hydrogen-bonding potential of Ser-148 in Gly-148 CAT. The three-dimensional structure of Ala-148 CAT at 2.34-A resolution is isosteric with that of wild-type CAT with two exceptions: the absence of the Ser-148 hydroxyl group and the loss of one poorly ordered water molecule from the active site region. The results are consistent with a catalytic role for Ser-148 rather than a structural one and support the hypothesis that Ser-148 is involved in transition-state stabilization. Ser-148 has also been replaced with cysteine and asparagine; the Ser-148----Cys mutation results in a 705-fold decrease in kcat and the Ser-148----Asn substitution in a 214-fold reduction in kcat. Removing the hydrogen bond donor (Ser-148----Ala or Gly) is less deleterious than replacing Ser-148 with alternative possible hydrogen bond donors (Ser-148----Cys or Asn).

Acetyl Coenzyme A↗
PubMed1989

Nucleotide sequence of the chloramphenicol acetyltransferase gene of Streptomyces acrimycini.

The nucleotide sequence of a gene (cat) encoding chloramphenicol acetyltransferase (CAT) in Streptomyces acrimycini was determined. The predicted amino acid sequence demonstrates extensive homology with those of CATs isolated from Gram-negative Enterobacteria, notably with the type III variant encoded by the IncK plasmid R387. Transcript mapping indicates a single cat mRNA with a 5' end coinciding with the AUG codon used for translational initiation in vivo. We also determined the extent of a spontaneous deletion in the 5'-noncoding DNA, which occurs when the gene is cloned in the BamHI site of pBR322 in a specific orientation and which results in constitutive cat expression in Escherichia coli from the tet promoter.

Amino Acid Sequence↗
PubMed1989

A flow cytometric technique for simultaneous analysis of human mononuclear cell surface antigens and DNA.

A method for dual staining of mononuclear cells for lymphocyte phenotypic markers and DNA is described. The cells were stained with fluorescein-conjugated monoclonal antibodies and then rendered permeable to propidium iodide using saponin. Propidium iodide stains DNA and, using flow cytometry, cell cycle analysis of individual lymphocyte subpopulations can be determined. Saponin acts within 1 min, preserves expression of surface antigens and is effective at all concentrations from 0.001% to 1%. This technique is simple, rapid and gives reproducible results.

Antigens, Surface↗
PubMed1988

Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate.

The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat(13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat(1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromo-acetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-19 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.

Alanine↗
PubMed1988

Nucleotide sequence analysis and overexpression of the gene encoding a type III chloramphenicol acetyltransferase.

The gene catIII, encoding a type III enterobacterial chloramphenicol acetyltransferase, was cloned from the transmissible plasmid R387 into pBR322 and bacteriophage M13 mp8. Nucleotide sequence analysis of 1160 bp of DNA identified an open reading frame encoding a protein of 213 amino acid residues and a calculated molecular mass of 24965 Da. The predicted N-terminal sequence is identical with that determined by Edman degradation of chloramphenicol acetyltransferase purified from Escherichia coli harbouring R387. Sequences equivalent to the consensus motifs for initiation and rho-factor-independent termination of transcription in E. coli occur 5' and 3' to the catIII open reading frame. In contrast with the catI gene, present on transposon Tn9 and many enterobacterial plasmids, expression of catIII is not subject to cyclic AMP-mediated catabolite repression in vivo and there is no sequence in the 5' non-coding DNA that resembles that deduced as the consensus for the binding of cyclic AMP receptor protein. Unique restriction-endonuclease cleavage sites were introduced adjacent to the catIII reading frame by using oligonucleotide-directed mutagenesis to facilitate insertion into E. coli expression vectors. Fully active chloramphenicol acetyltransferase represents 30-50% of the soluble protein component of cell-free extracts of E. coli containing the appropriate plasmids.

Acetyltransferases↗
PubMed1985

Regulation of antibiotic resistance in bacteria: the chloramphenicol acetyltransferase system.

The evaluations of antibiotic resistance has been a subject of interest to workers in several disciplines. Our current understanding of the molecular biology of plasmids, phages, and transposable elements provides a basis for appreciating the range of mechanisms likely to be involved in the horizontal spread of resistance determinants through microbial ecosystems. Rather less can be imagined with confidence about the origins of the genes or the constraints and selection pressures operating at the level of protein structure. The CAT system illustrates the extent of variation possible for an accessory gene product which is required infrequently and which is encoded by multicopy and promiscuous vectors which can cross taxonomic boundaries. Still less is known with certainty about the evolution of genetic control of the expression of antibiotic resistance. While there are sound reasons for looking in detail at prokaryotic antibiotic-producing organisms such as Streptomyces to find the progenitors of present resistance mechanisms (44, 45), it seems likely that controls of expression have been acquired during the "passage" of selectable markers through more distant bacterial genera. The CAT system is illustrative of the variety we may expect to find in control strategies used by microbial systems generally. It might indeed be a surprise to find an expression mechanism operating in the CAT system (or for any other family of resistance genes) which was not illustrative of a general strategy exploited by essential genes specifying biosynthetic or degradative functions. There may be some truth in referring to the cat structural gene as a "cartridge" for the isolation and manipulation of promoter functions. It would seem that nature has been at it for some time.

Acetyltransferases↗