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S J Cartwright

Publications and source records attributed to S J Cartwright.

11 recordsLinked to original sources
PubMed1994

Sequence analysis of the L1 metallo-beta-lactamase from Xanthomonas maltophilia.

The amino acid sequence deduced from the L1 beta-lactamase gene of Xanthomonas maltophilia shows a significant variation from that of the CphA and Blm metallo-beta-lactamases of Aeromonas hydrophila and Bacillus cereus, respectively. Whilst the N-terminus of the L1 protein shows some similarity, particularly at the histidine residues previously suggested as a zinc-binding motif, the C-terminus of the protein demonstrates very little similarity. Such differences amongst this group of enzymes would argue for at least three subclasses within the Group 3 beta-lactamases. However, in order to predict their phylogenetic ancestry more sequence data are required from other possible metallo-beta-lactamases.

Amino Acid Sequence↗
PubMed1989

Trapping the acyl-enzyme intermediate in beta-lactamase I catalysis.

Cryoenzymology techniques were used to facilitate trapping an acyl-enzyme intermediate in beta-lactamase I catalysis. The enzyme (from Bacillus cereus) was investigated in aqueous methanol cryosolvents over the 25 to -75 degrees C range, and was stable and functional in 70% (v/v) methanol at and below 0 degree C. The value of kcat. decreased linearly with increasing methanol concentration, suggesting that water is a reactant in the rate-determining step. In view of this, the lack of incorporation of methanol into the product means that the water molecule involved in the deacylation is shielded from bulk solvent in the enzyme-substrate complex. From the lack of adverse effects of methanol on the catalytic and structural properties of the enzyme we conclude that 70% methanol is a satisfactory cryosolvent system for beta-lactamase I. The acyl-enzyme intermediate from the reaction with 6-beta-(furylacryloyl)amidopenicillanic acid was accumulated in steady-state experiments at -40 degrees C and the reaction was quenched by lowering the pH to 2. H.p.l.c. experiments showed covalent attachment of the penicillin to the enzyme. Digestion by pepsin and trypsin yielded a single labelled peptide fragment; analysis of this peptide was consistent with Ser-70 as the site of attachment.

Acylation↗
PubMed1989

Permeability to cefsulodin of the outer membrane of Pseudomonas aeruginosa and discrimination between beta-lactamase-mediated trapping and hydrolysis as mechanisms of resistance.

A pair of strains of Pseudomonas aeruginosa (3-Pre: cefsulodin-sensitive, inducible beta-lactamase; and 3-Post: cefsulodin-resistant, elevated beta-lactamase, derived from 3-Pre by subculture in the presence of cefsulodin) were taken as representative of the class of bacteria resistant to third-generation cephalosporins due to elevated synthesis of the normally inducible, chromosomally encoded beta-lactamase. These two strains were used to differentiate between 'trapping' and 'hydrolytic' mechanisms of cefsulodin resistance by (a) measuring the outer-membrane permeabilities to cefsulodin, (b) measuring the kinetics of cefsulodin hydrolysis and the stoichiometry of cefsulodin trapping by the periplasmic beta-lactamase, and (c) comparing the predictions of the trapping and hydrolysis hypotheses with the minimum inhibitory concentrations (MIC) of cefsulodin. The MIC of cefsulodin for strains 3-Pre and 3-Post were 2.35 microM (1.25 micrograms ml-1) and 37.6 microM (20.0 micrograms ml-1) respectively. The permeability parameter for cefsulodin of the outer membrane of the resistant strain was 0.0034 cm3 min-1 mg dry mass-1, so the flux of cefsulodin across its outer membrane at the MIC was calculated to be 0.120 nmol min-1 mg dry mass-1. Hydrolysis of cefsulodin by the beta-lactamase in the periplasm occurred at a rate of 0.118 nmol min-1 mg dry mass-1 which can thus account for resistance by matching the above rate of inflow. Trapping by the beta-lactamase, even with a 1:1 stoichiometry, would require the enzyme to be synthesized at 5.0 micrograms protein min-1 mg dry mass-1 or about 40% of the dry mass/generation. We conclude that hydrolysis, but not trapping, adequately explains the resistance to cefsulodin in P. aeruginosa 3-Post. A similar calculation for latamoxef resistance, using data taken from the literature, led to the same conclusion.

Bacterial Outer Membrane Proteins↗
PubMed1987

Cryoenzymology of beta-lactamases.

The cryoenzymology of several different beta-lactamases has been investigated. Particular attention has been paid to the experimental pitfalls of the technique. These include such factors as false bursts at the start of the reaction, instability of the enzymes during turnover, and Km values so high that little of the enzyme is present as a complex. Many of the difficulties in cryoenzymology stem from the use of organic cryosolvents. A novel "salt" cryosolvent has been tested: ammonium acetate solutions can be used down to about -60 degrees C. The enzymes examined are readily soluble, and stable, in this solvent. Nevertheless, out of 17 beta-lactamase beta-lactam systems, only 4 proved suitable for detailed investigation. In two of these, the hydrolysis of nitrocefin or 7-(thienyl-2-acetamido)-3-[[2-[[4- (dimethylamino)phenyl]azo]pyridinio]-methyl]cephem-4-carboxylic acid (PADAC), by beta-lactamase I from Bacillus cereus, substrate was converted into product at a slow enough rate (at -60 or -55 degrees C, respectively) for it to be possible to do successive scans during the course of the reaction. The spectra were those of substrate and product, and no intermediate was detected. The results argue against the accumulation of intermediate acyl-enzyme. The hydrolysis of PADAC by the P99 beta-lactamase from Enterobacter cloacae again showed spectra characteristic of substrate and product, and there was, moreover, a break in the Arrhenius plot; it is possible that a conformational change is (at least partially) rate-determining. The hydrolysis of dinitrophenylpenicillin by the P99 beta-lactamase did show features suggesting the accumulation of acyl-enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus cereus↗
PubMed1986

6-beta-Iodopenicillanate as a probe for the classification of beta-lactamases.

An inactivator of serine beta-lactamases, 6 beta-iodopenicillanate, can be utilized as a probe in the classification of beta-lactamases. It is a substrate for class-B Zn2+-containing beta-lactamase II. Although it inactivates enzymes from both classes A and C, it is much more efficient for the former group, with which it sometimes interacts following a branched pathway. On the basis of these observations, predictions are made concerning the class to which several enzymes belong.

Hydrogen-Ion Concentration↗
PubMed1984

Purification of beta-lactamases by affinity chromatography on phenylboronic acid-agarose.

Several beta-lactamases, enzymes that play an important part in antibiotic resistance, have been purified by affinity chromatography on boronic acid gels. The procedure is rapid, appears to be selective for beta-lactamases, and allows a one-step purification of large amounts of enzyme from crude cell extracts. We have found the method useful for any beta-lactamase that is inhibited by boronic acids. Two kinds of boronic acid column have been prepared, the more hydrophobic one being reserved for those beta-lactamases that bind boronic acids relatively weakly. beta-Lactamase I from Bacillus cereus, P99 beta-lactamase and K 1 beta-lactamase from Gram-negative bacteria are among the better-known beta-lactamases that have been purified by this method. The procedure has also been used to purify a novel beta-lactamase from Pseudomonas maltophilia in high yield; the enzyme has an exceptionally broad substrate profile and hydrolyses monocyclic beta-lactams such as azthreonam and desthiobenzylpenicillin.

Chromatography, Affinity↗
PubMed1981

Cryoenzymology.

A detailed understanding of the mechanism of enzyme action requires a correspondingly detailed knowledge of the structures of the intermediates and transition states on the catalytic pathway, as well as the kinetics and thermodynamics of their interconversion. Cryoenzymology, the use of subzero temperatures and fluid cryosolvents, has the potential to supply this type of information. In this article recent investigations illustrating the advantages of cryoenzymology are reviewed. The major advantage lies in the ability to accumulate and stabilize productive enzyme-substrate intermediates for sufficient lengths of time to allow the collection of high resolution structural data, e.g. by X-ray diffraction. In addition, intermediates which are not detectable under normal conditions, due to too short lifetimes and/or low concentrations, may be detected at low temperatures, in some cases through changes in the rate-limiting step. The method seems of rather general applicability, judging by the number of different types of enzymes, including oligomeric and membrane-associated ones, which have successfully been studied using cryoenzymology. In some cases, at least, good agreement between observations at subzero temperatures and normal conditions has been found, demonstrating the relevance of the technique. Potential limitations of the techniques, as well as questions regarding the effects on the protein structure, are also discussed.

Animals↗
PubMedDate not supplied

beta-Lactamase inhibitors.

In summary, Table XVI shows the inhibition profiles of representative beta-lactamases from each major class of Richmond and Sykes. Either resistance (R) or sensitivity (S) is given as a general guide to the type of compounds likely to inhibit each class. Thus the (qualitative) statements regarding the effectiveness of clavulanic acid can be taken to represent those for the penam sulfones and similarly for MM4550 and the other olivanic acids, carpetimycins, PS series, and asparenomycins. This can also be said of cloxacillin and the other aromatic carboxamido penicillins. Compounds are also included which are specifically or particularly inhibitory to certain beta-lactamases.

Anti-Bacterial Agents↗