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A J Clarke

Publications and source records attributed to A J Clarke.

At least 37 records · Page 2Linked to original sources
PubMed1999

The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors.

The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.

Alanine↗
PubMed1998

Restoration of catalytic activity beyond wild-type level in glucoamylase from Aspergillus awamori by oxidation of the Glu400-->Cys catalytic-base mutant to cysteinesulfinic acid.

Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.

Alkylation↗
PubMed1998

Enzymatic properties of the cysteinesulfinic acid derivative of the catalytic-base mutant Glu400-->Cys of glucoamylase from Aspergillus awamori.

The pKa of the catalytic base was lowered and its distance to the general acid catalyst, Glu179, was increased in the glucoamylase from Aspergillus awamori by replacing the catalytic base Glu400 with cysteine followed by oxidation to cysteinesulfinic acid [Fierobe, H.-P., Mirgorodskaya, E., McGuire, K. A., Roepstorff, P., Svensson, B. and Clarke, A. J. (1998) Biochemistry 37, 3743-3752. 1H NMR spectroscopy demonstrated that the oxidized mutant Glu400-->Cys-SO2H glucoamylase, like the wild-type, catalyzed hydrolysis with inversion of the anomeric configuration of the product. Relative to the catalytic base mutant Glu400-->Cys, the Cys400-SO2H glucoamylase had 700 times higher kcat toward maltose, while K(m) was unchanged. Compared to wild-type glucoamylase, the Cys400-SO2H derivative had kcat values of 150-190% and 85-320% on malto- and isomaltooligosaccharides, respectively, while K(m) values were similar to those of wild-type with the two disaccharides and 3.5-5.5- and 1.8-2.5-fold higher for the longer malto- and isomaltooligosaccharides substrates, respectively. The pH-activity dependence at saturating concentration of maltose indicated that the pKa of the catalytic base Cys400-SO2H was about 0.5 pH unit lower than that of wild-type Glu400. The Ki of Cys400-SO2H glucoamylase for the pseudotetrasaccharide and potent inhibitor acarbose increased more than 10(4)-fold, but Ki values of the mono- and disaccharide analogues 1-deoxynojirimycin and beta-O-methylacarviosinide were unchanged, suggesting perturbation at binding subsites beyond the catalytic center. A distinct property of Cys400-SO2H glucoamylase was the catalysis of the condensation of beta-D-glucopyranosyl fluoride and subsequent hydrolysis of the product to beta-glucose, under conditions where this was not detected for the wild-type enzyme.

1-Deoxynojirimycin↗
PubMed1998

Gram-negative bacteria produce membrane vesicles which are capable of killing other bacteria.

Naturally produced membrane vesicles (MVs), isolated from 15 strains of gram-negative bacteria (Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Proteus, Salmonella, and Shigella strains), lysed many gram-positive (including Mycobacterium) and gram-negative cultures. Peptidoglycan zymograms suggested that MVs contained peptidoglycan hydrolases, and electron microscopy revealed that the murein sacculi were digested, confirming a previous modus operandi (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 174:2767-2774, 1996). MV-sensitive bacteria possessed A1alpha, A4alpha, A1gamma, A2alpha, and A4gamma peptidoglycan chemotypes, whereas A3alpha, A3beta, A3gamma, A4beta, B1alpha, and B1beta chemotypes were not affected. Pseudomonas aeruginosa PAO1 vesicles possessed the most lytic activity.

Antibiosis↗
PubMed1997

Identification of the catalytic nucleophile in the cellulase from Schizophyllum commune and assignment of the enzyme to Family 5, subtype 5 of the glycosidases.

Differential chemical modification of the cellulase from Schizophyllum commune with [N-methyl-3H]1-ethyl-3(4-azonia-4,4-dimethylpentyl)-carbodiimide in the presence and absence of substrate identified an active site glutamate residue within the peptide: Leu-Gln-Ala-Ala-Thr-Glu-Trp-Leu-(Lys). This Glu residue is proposed to participate in binding of substrate as amino acid sequence homology studies combined with mechanism-based inhibition of the cellulase with 4',5'-epoxypentyl-beta-D-cellobioside identified a neighboring Glu residue, which conforms to the Glu-X-Gly motif of Family 5 glycosidases, as the catalytic nucleophile. These data allow the assignment of the S. commune cellulase to Family 5, subtype 5 of the glycosidases.

Amino Acid Sequence↗
PubMed1997

The influence of porosity and pore size on the ultrasonic properties of bone investigated using a phantom material.

Ultrasonic propagation in bone has been investigated using the Leeds Ultrasonic Bone Phantom Material. Phantoms were produced with different porosities in the range of 45-83% and pore sizes of 1.3 and 0.6 mm. The phase velocity at 600 kHz was found to follow a second-order polynomial as a function of porosity. Phase velocity values between 1545 and 2211 m s-1 were measured and found to be largely independent of pore size for a given porosity. The slope of the phase velocity as a function of frequency (dispersion) decreases with increasing porosity. The values obtained from samples having different pore sizes were also similar. The attenuation coefficient and normalized broadband ultrasonic attenuation (nBUA) reached a maximum at about 50%. The normalized attenuation ranged from 6 to 25 dB cm-1 over the porosity range available and consistently showed higher values for the larger pore size. Similarly, the nBUA values were found to be between 14 and 53 dB MHz-1 cm-1, with the values for the larger pore size being roughly 10 dB MHz-1 cm-1 greater than those for the smaller pore size. These findings demonstrate that the Leeds phantom can be used to investigate the effect of structural changes in bone and to aid the understanding of quantitative ultrasound. The results support the assumption that the velocity in trabecular bone is not dependent on pore size but is influenced by the mechanical properties of the bone's constituents and the overall framework, whereas the attenuation and BUA are also influenced by structure.

Bone Density↗
PubMed1997

Characterization of gentamicin 2'-N-acetyltransferase from Providencia stuartii: its use of peptidoglycan metabolites for acetylation of both aminoglycosides and peptidoglycan.

The relationship between the acetylation of peptidoglycan and that of aminoglycosides in Providencia stuartii has been investigated both in vivo and in vitro. Adaptation of the assay for peptidoglycan N-->O-acetyltransferase permitted an investigation of the use of peptidoglycan as a source of acetate for the N acetylation of aminoglycosides by gentamicin N-acetyltransferase [EC 2.3.1.59; AAC(2')]. The peptidoglycan from cells of P. stuartii PR50 was prelabelled with 3H by growth in the presence of N-[acetyl-3H]glucosamine. Under these conditions, [3H]acetate was confirmed to be transferred to the C-6 position of peptidoglycan-bound N-acetylmuramyl residues. Isolated cells were subsequently incubated in the presence of various concentrations of gentamicin and tobramycin (0 to 5x MIC). Analysis of various cellular fractions from isolated cells and spent culture medium by the aminoglycoside-binding phosphocellulose paper assay revealed increasing levels of radioactivity associated with the filters used for whole-cell sonicates of cells treated with gentamicin up to 2 x MIC. Beyond this concentration, a decrease in radioactivity was observed, consistent with the onset of cell lysis. Similar results were obtained with tobramycin, but the increasing trend was less obvious. The transfer of radiolabel to either aminoglycoside was not observed with P. stuartii PR100, a strain that is devoid of AAC(2')-Ia. A high-performance anion-exchange chromatography-based method was established to further characterize the AAC(2')-Ia-catalyzed acetylation of aminoglycosides. The high-performance liquid chromatography (HPLC)-based method resolved a tobramycin preparation into two peaks, both of which were collected and confirmed by 1H nuclear magnetic resonance to be the antibiotic. Authentic standards of 2'-N-acetyltobramycin were prepared and were well separated from the parent antibiotic when subjected to the HPLC analysis. By applying this technique, the transfer of radiolabelled acetate from the cell wall polymer peptidoglycan to tobramycin was confirmed. In addition, isolated and purified AAC(2')-Ia was shown to catalyze in vitro the transfer of acetate from acetyl-coenzyme A, soluble fragments of peptidoglycan, and N-acetylglucosamine to tobramycin. These data further support the proposal that AAC(2')-Ia from P. stuartii may have a physiological role in its secondary metabolism and that its activity on aminoglycosides is simply fortuitous.

Acetates↗
PubMed1997

Bacterial genetic loci implicated in the Pseudomonas putida GR12-2R3--canola mutualism: identification of an exudate-inducible sugar transporter.

Pseudomonas putida GR12-2R3 promotes the emergence and growth of diverse plant species. Analyses of TnphoA insertion mutations are revealing bacterial characteristics pertinent to the plant-microbe interaction. Pseudomonas putida PG269 is a TnphoA insertion derivative of GR12-2R3 that expresses canola seed exudate-inducible alkaline phosphatase (PhoA) activity. It promoted the growth of canola roots, as well as strain GR12-2R3, and outgrew its parent when they were cocultured in the presence of canola roots or in liquid seed exudate medium. (In contrast, mutant PG126 failed to promote canola root growth and was outgrown by its parent strain.) The PhoA activity of strain PG269 was induced by glucosamine and other sugars; glucosamine inhibited the growth of strain GR12-2R3 and stimulated the growth of strain PG269. Strain PG269 contained two TnphoA insertions: seiA1::TnphoA and seiB1::TnphoA. Strain PG312, which contained only insertion seiA1::TnphoA, shared all aspects of the PG269 phenotype, except the ability to outcompete strain GR12-2R3 during coculture. Insertion seiA1::TnphoA interrupted an open reading frame related in sequence to members of the MalF family of sugar transporter subunits. The PhoA-inducing fraction of canola seed exudate was hydrophilic, low in molecular weight, and heat stable. It cochromatographed with basic amino acids and amino sugars, and was inactivated by strains GR12-2R3 and PG269. Gene seiA may encode a subunit of an ABC transporter with broad specificity for glucose and related sugars whose expression can be induced by exudate sugars.

Amino Acid Sequence↗
PubMed1997

Isolation, purification, and characterization of the major autolysin from Pseudomonas aeruginosa.

The major (26 kDa) autolysin from Pseudomonas aeruginosa was purified to apparent homogeneity by a combination of preparative electrophoresis, ion-exchange, and dye-ligand chromatographies. This purification was facilitated by the development of a spot-assay that involved the spotting and subsequent incubation of autolysin samples on polyacrylamide gels containing peptidoglycan. The pl of the 26-kDa autolysin was determined to be between 3.5 and 4 and disulfide bonds within the enzyme were essential for activity. The autolysin catalyzed the release of reducing sugars from the peptidoglycans of Pseudomonas aeruginosa and Escherichia coli indicating it to be a beta-glycosidase. It was ineffective at hydrolysing the peptidoglycan from Gram-positive bacteria and the O-acetylated peptidoglycans from either Proteus mirabilis or Staphylococcus aureus. The N-terminal sequence of the purified autolysin was determined to be His-Glu-Pro-Pro-Gly. The 26-kDa autolysin together with a 29-kDa autolysin was determined to be secreted into the medium by a mechanism that involves the production and release of surface membrane vesicles during normal growth, but the enzymes were not found free and active in culture broth supernatants.

Amino Acids↗
PubMed1996

The prevalence of gentamicin 2'-N-acetyltransferase in the Proteeae and its role in the O-acetylation of peptidoglycan.

The prevalence of aac(2')-Ia, a gene coding for gentamicin 2'-N-acetyltransferase in Providencia stuartii, among species of the Proteeae was investigated to determine if it is a common resistance factor and whether the correlation observed in P. stuartii between its expression and the levels of peptidoglycan O-acetylation represents a general feature of bacteria producing this form of modified peptidoglycan. An evaluation of the MICs of gentamicin for each of the species of the Proteeae did not reveal any apparent relationship between resistance and the degree of O-acetylation of peptidoglycan. The entire aac(2')-Ia gene was used as a probe in Southern hybridization experiments against genomic DNA from each species of the Proteeae. A sequence with strong homology to aac(2')-Ia was present only in Proteus penneri while weak hybridization was also observed to the restriction digested DNA from Providencia rettgeri. Other bacteria that O-acetylate peptidoglycan were also screened with this probe and a homologous DNA sequence was only found in Neisseria subflava. These data suggest that AAC(2')-Ia may contribute to the O-acetylation of peptidoglycan in P. stuartii, but a more specific enzyme must also be produced for this function.

Acetylation↗
PubMed1996

Ultrasonic measurement: an evaluation of three heel bone scanners compared with a bench-top system.

Three commercial ultrasound bone scanners designed for os calcis measurements (Lunar Achilles, C.U.B.A. "Research" and UBA 575) were compared using the Leeds Ultrasonic Bone Phantoms. The porosity of the phantoms ranged from 50% to 83% with velocities between 1490 and 1621 m s-1 and broadband ultrasound attenuation (BUA) values in the range 46-115 dB MHz-1. The three devices tested were able to discriminate porosity differences of at least 3%, although the values obtained for the propagation parameters varied widely. Velocity differences of up to 38 m s-1 and BUA variations of up to 33 dB MHz-1 were found, although a relationship was identified between the velocity and BUA measurements. In some cases, the variation can be attributed to differences in the measurement technique adopted, although there also seem to be detailed differences in the definition of the parameters themselves. The variation between different devices from the same manufacturer (Lunar) was also studied. Measurements taken from five devices showed variation in velocity values of up to 25 m s-1 (SD 10.8 m s-1) and in BUA values of up to 11 dB MHz-1 (SD 4.3 dB MHz-1). We conclude that the variation both between manufacturers and between nominally identical machines may be of clinical significance. Both users and manufacturers need to consider urgently the introduction of quality standards and consensus definition of terms and techniques. The fact that all machines studied have been superseded commercially does not invalidate these conclusions, since many of the devices tested remain in clinical use and there is no evidence of fundamental change in manufacturers' procedures.

Heel↗
PubMed1996

The measurement of the velocity of ultrasound in fixed trabecular bone using broadband pulses and single-frequency tone bursts.

The velocity of ultrasound in a series of 10 fixed os calces was measured using both short pulses and 750 kHz tonebursts. The values obtained from the pulse measurements differed from the toneburst values by up to 16% depending on the selection of the zero-crossing point used as a reference in the pulse measurements. It is demonstrated that the discrepancy between the values is itself a function of the frequency-dependent attenuation in the propagating medium and this is confirmed by theoretical modelling. The toneburst results are also compared with measurements using a cross-correlation technique, and these show a close agreement.

Aged↗
PubMed1996

The role of O-acetylation in the metabolism of peptidoglycan in Providencia stuartii.

The gentamicin 2'-N-acetyltransferase [EC 2.3.1.59; AAC(2')-Ia] of Providencia stuartii was shown to contribute to the O-acetylation of peptidoglycan and mutants that either under- or overexpress the aac(2')-Ia gene was characterized phenotypically to possess either lower or higher levels of peptidoglycan O-acetylation, respectively, compared to the wild-type. These mutants were subjected to scanning electron microscopy. P. stuartii PR100, with 42-44% peptidoglycan O-acetylation compared to 54% for the wild-type, appeared as irregular rods. In direct contrast, strains PR50.LM3 and PR51, with increased levels of peptidoglycan O-acetylation (63 and 65%, respectively), appeared as coccobacilli or chain formers, respectively. Zymogram analysis of the autolysins produced by another member of the closely related Proteeae group of bacteria, Proteus mirabilis, indicated the presence of three classes of enzymes: one that acts preferentially on native, O-acetylated peptidoglycan, a second that hydrolyses non-O-acetylated peptidoglycan, and a third that is not distinguished by the two forms of substrate. On the basis of the apparent morphological changes directly related to levels of O-acetylation combined with the presence of different classes of autolysins, a model is proposed that invokes the role of this modification in the control of autolysins for the maintenance of the structure of the peptidoglycan sacculus.

Acetylation↗
PubMed1996

The role of the nonconserved residues at position 167 of class A beta-lactamases in susceptibility to mechanism-based inhibitors.

Differences in specificities between the class A beta-lactamases for both substrate and inhibitors are known. The role of the nonconserved amino acid residue at position 167 of the class A enzyme, which forms a cis bond with the catalytically essential Glu-166 residue, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors, was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace thr-167 with the corresponding Staphylococcus aureus PC1 residue Ile. Kinetic data obtained from the purified Thr-167-Ile B. cereus 569/H beta-lactamase was compared to that obtained from the wild-type B. cereus and S. aureus enzymes and indicated that the replacement had little effect on the Michaelis parameters for the hydrolysis of S- and A-type penicillins. However, the Thr-167-Ile enzymes became more S. aureus PC1-like in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amidopenicillanic acid sulfone. A model for the role of this nonconserved residue at position 167 in the mechanism of inactivation by mechanism-based inhibitors is proposed.

Amino Acid Sequence↗
PubMed1996

A major autolysin of Pseudomonas aeruginosa: subcellular distribution, potential role in cell growth and division and secretion in surface membrane vesicles.

A 26-kDa murein hydrolase is the major autolysin of Pseudomonas aeruginosa PAO1, and its expression can be correlated with the growth and division of cells in both batch and synchronously growing cultures. In batch cultures, it is detected primarily during the mid-exponential growth phase, and in synchronous cultures, it is detected primarily during the cell elongation and division phases. Immunogold labeling of thin sections of P. aeruginosa using antibodies raised against the 26-kDa autolysin revealed that it is associated mainly with the cell envelope and in particular within the periplasm. It is also tightly bound to the peptidoglycan layer, since murein sacculi, isolated by boiling 4% sodium dodecyl sulfate treatment, could also be immunogold labeled. Since division is due to cell constriction in this P. aeruginosa strain (septa are rarely seen), we cannot comment on the autolysin's contribution to septation, although constriction sites were always heavily labeled. Some labeling was also found in the cytoplasm, and this was thought to be due to the de novo synthesis of the enzyme before translocation to the periplasm. Interestingly, the autolysin was also found to be associated with natural membrane vesicles which blebbed from the surface during cell growth; the enzyme is therefore part of the complex makeup of these membrane packages of secreted materials (J. L. Kadurugamuwa and T. J. Beveridge, J. Bacteriol. 177:3998-4008, 1995). The expression of these membrane vesicles was correlated with the expression of B-band lipopolysaccharide.

Antibody Specificity↗
PubMed1996

Value of intraoperative image intensifier prints in trauma surgery.

We have studied the use of image intensification in a trauma theatre over a period of 6 months with particular reference to the acquisition of intraoperative image intensifier thermal prints instead of formal radiographs. The quality of the prints and the savings generated have been assessed. During the study period, 476 patients underwent orthopaedic trauma procedures. The image intensifier was used for intraoperative screening in 280 patients. In 278 of these a thermal print was obtained. This was used, instead of formal check radiographs, to plan further management in 210 patients (75%). In 68 patients, the printout was insufficient in its coverage of the operated area, and a check radiograph was also obtained. In no case did the clarity of the thermal image hinder accurate interpretation. We believe that thermal images are a useful substitute for formal postoperative radiographs in many trauma cases, and that, with notable exceptions, their use could decrease costs, reduce patient discomfort and radiation dose and spare overloaded radiology services.

Bone and Bones↗
PubMed1995

Identification of an essential tyrosyl residue in the binding site of Schizophyllum commune xylanase A.

Ultraviolet difference spectroscopy studies with the Schizophyllum commune xylanase in the presence of inhibitors and substrates indicated the participation of one or more tyrosyl residues in the binding of substrates to xylanase. Chemical modification experiments with group-specific reagents in the absence and presence of substrates confirmed the essential role of a tyrosyl residue in substrate binding while discounting the participation of tryptophan. A fourth-derivative absorbance spectroscopic method was developed to facilitate the quantitation of modified tyrosyl and tryptophanyl residues. This analysis showed that two tyrosyl residues of the xylanase are modified by tetranitromethane in the absence of substrate with the concomitant loss of catalytic activity. Protection of the xylanase with xylooligosaccharides resulted in the nitration of only one residue, and such enzyme derivatives retained 94% catalytic activity. Differential modification of the xylanase with tetranitromethane generated an enzyme derivative with the characteristic absorbance at 428 nm of 3-nitrotyrosine. Amino acid analysis and N-terminal sequencing of peptides with strong absorbance at 428 nm isolated from the protease-digested modified enzyme by reverse-phase HPLC identified the essential residue as Tyr97. Alignment of the S. commune xylanase amino acid sequence with those of the 18 other known family G xylanases revealed that Tyr97 is a conserved aromatic residue, further suggesting its essential role in substrate binding.

Amino Acid Sequence↗