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T Palzkill

Publications and source records attributed to T Palzkill.

36 records · Page 2Linked to original sources
PubMed1998

Characterization of a PSE-4 mutant with different properties in relation to penicillanic acid sulfones: importance of residues 216 to 218 in class A beta-lactamases.

Class A beta-lactamases are inactivated by the suicide inactivators sulbactam, clavulanic acid, and tazobactam. An examination of multiple alignments indicated that amino acids 216 to 218 differed among class A enzymes. By random replacement mutagenesis of codons 216 to 218 in PSE-4, a complete library consisting of 40,864 mutants was created. The library of mutants with mutations at positions 216 to 218 in PSE-4 was screened on carbenicillin and ampicillin with the inactivator sulbactam; a collection of 14 mutants was selected, and their bla genes were completely sequenced. Purified wild-type and mutant PSE-4 beta-lactamases were used to measure kinetic parameters. One enzyme, V216S:T217A:G218R, was examined for its peculiar pattern of inhibition. There was an increase in the Km from 68 microM for the wild type to 271 microM for the mutant for carbenicillin and 33 to 216 microM for ampicillin. Relative to the wild-type PSE-4 enzyme, 37- and 30-fold increases in Ki values were observed for the mutant enzyme for sulbactam and tazobactam, respectively. The results that were obtained suggested that positions 216 to 218 are important for interactions with penicillanic acid sulfone inhibitors. In contrast, V216 and A217 in the TEM-1 class A beta-lactamase do not tolerate amino acid residue substitutions. However, for the PSE-4 beta-lactamase, 11 of 14 mutants from the library of mutants with mutations at positions 216 to 218 whose sequences were determined had substitutions at position 216 (G, R, A, S) and position 217 (A, S). The data showed the importance of residues 216 to 218 in their atomic interactions with inactivators in the PSE-4 beta-lactamase structure.

Binding Sites↗
PubMed1997

Cephalosporin substrate specificity determinants of TEM-1 beta-lactamase.

beta-lactamase is a bacterial enzyme that catalyzes the hydrolysis of beta-lactam antibiotics such as penicillins and cephalosporins. TEM-1 beta-lactamase is a prevalent beta-lactamase found in Gram-negative bacteria and is capable of hydrolyzing both penicillins and cephalosporins, except for the extended-spectrum cephalosporins. To identify the sequence determinants in the active site for a given antibiotic substrate, random libraries were constructed that each contain all possible amino acid combinations for the designated region of TEM-1 beta-lactamase. To establish the determinants of substrate specificity for cephalosporins versus those for penicillins, these active site libraries have been screened for mutants with high levels of activity for the second generation cephalosporin cephaloridine. Based on the sequence results, substitutions of W165S, A237T, and E240C were identified as cephalosporin-specific. Kinetic analysis of these mutants was done to determine whether each is capable of distinguishing between the two classes of antibiotics. Both the A237T and E240C substitutions, alone or in combination, exhibited increased cephalosporinase activity and decreased penicillinase activity relative to the wild-type enzyme. A sequence comparison between functional mutants selected for cephaloridine hydrolytic activity and functional mutants previously selected for ampicillin hydrolytic activity suggests that TEM-1 beta-lactamase has greater restrictions in maintaining cephalosporinase activity versus maintaining penicillinase activity.

Amino Acid Sequence↗
PubMed1997

A natural polymorphism in beta-lactamase is a global suppressor.

A M182T substitution was discovered as a second-site suppressor of a missense mutation in TEM-1 beta-lactamase. The combination of the M182T substitution with other substitutions in the enzyme indicates the M182T substitution is a global suppressor of missense mutations in beta-lactamase. The M182T substitution also is found in natural variants of TEM-1 beta-lactamase with altered substrate specificity that have evolved in response to antibiotic therapy. The M182T substitution may have been selected in natural isolates as a suppressor of folding or stability defects resulting from mutations associated with drug resistance. This pathway of protein evolution may occur in other targets of antimicrobial drugs such as the HIV protease.

Enzyme Stability↗
PubMed1996

Selection and characterization of amino acid substitutions at residues 237-240 of TEM-1 beta-lactamase with altered substrate specificity for aztreonam and ceftazidime.

Recently, natural variants of TEM-1 beta-lactamase with amino acid substitutions at residues 237-240 have been identified that have increased hydrolytic activity for extended-spectrum antibiotics such as ceftazidime. To identify the sequence requirements in this region for a given antibiotic, a random library was constructed that contained all possible amino acid combinations for the 3-residue region 237-240 (ABL numbering system) of TEM-1 beta-lactamase. An antibiotic disc diffusion method was used to select mutants with wild-type level activity or greater for the extended-spectrum cephalosporin ceftazidime and the monobactam aztreonam. Mutants that were selected for optimal ceftazidime hydrolysis contained a conserved Ala at position 237, a Ser for Gly substitution at position 238, and a Lys for Glu at position 240. Mutants selected for aztreonam hydrolysis exhibited a Gly for Ala substitution at position 237, a Ser for Gly substitution at position 238, and a Lys/Arg for Glu at position 240. The role of the A237G substitution in differentiating between ceftazidime and aztreonam was further investigated by kinetic analysis of the A237G, E240K, G238S:E240K, and A237G:G238S:E240K enzymes. The A237G single mutant and the G238S:E240K double mutant exhibited increases in catalytic efficiency for both ceftazidime and aztreonam. However, the triple mutant A237G:G238S:E240K, displayed a 12-fold decrease in catalytic efficiency for ceftazidime but a 3-fold increase for aztreonam relative to the G238S:E240K double mutant. Thus, the A237G substitution increases ceftazidime hydrolysis when present alone but antagonizes ceftazidime hydrolysis when it is combined with the G238S:E240K substitutions. In contrast, the A237G substitution acts additively with the G238S:E240K substitutions to increase aztreonam hydrolysis.

Amino Acid Sequence↗
PubMed1996

Amino acid sequence determinants of beta-lactamase structure and activity.

TEM-1 beta-lactamase catalyzes the hydrolysis of beta-lactam antibiotics such as the penicillins and cephalosporins, thus providing for bacterial resistance to these compounds. To determine the amino acid residues critical for the structure and function of TEM-1 beta-lactamase, the codons for each of the 263 amino acid residues that constitute the mature form of the enzyme were randomized using a site-directed mutagenesis procedure. Functional random mutants were selected based on their ability to confer ampicillin resistance to Escherichia coli. The DNA sequence of several functional mutants was determined for each set of random mutants. It was found that 43 out of the 263 amino acid residues do not tolerate substitutions and therefore are critical for the structure and activity of the enzyme. In addition, a comparison of conserved residue positions among functional beta-lactamase mutants with conserved residues in the beta-lactamase gene family identified many positions which did not tolerate substitutions in the mutagenesis studies but are freely substituted among members of the gene family. This observation may be due to the accumulation of compensating mutations among members of the gene family. Finally, the sequence variability at residue positions among functional mutants was quantitated by calculating the effective number of substitutions at each position using information-theoretical entropy. These values were used to obtain a quantitative estimate of the correlation between the sequence variability at a position and the fractional accessible surface area of the residue. The correlation is found to be statistically significant in that buried residues tend to exhibit low variability and invariant residues tend to exhibit low solvent exposure. However, the correlation is weak because most residues are neither completely buried nor invariant.

Amino Acid Sequence↗
PubMed1996

The rate-limiting step in the folding of the cis-Pro167Thr mutant of TEM-1 beta-lactamase is the trans to cis isomerization of a non-proline peptide bond.

The stability and kinetics of unfolding and refolding of the P167T mutant of the TEM-1 beta-lactamase have been investigated as a function of guanidine hydrochloride concentration. The activity of the mutant enzyme was not significantly modified, which strongly suggests that the Glu166-Thr167 peptide bond, like the Glu166-Pro167, is cis. The mutation, however, led to a significant decrease in the stability of the native state relative to both the thermodynamically stable intermediate and the fully unfolded state of the protein. In contrast to the two slower phases seen in the refolding of the wild-type enzyme, only one phase was detected in the refolding of the mutant, indicating a determining role of proline 167 in the kinetics of folding of the wild-type enzyme. The former phases are replaced by rapid refolding when the enzyme is unfolded for short periods of time, but the latter is independent of the time of unfolding. The monophasic refolding reaction of the mutant is proposed to reflect mainly the trans-->cis isomerization of the Glu166-Thr167 peptide bond.

Anti-Bacterial Agents↗
PubMed1996

Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants.

Amino Acid Sequence↗
PubMed1994

Characterization of TEM-1 beta-lactamase mutants from positions 238 to 241 with increased catalytic efficiency for ceftazidime.

Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins such as cefotaxime and ceftazidime. To identify other amino acid substitutions that alter the activity of TEM-1 toward extended-spectrum cephalosporins, a random library was constructed that contained all possible amino acid substitutions over the 3-residue window of 238-241 (ABL numbering). Mutants were selected for 100-fold greater ceftazidime resistance than wild-type. All mutants had a serine substitution at position 238, a lysine or arginine at position 240, and a small amino acid at position 241. The role of each substitution was investigated by constructing individual G238S, E240K, and R241G substitutions as well as the G238S:E240K double mutant. Each enzyme was purified to homogeneity and the kinetic parameters kcat and Km were determined using several substrates. The G238S substitution increases catalytic efficiency for both ceftazidime and cefotaxime. However, to achieve large increases in catalytic efficiency, both G238S and the E240K substitutions are required. The R241G substitution results in a small increase in catalytic efficiency for only ceftazidime. The contribution of each residue to the transition-state stabilization energy was found to be additive indicating that the substitutions act independently to change the catalytic properties of the enzyme.

Anti-Bacterial Agents↗
PubMed1994

Systematic mutagenesis of the yeast mating pheromone receptor third intracellular loop.

Signal transduction in the mating pathway of the yeast Saccharomyces cerevisiae is initiated by binding of a peptide pheromone to a G protein-coupled receptor (Ste2). We systematically have mutated the third intracellular loop of the Ste2 receptor to investigate its functional significance. We substituted each of the 13 amino acids in the loop with alanine individually or together with one other residue. In addition, we used a site-directed random replacement mutagenesis technique to replace a region encoding three amino acids in the loop with random sequence. Over 80 such Ste2 mutants have been analyzed by several functional and biochemical criteria in a yeast strain that carries a genomic deletion of the STE2 gene. The mutant phenotypes range from fully functional to severely compromised in signaling. The observation that amino acid substitutions in the third intracellular loop of the Ste2 receptor can affect activation of the yeast mating response implicates the loop in this signal transduction pathway. The types of mutations that compromise the function of the receptor may provide clues to the physical interaction between the receptor and the G protein.

Amino Acid Sequence↗
PubMed1994

Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta-lactamase.

In order to understand how TEM-1 beta-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161-170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified. Characterization of altered beta-lactamase enzymes indicated that while their catalytic efficiency (kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 beta-lactamase.

Amino Acid Sequence↗
PubMed1994

Effect of threonine-to-methionine substitution at position 265 on structure and function of TEM-1 beta-lactamase.

TEM beta-lactamase variants with the amino acid substitutions R164S, E104K, G238S, and E240K (ABL numbering) display increased activity toward extended-spectrum cephalosporins. The T265M substitution is frequently found to be associated with the above substitutions in extended-spectrum beta-lactamases. However, the residue is located away from the active site in the three-dimensional structure and has been assumed to have no effect on catalysis. To examine the effect of the substitution on the structure and function of TEM beta-lactamase we constructed the following mutants: G238S, T265M, T265M:G238S, and T265M:G238S:E240K. Each enzyme was purified to homogeneity and the kinetic parameters kcat, Km and kcat/Km were determined for cefotaxime, ceftazidime, cephaloridine, and ampicillin. The results indicate that the T265M mutation has little effect on hydrolysis. In addition, we used immunoblotting to show that the substitution has little or no effect on the in vivo steady-state levels of beta-lactamase.

Anti-Bacterial Agents↗
PubMed1994

Selection of functional signal peptide cleavage sites from a library of random sequences.

The export of proteins to the periplasmic compartment of bacterial cells is mediated by an amino-terminal signal peptide. After transport, the signal peptide is cleaved by a processing enzyme, signal peptidase I. A comparison of the cleavage sites of many exported proteins has identified a conserved feature of small, uncharged amino acids at positions -1 and -3 relative to the cleavage site. To determine experimentally the sequences required for efficient signal peptide cleavage, we simultaneously randomized the amino acid residues from positions -4 to +2 of the TEM-1 beta-lactamase enzyme to form a library of random sequences. Mutants that provide wild-type levels of ampicillin resistance were then selected from the random-sequence library. The sequences of 15 mutants indicated a bias towards small amino acids. The N-terminal amino acid sequence of the mature enzyme was determined for nine of the mutants to assign the new -1 and -3 residues. Alanine was present in the -1 position for all nine of these mutants, strongly supporting the importance of alanine at the -1 position. The amino acids at the -3 position were much less conserved but were consistent with the -3 rules derived from sequence comparisons. Compared with the wild type, two of the nine mutants have an altered cleavage position, suggesting that sequence is more important than position for processing of the signal peptide.

Amino Acid Sequence↗
PubMed1994

Alanine-scanning mutagenesis reveals residues involved in binding of pap-3-encoded pili.

In order to identify functionally important residues in the pap-3-encoded adhesin, oligonucleotide-directed mutagenesis was used to substitute alanine(s) at sixteen positions in the adhesion. These alanine substitutions span nearly every domain and hydrophilic peak of the protein. The effects of these substitutions were measured by evaluating the patterns of hemagglutination exhibited by the mutant strains. It was found that strains harboring alanine substitutions at positions 88 and 89, 128 to 130, and 316 had lost the capacity to hemagglutinate. The presence of the mutated adhesin in the assembled pilus structure was verified by the reactions of purified pili with an adhesin-specific monoclonal antibody in an enzyme-linked immunosorbent assay and with a polyclonal antibody in Western blotting (immunoblotting). Alanine substitutions at positions 68, 110 and 111, and 143 to 146 had no effect upon hemagglutination, whereas substitutions at positions 203 and 204 and position 291 resulted in diminished binding. Thus, the residues necessary for hemagglutination are scattered throughout the adhesin in both the amino and carboxy regions. Delineation of these residues may prove useful in designing a preventive treatment that would cross-react with the essential binding residues from the adhesins of several different pyelonephritis-causing strains.

Adhesins, Escherichia coli↗
PubMed1992

Probing beta-lactamase structure and function using random replacement mutagenesis.

A new analytical mutagenesis technique is described that involves randomizing the DNA sequence of a short stretch of a gene (3-6 codons) and determining the percentage of all possible random sequences that produce a functional protein. A low percentage of functional random sequences in a complete library of random substitutions indicates that the region mutagenized is important for the structure and/or function of the protein. Repeating the mutagenesis over many regions throughout a protein gives a global perspective of which amino acid sequences in a protein are critical. We applied this method to 66 codons of the gene encoding TEM-1 beta-lactamase in 19 separate experiments. We found that TEM-1 beta-lactamase is extremely tolerant of amino acid substitutions: on average, 44% of all mutants with random substitutions function and 20% of the substitutions are expressed, secreted, and fold well enough to function at levels similar to those for the wild-type enzyme. We also found a few exceptional regions where only a few random sequences function. Examination of the X-ray structures of homologous beta-lactamases indicates that the regions most sensitive to substitution are in the vicinity of the active site pocket or buried in the hydrophobic core of the protein. DNA sequence analysis of functional random sequences has been used to obtain more detailed information about the amino acid sequence requirements for several regions and this information has been compared to sequence conservation among several related beta-lactamases.

Amino Acid Sequence↗
PubMed1992

Outbreak of ceftazidime resistance due to a novel extended-spectrum beta-lactamase in isolates from cancer patients.

Ceftazidime is widely used in the therapy of infectious complications in neutropenic patients. We studied an outbreak of ceftazidime-resistant gram-negative bacillary infections in pediatric cancer patients receiving empirical ceftazidime therapy for neutropenic fever. Fourteen isolates (12 Klebsiella pneumoniae and 2 Escherichia coli) from 13 patients were studied. Specimens were obtained from multiple clinical sites including blood, urine, throat, and lung. The organisms were resistant to ceftazidime, aztreonam, and penicillins but remained susceptible to cephamycins and imipenem. All resistant isolates produced a novel beta-lactamase (TEM-26) with a pI of approximately 5.58, which was transferred by transformation to E. coli on a 7.9-kb nonconjugative plasmid which cotransferred resistance to trimethoprim-sulfamethoxazole. This enzyme readily hydrolyzed ceftazidime, aztreonam, and penicillins in a spectrophotometric assay. DNA sequencing data suggest that TEM-26 is derived from TEM-1.

Bacteria↗
PubMed1992

Identification of amino acid substitutions that alter the substrate specificity of TEM-1 beta-lactamase.

TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins. To identify other amino acid substitutions that alter the activity of TEM-1 towards extended-spectrum cephalosporins, we probed regions around the active-site pocket by random-replacement mutagenesis. This mutagenesis technique involves randomizing the DNA sequence of three to six codons in the blaTEM-1 gene to form a library containing all or nearly all of the possible substitutions for the region randomized. In total, 20 different residue positions that had been randomized were screened for amino acid substitutions that increased enzyme activity towards the extended-spectrum cephalosporin cefotaxime. Substitutions at positions 104, 168, and 238 in the TEM-1 beta-lactamase that resulted in increased enzyme activity towards extended-spectrum cephalosporins were found. In addition, small deletions in the loop containing residues 166 to 170 drastically altered the substrate specificity of the enzyme by increasing activity towards extended-spectrum cephalosporins while virtually eliminating activity towards ampicillin.

Amino Acid Sequence↗
PubMed1983

Enzymes involved in oxygen detoxification during development of Drosophila melanogaster.

Activities of superoxide dismutase (SOD), catalase (CAT), and peroxidases (PER) were examined at 24-h intervals during Drosophila development. SOD activities show a U-shaped curve from egg to adult stages whereas CAT and PER are consistently low in egg through larval stages. Male and female larvae have similar activities of SOD, CAT, and PER whereas male adults have elevated activities of these enzymes. Larvae are more sensitive to H2O2 and 3-amino-1,2,4-triazole (an inhibitor of CAT) than adults.

Aging↗