Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0276640 (TEM)
 20,729 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CAZ-2, CAZ-6, and CAZ-7 are plasmid-mediated beta-lactamases that are markedly active against ceftazidime. The corresponding structural genes were amplified by the polymerase chain reaction. Nucleotide sequences were determined by direct sequencing of the amplified products. Analysis of the nucleotide and the deduced amino acid sequences showed that CAZ-2, CAZ-6, and CAZ-7 are derived from TEM-2 by three, four, and two amino acid substitutions, respectively. All these substitutions are located at positions 102, 162, 235, 236, and 237 (Sutcliffe numbering), which are known to extend the substrate range of beta-lactamases. These substitutions are Lys-102, Ser-162, and Ser-236 in CAZ-2; Lys-102, Ser-162, Thr-235, and Lys-237 in CAZ-6; and Lys-102 and His-162 in CAZ-7. These results indicate that the nucleotide sequence of CAZ-2 is identical to that of TEM-8. The nucleotide sequence of CAZ-7 possesses the two mutations described in TEM-16 by the oligotyping method. In contrast, the combination of mutations encountered in CAZ-6 has not yet been described, and this enzyme was designated TEM-24.
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PMID:Nucleotide sequences of CAZ-2, CAZ-6, and CAZ-7 beta-lactamase genes. 141 73

The nucleotide sequence of PSE-2 beta-lactamase, an enzyme that readily hydrolyzes both carbenicillin and oxacillin, has been determined. The deduced sequence of 266 amino acids contained 93 residues identical to those of OXA-2 beta-lactamase and the Ser-Thr-Phe-Lys tetrad also found in the active site of TEM-1 beta-lactamase.
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PMID:Sequence of PSE-2 beta-lactamase. 312 5

Oligonucleotide-directed site-specific mutagenesis was used to study the structure-function relationship of the positively charged amino terminus of the Escherichia coli outer membrane protein OmpA signal peptide. Mutations were isolated which reduced the overall charge of the amino-terminal region from +2 (wild type) to +1, 0, and -1, as well as one mutation from Thr to Ser at position 4. DNA encoding the wild type and mutant OmpA signal peptides was then fused in-frame to DNA encoding the mature regions of Staphylococcus aureus nuclease A and TEM beta-lactamase. In the case of both the beta-lactamase and nuclease fusions, normal processing was no longer observed when the charge at the amino terminus was reduced to zero or made negative. Differences between the two hybrid proteins were observed in the case of the Thr to Ser mutation. As expected, this mutation had no effect on the beta-lactamase hybrid; however, the processing rate of the nuclease hybrid protein was reduced to nearly one-half. Furthermore, this effect was essentially reversed when a Lys residue at position 3 was deleted. A model is presented which explains the differing effects of a signal peptide mutation on the secretion of different hybrid proteins based on kinetic differences in the translocation of the nuclease and beta-lactamase proteins.
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PMID:Modulation of the effects of mutations in the basic region of the OmpA signal peptide by the mature portion of the protein. 329 25

A new extended-spectrum beta-lactamase was detected in a lactose-positive Salmonella enterica subsp. enterica strain that caused a nosocomial outbreak involving eight patients in a pediatric cardiology unit. This strain showed high levels of resistance to ceftazidime and aztreonam and relatively low levels of resistance to cefotaxime and ceftriaxone. Resistance was associated with a conjugative plasmid of 59 kb, which encoded a new beta-lactamase with an isoelectric point of 5.9 that strongly hydrolyzed ceftazidime and to a much lesser extent hydrolyzed cefotaxime. The enzyme activity was inhibited by clavulanate. The corresponding bla gene was cloned and sequenced. The deduced amino acid sequence showed three significant amino acid replacements with respect to the TEM-1 sequence: Arg-164-->His, Glu-240-->Lys, and Thr-265-->Met. This combination is unique among extended-spectrum beta-lactamases and served to characterize the new enzyme, TEM-27.
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PMID:New extended-spectrum TEM-type beta-lactamase from Salmonella enterica subsp. enterica isolated in a nosocomial outbreak. 772 15

A gene, pkn2, encoding a Myxococcus xanthus protein with significant similarities to eukaryotic protein serine/threonine kinases, was cloned using the polymerase chain reaction. The open reading frame for the protein, beginning with a GUG initiation codon, consists of 830 amino acids. The amino-terminal 279 residues show 37% identity to catalytic domain of Pkn1, another protein serine/threonine kinase expressed during the development at the onset of sporulation. The catalytic domain of Pkn2 contains 27% and 25% identity to rat Ca2+/calmodulin-dependent protein kinase and Bos taurus rhodopsin kinase, respectively. In the middle of the carboxy-terminal regulatory domain, there is a typical transmembrane domain consisting of 18 hydrophobic residues. The gene product, Pkn2, produced in Escherichia coli under a T7 promoter was phosphorylated at both serine and threonine residues. TEM-beta-lactamase produced in E. coli was found to serve as an effective substrate for Pkn2, phosphorylated only at threonine residues, shifting its apparent molecular mass from 29 to 44 kD. The phosphorylated beta-lactamase was unable to be secreted into the periplasmic space and localized in the cytoplasmic and membrane fractions. Analysis of phoA fusions with pkn2 demonstrated that Pkn2 is a transmembrane protein with the kinase domain in the cytoplasm and the 207-residue carboxy-terminal domain outside the cytoplasmic membrane. Disruption of pkn2 showed no effect on vegetative growth but reduced the yield of myxospores by 30%-50%. On the basis of the present results, we propose that Pkn2 is a transmembrane protein serine/threonine kinase that regulates the activity of endogenous beta-lactamase or related enzymes in response to an external signal yet to be identified.
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PMID:Myxococcus xanthus, a gram-negative bacterium, contains a transmembrane protein serine/threonine kinase that blocks the secretion of beta-lactamase by phosphorylation. 777 14

Residue 104 is frequently mutated from a glutamic acid to a lysine in the extended-spectrum TEM beta-lactamases responsible for the resistance to third-generation cephalosporins in clinical Gram negative strains. Among class A beta-lactamases, it is the most variable residue within a highly conserved loop which delineates one side of the active site of the enzymes. To investigate the role of this residue in the extended-spectrum phenotype, it has been replaced by serine, threonine, lysine, arginine, tyrosine and proline. All these substitutions yield active enzymes, with no drastic changes in kinetic properties compared with the wild-type enzyme, except with cefaclor, but an overall improved affinity for second- and third-generation cephalosporins. Only mutant E104K exhibits a significant ability to hydrolyse cefotaxime. Molecular modelling shows that the substitutions have generally no impact on the conformation of the 101-111 loop as the side chains of residues at position 104 are all turned towards the solvent. Unexpectedly, the E104P mutant turns out to be the most efficient enzyme. All our results argue in favour of an indirect role for this residue 104 in the substrate specificity of the class A beta-lactamases. This residue contributes to the precise positioning of residues 130-132 which are involved in substrate binding and catalysis. Changing residue 104 could also modify slightly the local electrostatic potential in this part of the active site. The limited kinetic impact of the mutations at this position have to be analysed in the context of the microbiological problem of resistance to third-generation cephalosporins. Although mutation E104K improves the ability of the enzyme to hydrolyse these compounds, it is not sufficient to confer true resistance, and is always found in clinical isolates associated with at least one mutation at another part of the active site. It is the combined effect of the two mutations that synergistically enhances the hydrolytic capability of the enzyme towards third-generation cephalosporins.
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PMID:Multiple substitutions at position 104 of beta-lactamase TEM-1: assessing the role of this residue in substrate specificity. 782 50

From sequence alignments, two groups can be defined for the carbenicillin-hydrolysing beta-lactamases (CARB enzymes). One group includes the Pseudomonas-specific enzymes PSE-1, PSE-4, CARB-3, CARB-4 and also the Proteus mirabilis GN79, for which the well-conserved residue Lys 234 in all class-A beta-lactamases is changed to an arginine residue. The second group includes the enzymes PSE-3 and AER-1 which have an arginine or a lysine residue at position 165. All these enzymes also have leucine at position 68, threonine at position 104 and glycine at position 240. We engineered these mutations into the TEM-1 beta-lactamase to study their potential role in defining the substrate profile of the CARB enzymes. The mutations K234R and E240G in TEM-1 noticeably increased the hydrolysis of carboxypenicillins relative to other penicillins by approximately sixfold and twofold, respectively. The variant E240G also demonstrated an improved rate of second-generation cephalosporin and cefotaxime hydrolysis. In contrast, the substitution of Trp165 by arginine does not extend the substrate profile to alpha-carboxypenicillins nor does it noticeably modify the kinetic behavior of the enzyme. The mutations M68L and E104T do not have a large effect on the hydrolysis rate but the mutation E104T enhances the affinity of the enzyme for third-generation cephalosporins. As the mutation K234R resulted in a severe decrease in the affinity for carboxypenicillins, the double mutant E240G/K234R was constructed in an attempt to enhance the CARB character of the enzyme. Contrary to what could be expected, the additional mutation E240G for the TEM-1 K234R enzyme increases neither the catalytic constant for the carboxypenicillins nor the affinity towards these substrates. Consequently, this study strongly suggests that the three-dimensional structures of the active site of the TEM-1 enzyme and PSE-3, PSE-4 or other related enzymes are significantly different. This probably explains the discrepancy of the substrate profile between the CARB enzymes and the TEM-1 protein variants.
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PMID:Site-directed mutagenesis of beta-lactamase TEM-1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes. 822 51

A clinical Escherichia coli strain highly resistant to the combinations of amoxicillin-clavulanate, ampicillin-sulbactam, and piperacillin-tazobactam was isolated from a patient with a community-acquired urinary tract infection who was previously treated with amoxicillin-clavulanate. These resistances were carried by a 45-kb conjugative plasmid encoding for a single beta-lactamase with a pI of 5.4. Cloning and sequencing of the new beta-lactamase, IRT-3, revealed identity with the blaT1 gene encoding the TEM-1 beta-lactamase except for a replacement of the methionine residue at position 67 by isoleucine and of the methionine residue at position 180 by threonine. Both mutations were segregated by the construction of hybrid genes, and only the mutation at methionine at position 67 was related to resistance to the suicide inhibitors. The inhibitory effects of clavulanate, sulbactam, and tazobactam on the TEM-1 enzyme were substantially decreased in comparison with those on IRT-3, as indicated by the 50% inhibitory concentrations.
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PMID:Characterization of a new TEM-type beta-lactamase resistant to clavulanate, sulbactam, and tazobactam in a clinical isolate of Escherichia coli. 825 23

Among Escherichia coli organisms isolated at St. Thomas's Hospital during the years 1990 to 1994, the frequency of resistance to amoxicillin-clavulanic acid (tested by disk diffusion in a ratio of 2:1) remained constant at about 5% of patient isolates (10 to 15% of the 41 to 45% that were amoxicillin resistant). Mechanisms of increased resistance were determined for 72 consecutively collected such amoxicillin-clavulanic acid-resistant isolates. MICs of the combination were 16-8 micrograms/ml for 51 (71%) of these and > or = 32-16 micrograms/ml for the remainder. The predominant mechanism was hyperproduction of enzymes isoelectrically cofocusing with TEM-1 (beta-lactamase activities, > 200 nmol of nitrocefin hydrolyzed per min per mg of protein) which was found in 44 isolates (61%); two isolates produced smaller amounts (approximately 150 nmol/min/mg) of such enzymes, and two isolates hyperproduced enzymes cofocusing with TEM-2. Eleven isolates produced enzymes cofocusing with OXA-1 beta-lactamase, which has previously been associated with resistance to amoxicillin-clavulanic acid. Ten isolates produced increased amounts of chromosomal beta-lactamase, and four of these additionally produced TEM-1 or TEM-2. Three isolates produced inhibitor-resistant TEM-group enzymes. In one of the enzymes (pI, 5.4), the amino acid sequence change was Met-67-->Val, and thus the enzyme is identical to TEM-34. Another (pI, 5.4) had the substitution Met-67-->Ile and is identical to IRT-I67, which we propose now be given the designation TEM-40. The third (pI, 5.2) had the substitution Arg-241-->Thr; this enzyme has not been reported previously and should be called TEM-41. The rarity and diversity of inhibitor-resistant TEM-group enzymes suggest that they are the result of spontaneous mutations that have not yet spread.
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PMID:Incidence and mechanisms of resistance to the combination of amoxicillin and clavulanic acid in Escherichia coli. 858 29

Escherichia coli TUH12191, which is resistant to piperacillin, cefazolin, cefotiam, ceftizoxime, cefuzonam, and aztreonam but is susceptible to cefoxitin, latamoxef, flomoxef, and imipenem, was isolated from the urine of a patient treated with beta-lactam antibiotics. The beta-lactamase (Toho-1) purified from the bacteria had a pI of 7.8, had a molecular weight of about 29,000, and hydrolyzed beta-lactam antibiotics such as penicillin G, ampicillin, oxacillin, carbenicillin, piperacillin, cephalothin, cefoxitin, cefotaxime, ceftazidime, and aztreonam. Toho-1 was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid and tazobactam. Resistance to beta-lactams, streptomycin, spectinomycin, sulfamethoxazole, and trimethoprim was transferred by conjugational transfer from E. coli TUH12191 to E. coli ML4903, and the transferred plasmid was about 58 kbp, belonging to incompatibility group M. The cefotaxime resistance gene for Toho-1 was subcloned from the 58-kbp plasmid by transformation of E. coli MV1184. The sequence of the gene for Toho-1 was determined, and the open reading frame of the gene consisted of 873 or 876 bases (initial sequence, ATGATG). The nucleotide sequence of the gene (DDBJ accession number D37830) was found to be about 73% homologous to the sequence of the gene encoding a class A beta-lactamase produced by Klebsiella oxytoca E23004. According to the amino acid sequence deduced from the DNA sequence, the precursor consisted of 290 or 291 amino acid residues, which contained amino acid motifs common to class A beta-lactamases (70SXXK, 130SDN, and 234KTG). Toho-1 was about 83% homologous to the beta-lactamase mediated by the chromosome of K. oxytoca D488 and the beta-lactamase mediated by the plasmid of E. coli MEN-1. Therefore, the newly isolated beta-lactamase Toho-1 produced by E. coli TUH12191 is similar to beta-lactamases produced by K. oxytoca D488, K. oxytoca E23004, and E. coli MEN-1 rather than to mutants of TEM or SHV enzymes. Toho-1 has shown the highest degree of similarity to K. oxytoca class A beta-lactamase. Detailed comparison of Toho-1 with other beta-lactamases implied that replacement of Asn-276 by Arg with the concomitant substitution of Thr for Arg-244 is an important mutation in the extension of the substrate specificity.
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PMID:Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A beta-lactamase isolated from Escherichia coli. 1118 30


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