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Query: UMLS:C0276640 (TEM)
 20,729 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

On the basis of the biophysical studies on the synthetic mutant (Ile-8----Asn) OmpA signal peptide in the preceding paper (Hoyt, D. C., and Gierasch, L.M. (1991) J. Biol. Chem. 266, 14406-14412), the in vivo effects of the same mutation were examined by fusing the mutant OmpA signal sequence to Staphylococcus aureus nuclease or TEM beta-lactamase. The mutation in which the isoleucine residue at position 8 of the OmpA signal sequence of Escherichia coli was replaced with a neutral polar residue, asparagine, resulted in a defective signal peptide. The mutant signal sequence was unable to be processed, and the precursor molecule accumulated in the cytoplasmic as well as in the membrane fractions, indicating that the Ile-8----Asn OmpA signal sequence is not competent for translocating nuclease A or beta-lactamase across the membrane. This result is consistent with the in vitro studies on the Ile-8----Asn OmpA signal peptide, which indicated that the mutant signal peptide was unable to penetrate into the hydrophobic core of the lipid bilayer. Other asparagine or glutamine substitution mutations in the hydrophobic region of the OmpA signal sequence were also examined. Interestingly, the OmpA signal sequence with either Ile-8----Gln, Val-10----Asn, or Leu-12----Asn mutation was completely defective as the Ile-8----Asn OmpA signal sequence, while the Ile-6----Asn and Ala-9----Asn OmpA nucleases were able to be processed to secrete nuclease, although the processing occurred at a much slower rate than the wild-type OmpA nuclease. These results indicate that the defects depend on the position of the lesion in the hydrophobic core of the OmpA signal sequence.
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PMID:In vivo effect of asparagine in the hydrophobic region of the signal sequence. 186 Aug 48

The hydrophobic region of the signal peptide of the OmpA protein of the Escherichia coli outer membrane was extensively altered in its hydrophobicity and predicted secondary structure by site-specific mutagenesis. The mutated signal peptides were fused to nuclease A from Staphylococcus aureus, and the function of the signal peptide was examined by measuring the rate of processing of the signal peptide. Six of the 12 mutated signal peptides in the nuclease hybrid were processed faster than the wild-type. In particular, the processing of the mutated signal peptide in which the alanine residue at position 9 was substituted with a valine residue was enhanced almost twofold over the processing of the wild-type signal peptide. In addition, the production of nuclease A fused with this mutated signal peptide also increased twofold. However, these effects were not observed when the mutated signal peptide was fused to TEM beta-lactamase. Analysis of the present mutations suggests that both overall hydrophobicity and distinct structural requirements in the hydrophobic region have important roles in signal peptide function.
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PMID:Enhancement of protein translocation across the membrane by specific mutations in the hydrophobic region of the signal peptide. 240 17

The reactions of a set of amino acid and peptidyl C10-esters of deacetylcephalothin (1-5) have been examined with purified enzymes in vitro. Each of the compounds examined is a substrate for the Escherichia coli TEM-2 beta-lactamase, and enzyme-catalyzed hydrolysis of the lactam bond gives release of an amino acid or a peptidyl fragment from a cephem nucleus. 7 beta-(2-Thienylacetamido)-3-[[(beta-chloro-L-alanyl)oxy]methyl]-3- cephem-4-carboxylate (4) gives time-dependent inactivation of E. coli JSR-O alanine racemase in a process that requires beta-lactamase for the initial liberation of beta-chloro-L-alanine from the cephalosporin. Alanine racemase is similarly inactivated by 7 beta-(2-thienylacetamido)-3-[[[(beta-chloro-L-alanyl)-beta-chloro- L- alanyl]oxy]methyl]-3-cephem-4-carboxylate (1), but this inhibition requires the sequential action of both beta-lactamase and alanine aminopeptidase. Analysis of the enzymatic transformations of 7 beta-(2-thienylacetamido)-3-[[[(beta-chloro-L-alanyl)-L- alanyl]oxy]methyl]-3-cephem-4-carboxylate (3), monitored by high-field 1H NMR, reveals that (1) beta-lactamase releases the dipeptide beta-chloro-L-alanyl-L-alanine from 3 and (2) leucine aminopeptidase effects stoichiometric hydrolysis of the dipeptide to beta-chloro-L-alanine and L-alanine. These biochemical findings are discussed with reference to the mechanism of antibacterial action of 1 against beta-lactamase-producing, penicillin-resistant microorganisms [Mobashery, S., Lerner, S. A., & Johnston, M. (1986) J. Am. Chem. Soc. 108, 1685].
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PMID:Inactivation of alanine racemase by beta-chloro-L-alanine released enzymatically from amino acid and peptide C10-esters of deacetylcephalothin. 311 51

Oligonucleotide-directed site-specific mutagenesis was used to systematically shorten the hydrophobic region within the signal peptide of the Escherichia coli outer membrane protein OmpA. DNA encoding the wild type and mutant OmpA signal peptides were then fused in frame to DNA encoding the mature regions of Staphylococcus aureus nuclease A and TEM beta-lactamase. The ability of these signal peptides to direct processing of the resulting hybrid proteins was dependent on both their length and the protein to which they were fused. Deletion of two or more residues progressively slowed processing of pro-OmpA-nuclease. By contrast, pro-OmpA-beta-lactamase was less sensitive to the length of the hydrophobic region than to the nature of the deleted residue(s). Deletion of an Ala residue tended to reduce processing efficiency of pro-OmpA-beta-lactamase, while deletion of an Ile residue, together with the Ala residue, resulted in improvement. The loss of either 3 or 4 residues abolished processing of both hybrids. These data indicate that both the length as well as the identity of residues in the hydrophobic region are important. The relative importance of these two factors depends on the mature region of the protein being secreted.
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PMID:The differential effect on two hybrid proteins of deletion mutations within the hydrophobic region of the Escherichia coli OmpA signal peptide. 354 10

From the crystal structure of the Bacillus licheniformis 749/C beta-lactamase, energy-minimized structures for the precatalytic, the acyl-enzyme intermediate, and the acylated linear inactivating species for sulbactam--a clinically useful mechanism-based inactivator for class A beta-lactamases--were generated. The effect of individual Ser-235-Ala and Arg244-Ser point mutations on the inactivation and turnover processes was consistent with the existence of hydrogen bonds between the side chains of these residues and the sulbactam species. The departure of the sulfinate leaving group from the acyl-enzyme intermediate of sulbactam is believed to be a prerequisite for the inactivation process. In order to explore the influence of the leaving group, penicillanic acid (2), penicillanic acid alpha-S-oxide (3), and penicillanic acid beta-S-oxide (4) were synthesized and studied in kinetic experiments with the TEM-1 beta-lactamase. Penicillanic acid is only a substrate, but penicillanic acid S-oxides were both substrates and inactivators for the enzyme. An argument is presented to rationalize these observations on the basis of the leaving ability of thiolate, sulfenate, and sulfinate from the acyl-enzyme intermediates of penicillanic acid (2), the penicillanic acid S-oxides (3 and 4), and sulbactam, respectively. The departure of the leaving group does not appear to be rate limiting in the inactivator process, but is an indispensable component of the irreversible inactivation of the enzyme. Molecular dynamics calculations of the putative inactivating species suggest that Lys-73, Lys-234, and Ser-130 are three likely residues that may be modified in the course of the inactivation chemistry. A discussion is presented of the mechanism of formation of the transiently inhibited enzyme species, which comes about as a consequence of the tautomerization of the double bond of the inactivating iminium moiety. In addition, the mechanistic details presented for sulbactam are compared and contrasted with those of clavulanic acid, another clinically used inactivator for class A beta-lactamases.
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PMID:A structure-based analysis of the inhibition of class A beta-lactamases by sulbactam. 818 Jan 99

The mutant 554 of TEM-2 beta-lactamase was selected for a decrease in the resistance to carbenicillin of an Escherichia coli K12 carrier. The amino acid sequence of the mutant beta-lactamase was determined by manual Edman degradation analysis of proteolytic peptides. A single substitution Val for Ala was localized at position 237. The mutant exhibited only 2% of the catalytic efficiency of the wild-type enzyme towards carbenicillin and ticarcillin, whereas it retained 30-60% of the hydrolytic activity towards other penicillin and cephalosporin substrates. Carfecillin, the phenyl ester of the side-chain carboxyl group of carbenicillin, was hydrolysed as a good substrate. This suggests that the behaviour of the mutant enzyme towards carbenicillin may result from ionic rather than steric constraints. A molecular model of the Val-237 TEM-2 mutant suggests possible electrostatic interaction between Glu-171 and the carboxylic group of the side chain of carbenicillin.
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PMID:Val-237 for Ala substitution in the TEM-2 beta-lactamase dramatically alters the catalytic efficiencies towards carbenicillin and ticarcillin. 820 May 9

The role of Ser-235 in the catalytic mechanism of the TEM-1 beta-lactamase has been explored by the study of a mutant enzyme in which Ser-235 has been substituted by alanine (Ala-235 mutant enzyme). A comparative kinetic analysis of both the wild-type and the Ala-235 TEM-1 enzymes revealed little effect of this substitution of residue 235 on the turnover of penicillins but a greater effect on the turnover of cephalosporins. Susceptibility testing of Escherichia coli strains harboring the wild-type TEM-1 beta-lactamase and the Ala-235 mutant enzyme revealed an effect of the mutation similar to that observed in the enzymological studies. The MICs of two representative cephalosporins for the strain containing the mutant enzyme were much lower than those for the isogenic strain bearing the wild-type TEM-1 beta-lactamase. On the other hand, the strain with the mutant enzyme was still highly resistant to penicillins.
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PMID:Critical hydrogen bonding by serine 235 for cephalosporinase activity of TEM-1 beta-lactamase. 828 30

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.
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PMID:Selection of functional signal peptide cleavage sites from a library of random sequences. 830 May 11

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.
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PMID:Selection and characterization of amino acid substitutions at residues 237-240 of TEM-1 beta-lactamase with altered substrate specificity for aztreonam and ceftazidime. 879 21

A clinical strain of Pseudomonas aeruginosa, PAe1100, was found to be resistant to all antipseudomonal beta-lactam antibiotics and to aminoglycosides, including gentamicin, amikacin, and isepamicin. PAe1100 produced two beta-lactamases, TEM-2 (pI 5.6) and a novel, TEM-derived extended-spectrum beta-lactamase called TEM-42 (pI 5.8), susceptible to inhibition by clavulanate, sulbactam, and tazobactam. Both enzymes, as well as the aminoglycoside resistance which resulted from AAC(3)-IIa and AAC(6')-I production, were encoded by an 18-kb nonconjugative plasmid, pLRM1, that could be transferred to Escherichia coli by transformation. The gene coding for TEM-42 had four mutations that led to as many amino acid substitutions with respect to TEM-2: Val for Ala at position 42 (Ala42), Ser for Gly238, Lys for Glu240, and Met for Thr265 (Ambler numbering). The double mutation Ser for Gly238 and Lys for Glu240, which has so far only been described in SHV-type but not TEM-type enzymes, conferred concomitant high-level resistance to cefotaxime and ceftazidime. The novel, TEM-derived extended-spectrum beta-lactamase appears to be the first of its class to be described in P. aeruginosa.
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PMID:A TEM-derived extended-spectrum beta-lactamase in Pseudomonas aeruginosa. 891 51


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