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)

A five-step procedure has been developed for purifying Pitton's type 2 plasmid-mediated beta-lactamase (PIT-2, also called SHV-1) from cultures of a hyperproducing variant of an Escherichia coli K12 strain carrying the plasmid p453. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a single protein band with a molecular weight of about 27,500. The amino acid composition of the protein and the amino acid sequence of the NH2-terminal region have been determined. PIT-2 enzyme contains 272 amino acid residues with 2 cysteines. Studies of the S-carboxymethylated protein (previously reduced or unreduced) suggest that these two residues are presumably in the form of free sulphydryl groups in the native protein. Conversely, TEM-2 beta-lactamase contains 2 cysteine residues which are in the form of a disulphide bond. Comparison of PIT-2 with other beta-lactamases was made using the difference index (DI) of Metzger et al. The PIT-2 enzyme appeared more closely related to the TEM-type penicillinases (DI of 6.5).
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PMID:Purification and biochemical properties of Pitton's type 2 beta-lactamase (SHV-1). 332 41

Both a penicillinase and a cephalosporinase are present in a strain of Citrobacter freundii (GN7391) resistant to beta-lactam antibiotics. The penicillinase was identical to the type Ia penicillinases (Type III by Richmond classification), mediated by Rms212 and R-TEM. A cephalosporinase, typical of enterobacteriaceae chromosomal beta-lactamase (Type I by Richmond classification), was purified from the strain. It gave a single protein band on polyacrylamide gel electrophoresis and immunoelectrophoresis; the pI was 8.6 and its molecular weight was approximately 38 000. Cysteine was not found among its amino acids. The specific activity was 388 units (mg protein)-1 for the hydrolysis of cephaloridine, and the optimal pH was 8.0. Rabbit antiserum obtained against the purified enzyme showed cross-reaction with cephalosporinases produced by strains of Enterobacter cloacae in a neutralization test.
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PMID:Purification and properties of chromosomally mediated beta-lactamase from Citrobacter freundii GN7391. 697 12

Two blaTEM-like genes were characterized that encoded IRT beta-lactamases (previously called TRI) in clinical isolates of Escherichia coli resistant to amoxycillin alone and to combinations of amoxycillin with beta-lactamase inhibitors. Plasmids carrying this resistance were isolated from E. coli K 12 transconjugants and the genes were sequenced after amplification of defined fragments, using TEM-1-specific primers. The gene for IRT-1 beta-lactamase resembled the blaTEM-1B gene, and that for IRT-2 resembled blaTEM-2. However, both IRT enzymes have a glutamine residue at position 37, which is characteristic of TEM-1. The unique nucleotide difference with parental genes corresponding to amino acid variation was observed at nucleotide position 929. The consequence of C to T transition in the blaIRT-1 gene and C to A transversion in the blaIRT-2 gene was the substitution of arginine 241 in the native protein by cysteine and serine, respectively, in the mutants. Thus, the nature of amino acid 241 is critical in conferring resistance or susceptibility to beta-lactamase inhibitors. Furthermore, these basic to neutral amino acid replacements explain the more acidic pI (pI = 5.2) of these IRT enzymes compared to that of TEM-1 (pI = 5.4). The presence of cysteine-241 in IRT-1 also explains the selective sensitivity of this beta-lactamase to inhibition by p-chloromercuribenzoate.
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PMID:Nucleotide sequences of the genes coding for the TEM-like beta-lactamases IRT-1 and IRT-2 (formerly called TRI-1 and TRI-2). 805 97

Similar to other proteins of the periplasm of Escherichia coli, TEM 1 beta-lactamase contains only a single disulfide bond. It can fold to its native conformation in both the presence and the absence of this disulfide bond. The GdmC1-dependent equilibrium unfolding of beta-lactamase in vitro is well described by a N reversible I reversible U three-state model in which the native protein (N) first reacts to an intermediate of the molten globule type (I) and then to the unfolded state (U). We find that the disulfide bond of beta-lactamase stabilizes I relative to U, but does not change the stability of N relative to I. The I reversible U transition is an extremely rapid reaction for both reduced and oxidized beta-lactamase, but the N reversible I folding kinetics are slow and identical in the presence and the absence of the disulfide bond. This insensitivity of the N reversible I equilibrium and kinetics suggests that the region around the disulfide bond is already native-like folded and is presumably buried in the intermediate I, prior to the slow and rate-limiting events of folding. This was confirmed by measuring the stability of the disulfide bond, which, to a first approximation, is identical in N and I. In native, reduced beta-lactamase, the thiol groups are inaccessible for oxidation by DsbA, but at the stage of the molten globule intermediate I oxidation is still possible, because I is in fast exchange with the unfolded protein U. The introduction of the disulfide bond into beta-lactamase by DsbA competes with conformational folding at the stage of the final slow steps in the folding of the reduced protein. The major problem in the oxidation of proteins with one or two disulfide bonds (such as beta-lactamase) is not the formation of incorrect disulfide bonds, but the premature burial of the thiol groups by the rapid conformational folding of the reduced protein. DsbA, the major thiol/ disulfide isomerase of the bacterial periplasm, meets this problem. It is a very strong oxidant, and its reaction with cysteine residues in unfolded proteins is extremely fast.
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PMID:Competition between DsbA-mediated oxidation and conformational folding of RTEM1 beta-lactamase. 878 94

Class A beta-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 beta-lactamase, this bond was removed by introducing a Cys-77-->Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N<-->H<--U). Both the folded mutant protein (N) and, to a lesser extent the thermodynamically stable intermediate, H. were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.
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PMID:Kinetic and thermodynamic consequences of the removal of the Cys-77-Cys-123 disulphide bond for the folding of TEM-1 beta-lactamase. 902 Aug 74

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.
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PMID:Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy. 1179 Jan 1

Hydrogen peroxide and hydroxyl-free radicals determine a diffuse aggregation of porcine fumarase and a loss of its enzymatic activity. In this study, hydroxyl-free radicals were generated "in situ" by irradiation with ultrasound (US) at 38 kHz. The structural characteristics of aggregated fumarase were studied using circular dichroism spectroscopy (CD) and steady state fluorescence spectroscopy. Enzyme aggregation is caused by the formation of intermolecular disufide bridges, originated by the oxidation of cysteine residues, together with a diffuse increase in beta-turn in the protein's secondary structure. These conformational changes lead to a fibrous, amyloid-like aggregation which appears ordered and regular under TEM microscopy.
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PMID:Low frequency ultrasound induces aggregation of porcine fumarase by free radicals production. 1545 Mar 73

This study deals with the synthesis of cysteine capped gold nanoparticles with an average size of 12 nm by borohydride reduction and spectroscopic identification of SAu interaction. We have studied the interaction of thiol with gold nanoparticles in aqueous medium by employing UV-vis, Raman, NMR, and FT-IR spectroscopy. The shifting of gold plasmon resonance in the UV-vis spectra shows the stabilization of gold nanoparticles by cysteine. The disappearance of S-H stretching in both the IR and Raman spectra and the shifting of the NMR signals of the protons in close proximity to the metal center supported the existence of the S-Au interaction in cysteine capped gold nanoparticles. The TEM images shows cysteine capped gold nanoparticles as distinct and spherical entities as compared to free colloidal gold nanoparticles.
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PMID:Spectroscopic identification of S-Au interaction in cysteine capped gold nanoparticles. 1595 26

The increasing emergence of new bacterial beta-lactamases that can efficiently hydrolyze beta-lactam antibiotics to clinically inactive carboxylic acids has created an intractable problem in the treatment of bacterial infections, and it is highly desirable to develop a useful tool that can rapidly screen bacteria for beta-lactamases against a variety of antibiotic candidates in a high-throughput manner. This paper describes the use of a fluorescein-labeled beta-lactamase mutant (E166Cf) as a convenient fluorescent tool to screen beta-lactamases, including the Bacillus cereus beta-lactamase I (PenPC), B. cereus beta-lactamase II, Bacillus licheniformis PenP, Escherichia coli TEM-1, and Enterobacter cloacae P99 against various beta-lactam antibiotics (penicillin G, penicillin V, ampicillin, cefuroxime, cefoxitin, moxalactam, cephaloridine), using a 96-well microplate reader. The E166Cf mutant was constructed by replacing Glu166 on the flexible Omega-loop, which is close to the enzyme's active site, with a cysteine residue on a class A beta-lactamase (B. cereus PenPC) and subsequently labeling the mutant with thiol-reactive fluorescein-5-maleimide. Such modifications significantly impaired the hydrolytic activity of the E166Cf mutant compared to that of the wild-type enzyme. The fluorescence intensity of the E166Cf mutant increases in the presence of beta-lactam antibiotics. For antibiotics that are resistant to hydrolysis by the E166Cf mutant (cefuroxime, cefoxitin, moxalactam), the fluorescence signal slowly increases until it reaches a plateau. For antibiotics that can be slowly hydrolyzed by the E166Cf mutant (penicillin G, penicillin V, ampicillin), the fluorescence signal rapidly increases to the plateau and then declines after a prolonged incubation. The E166Cf mutant retains its characteristic pattern of fluorescence signals in the presence of both bacterial beta-lactamases and beta-lactamase-resistant antibiotics. In contrast, in the presence of both bacterial beta-lactamases and beta-lactamase-sensitive antibiotics, the fluorescence signals of the E166Cf mutant were decreased. The fluorescence signals from the E166Cf mutant allow an unambiguous differentiation of beta-lactamase-resistant antibiotics from beta-lactamase-sensitive ones in the screening of bacterial beta-lactamases against a panel of antibiotic candidates. This simple method may provide an alternative tool in choosing potent beta-lactam antibiotics for treatment of bacterial infections.
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PMID:Fluorescein-labeled beta-lactamase mutant for high-throughput screening of bacterial beta-lactamases against beta-lactam antibiotics. 1609 68

The production of class A beta-lactamases is a major cause of clinical resistance to beta-lactam antibiotics. Some of class A beta-lactamases are known to have a disulfide bridge. Both narrow spectrum and extended spectrum beta-lactamases of TEM and the SHV enzymes possess a disulfide bond between Cys77 and Cys123, and the enzymes with carbapenem-hydrolyzing activity have a well-conserved disulfide bridge between Cys69 and Cys238. We produced A77C/G123C mutant of the extended-spectrum beta-lactamase Toho-1 in order to introduce a disulfide bond between the cysteine residues at positions 77 and 123. The result of 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) titrations confirmed formation of a new disulfide bridge in the mutant. The results of irreversible heat inactivation and circular dichroism (CD) melting experiments indicated that the disulfide bridge stabilized the enzyme significantly. Though kinetic analysis indicated that the catalytic properties of the mutant were quite similar to those of the wild-type enzyme, E. coli producing this mutant showed drug resistance significantly higher than E. coli producing the wild-type enzyme. We speculate that the stability of the enzymes provided by the disulfide bond may explain the wide distribution of TEM and SHV derivatives and explain how various mutations can cause broadened substrate specificity without loss of stability.
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PMID:Effect of disulfide-bond introduction on the activity and stability of the extended-spectrum class A beta-lactamase Toho-1. 1689 32


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