Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Implication of Ile-69 and Thr-182 residues in kinetic characteristics of IRT-3 (TEM-32) beta-lactamase.

ABSTRACT: The substitution of a methionine for an isoleucine at position 69 (Met69Ile), which causes inhibitor resistance to TEM-type beta-lactamases (IRT-3 and IRT-I69), altered the positions of the Asn-170 and Glu-166 side chains as well as the position of the catalytic water molecule. A novel hydrogen bond between the hydroxyl of Thr-182 and the carbonyl of Glu-64 was expected to be responsible for the increase in the catalytic activity of the IST-T182 and IRT-3 enzymes compared with those of TEM-1 and IRT-169, respectively.

SUBMITTER: Farzaneh S

PROVIDER: S-EPMC163551 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Implication of Ile-69 and Thr-182 residues in kinetic characteristics of IRT-3 (TEM-32) beta-lactamase.

Farzaneh S S Chaibi E B EB Peduzzi J J Barthelemy M M Labia R R Blazquez J J Baquero F F

Antimicrobial agents and chemotherapy 19961001 10

The substitution of a methionine for an isoleucine at position 69 (Met69Ile), which causes inhibitor resistance to TEM-type beta-lactamases (IRT-3 and IRT-I69), altered the positions of the Asn-170 and Glu-166 side chains as well as the position of the catalytic water molecule. A novel hydrogen bond between the hydroxyl of Thr-182 and the carbonyl of Glu-64 was expected to be responsible for the increase in the catalytic activity of the IST-T182 and IRT-3 enzymes compared with those of TEM-1 and ...[more]

PMID: 8891161

Similar Datasets

TEM-109 (CMT-5), a natural complex mutant of TEM-1 beta-lactamase combining the amino acid substitutions of TEM-6 and TEM-33 (IRT-5).

Project description:Escherichia coli CF349 exhibited a complex beta-lactam resistance phenotype, including resistance to amoxicillin and ticarcillin alone and in combination with clavulanate and to some extended-spectrum cephalosporins. The double-disk synergy test was positive. CF349 harbored an 85-kb conjugative plasmid which encoded a beta-lactamase of pI 5.9. The corresponding bla gene was identified by PCR and sequencing as a bla(TEM) gene. The deduced protein sequence revealed a new complex mutant of TEM-1 beta-lactamase designated TEM-109 (CMT-5). TEM-109 contained both the substitutions Glu104Lys and Arg164His of the expanded-spectrum beta-lactamase (ESBL) TEM-6 and Met69Leu of the inhibitor-resistant TEM-33 (IRT-5). TEM-109 exhibited hydrolytic activity against ceftazidime similar to that of TEM-6 (k(cat), 56 s(-1) and 105 s(-1), respectively; K(m) values, 226 and 247 microM, respectively). The 50% inhibitory concentrations of clavulanate and tazobactam (0.13 microM and 0.27 microM, respectively) were 5- to 10-fold higher for TEM-109 than for TEM-6 (0.01 and 0.06 microM, respectively) but were almost 10-fold lower than those for TEM-33. The characterization of this novel CMT, which exhibits a low level of resistance to inhibitors, highlights the emergence of this new ESBL type.

| S-EPMC1280126 | biostudies-literature

Molecular characterization of TEM-59 (IRT-17), a novel inhibitor-resistant TEM-derived beta-lactamase in a clinical isolate of Klebsiella oxytoca.

Project description:A clinical isolate of Klebsiella oxytoca (Kox 443) was found to have a low-level resistance to broad-spectrum penicillins (MICs of amoxicillin and ticarcillin, 256 and 32 microg/ml, respectively), without substantial potentiation by 2 microg of clavulanic acid per ml (amoxicillin- and ticarcillin-clavulanate, 128 and 8 microg/ml, respectively), while being fully susceptible to cephalosporins and other beta-lactam antibiotics. These resistances were carried by a ca. 50-kb conjugative plasmid that encodes a single beta-lactamase with a pI of 5.6. Compared to TEM-2, this enzyme exhibited a 3- to 30-fold higher Km and a decreased maximal hydrolysis rate for beta-lactams; higher concentrations of suicide inactivators (5- to 500-fold higher concentrations giving a 50% reduction in hydrolysis) were required for inhibition. Nucleotide sequence analysis revealed identity between the blaTEM gene of Kox 443 and the blaTEM-2 gene, except for a single A-to-G change at position 590, leading to the amino acid change from Ser-130 Gly. This mutation has not been reported previously in the TEM type beta-lactamases produced by clinical strains, and the novel enzyme was called TEM-59 (alternative name IRT-17). This is the first description of an inhibitor-resistant TEM-derived enzyme in the species K. oxytoca.

| S-EPMC89339 | biostudies-literature

TEM-89 beta-lactamase produced by a Proteus mirabilis clinical isolate: new complex mutant (CMT 3) with mutations in both TEM-59 (IRT-17) and TEM-3.

Project description:TEM-89 (CMT-3) is the first complex mutant beta-lactamase produced by a clinical strain of Proteus mirabilis (strain Pm 631). This new enzyme, which has a pI of 6.28, is derived from TEM-3 and has a single amino acid substitution also encountered in TEM-59 (inhibitor-resistant TEM beta-lactamase IRT-17): Ser-130 to Gly. TEM-89 hydrolyzed penicillins to the same extent that TEM-3 did but lost almost all hydrolytic activity for cephalosporins and, like TEM-59, was highly resistant to inhibitors.

| S-EPMC90874 | biostudies-literature

The Role of Rigid Residues in Modulating TEM-1 β-Lactamase Function and Thermostability.

Project description:The relationship between protein motions (i.e., dynamics) and enzymatic function has begun to be explored in β-lactamases as a way to advance our understanding of these proteins. In a recent study, we analyzed the dynamic profiles of TEM-1 (a ubiquitous class A β-lactamase) and several ancestrally reconstructed homologues. A chief finding of this work was that rigid residues that were allosterically coupled to the active site appeared to have profound effects on enzyme function, even when separated from the active site by many angstroms. In the present work, our aim was to further explore the implications of protein dynamics on β-lactamase function by altering the dynamic profile of TEM-1 using computational protein design methods. The Rosetta software suite was used to mutate amino acids surrounding either rigid residues that are highly coupled to the active site or to flexible residues with no apparent communication with the active site. Experimental characterization of ten designed proteins indicated that alteration of residues surrounding rigid, highly coupled residues, substantially affected both enzymatic activity and stability; in contrast, native-like activities and stabilities were maintained when flexible, uncoupled residues, were targeted. Our results provide additional insight into the structure-function relationship present in the TEM family of β-lactamases. Furthermore, the integration of computational protein design methods with analyses of protein dynamics represents a general approach that could be used to extend our understanding of the relationship between dynamics and function in other enzyme classes.

| S-EPMC7999226 | biostudies-literature

Saturation Mutagenesis and Molecular Modeling: The Impact of Methionine 182 Substitutions on the Stability of β-Lactamase TEM-1.

Project description:Serine β-lactamase TEM-1 is the first β-lactamase discovered and is still common in Gram-negative pathogens resistant to β-lactam antibiotics. It hydrolyzes penicillins and cephalosporins of early generations. Some of the emerging TEM-1 variants with one or several amino acid substitutions have even broader substrate specificity and resistance to known covalent inhibitors. Key amino acid substitutions affect catalytic properties of the enzyme, and secondary mutations accompany them. The occurrence of the secondary mutation M182T, called a "global suppressor", has almost doubled over the last decade. Therefore, we performed saturating mutagenesis at position 182 of TEM-1 to determine the influence of this single amino acid substitution on the catalytic properties, thermal stability, and ability for thermoreactivation. Steady-state parameters for penicillin, cephalothin, and ceftazidime are similar for all TEM-1 M182X variants, whereas melting temperature and ability to reactivate after incubation at a higher temperature vary significantly. The effects are multidirectional and depend on the particular amino acid at position 182. The M182E variant of β-lactamase TEM-1 demonstrates the highest residual enzymatic activity, which is 1.5 times higher than for the wild-type enzyme. The 3D structure of the side chain of residue 182 is of particular importance as observed from the comparison of the M182I and M182L variants of TEM-1. Both of these amino acid residues have hydrophobic side chains of similar size, but their residual activity differs by three-fold. Molecular dynamic simulations add a mechanistic explanation for this phenomenon. The important structural element is the V159-R65-E177 triad that exists due to both electrostatic and hydrophobic interactions. Amino acid substitutions that disturb this triad lead to a decrease in the ability of the β-lactamase to be reactivated.

| S-EPMC11276661 | biostudies-literature

The desoxazoline asidiacyclamide analogue cyclo(Gly-Thr-D-Val-Thz-Ile-Thr-D-Val-Thz) acetonitrile monosolvate.

Project description:THE TITLE PEPTIDE [SYSTEMATIC NAME: 4-(butan-2-yl)-7,20-bis-(1-hy-droxy-eth-yl)-10,23-bis-(propan-2-yl)-12,25-dithia-3,6,9,16,19,22,27,28-octa-aza-tricyclo-[22.2.1.1(11,14)]octa-cosa-1(26),11(28),13,24(27)-tetra-ene-2,5,8,15,18,21-hexone acetonitrile monosolvate], C(32)H(48)N(8)O(8)S(2)·CH(3)CN, an analogue of ascidiacyclamide (ASC) [cyclo(-Ile-Oxz-D-Val-Thz-)(2)], lies about a twofold rotation axis, so that the glycine (Gly) and isoleucine (Ile) residues are each disordered over two sites with equal occupancies. The acetonitrile mol-ecule is also located on a twofold axis passing through the C and N atoms. In the peptide, the thia-zole rings are faced to each other with a dihedral angle of 9.63 (15)° and intra-molecular N-H⋯O and O-H⋯O hydrogen bonds are observed. A bifurcated N-H⋯(O,O) hydrogen bond links the peptide mol-ecules into a layer parallel to the ab plane.

| S-EPMC3254412 | biostudies-literature

Properties of IRT-14 (TEM-45), a newly characterized mutant of TEM-type beta-lactamases.

Project description:IRT-14 (TEM-45) is a new mutant TEM-type beta-lactamase that was isolated from clinical Escherichia coli P37 and that confers resistance to broad-spectrum penicillins with reduced sensitivity to beta-lactamase inhibitors. The MICs of amoxicillin alone and of amoxicillin combined with 2 micrograms of clavulanic acid or 2 micrograms of tazobactam per ml were 4,096, 2,048, and 1,024 micrograms/ml, respectively. The strain was susceptible to cephalosporins, aztreonam, moxalactam, and imipenem. The enzyme was purified to homogeneity, and values of the kinetic parameters Kcat, Km, and Kcat/Km were determined for different substrates. This enzyme, with a pI of 5.2, was found to have reduced affinity for broad-spectrum penicillins and cephalosporins. The values of 50% inhibitory concentrations of clavulanic acid, sulbactam, tazobactam, and brobactam are correlated with the higher KmS for substrates. The resistance of E. coli P37 to mechanism-based inactivators results from a higher level of production of the TEM-derived enzyme due to the G-to-T substitution at position 162 (G-162-->T) in the promoter region of blaTEM and from the structural modifications resulting from the Met-69-->Leu and Arg-275-->Gln substitutions that characterize IRT-14 beta-lactamase.

| S-EPMC163716 | biostudies-other

TEM-121, a novel complex mutant of TEM-type beta-lactamase from Enterobacter aerogenes.

Project description:Enterobacter aerogenes clinical isolate LOR was resistant to penicillins and ceftazidime but susceptible to cefuroxime, cephalothin, cefoxitin, cefotaxime, ceftriaxone, and cefepime. PCR and cloning experiments from this strain identified a novel TEM-type beta-lactamase (TEM-121) differing by five amino acid substitutions from beta-lactamase TEM-2 (Glu104Lys, Arg164Ser, Ala237Thr, Glu240Lys, and Arg244Ser) and by only one amino acid change from the extended-spectrum beta-lactamase (ESBL) TEM-24 (Arg244Ser), with the last substitution also being identified in the inhibitor-resistant beta-lactamase IRT-2. Kinetic parameters indicated that TEM-121 hydrolyzed ceftazidime and aztreonam (like TEM-24) and was inhibited weakly by clavulanic acid and strongly by tazobactam. Thus, TEM-121 is a novel complex mutant TEM beta-lactamase (CMT-4) combining the kinetic properties of an ESBL and an inhibitor-resistant TEM enzyme.

| S-EPMC529203 | biostudies-literature

TEM-184, a Novel TEM-Derived Extended-Spectrum ?-Lactamase with Enhanced Activity against Aztreonam.

Project description:TEM-184, a novel TEM-derived extended-spectrum ?-lactamase (ESBL), was isolated from an Escherichia coli ST354 clinical strain. Compared to TEM-1, TEM-184 contains the mutations Q6K, E104K, I127V, R164S, and M182T. Kinetic analysis of this enzyme revealed extended-spectrum activity against aztreonam in particular. TEM-184 was also susceptible to inhibitors, including clavulanic acid, tazobactam, and avibactam.

| S-EPMC6125571 | biostudies-literature

Inversion of the Side-Chain Stereochemistry of Indvidual Thr or Ile Residues in a Protein Molecule: Impact on the Folding, Stability, and Structure of the ShK Toxin.

Project description:ShK toxin is a cysteine-rich 35-residue protein ion-channel ligand isolated from the sea anemone Stichodactyla helianthus. In this work, we studied the effect of inverting the side chain stereochemistry of individual Thr or Ile residues on the properties of the ShK protein. Molecular dynamics simulations were used to calculate the free energy cost of inverting the side-chain stereochemistry of individual Thr or Ile residues. Guided by the computational results, we used chemical protein synthesis to prepare three ShK polypeptide chain analogues, each containing either an allo-Thr or an allo-Ile residue. The three allo-Thr or allo-Ile-containing ShK polypeptides were able to fold into defined protein products, but with different folding propensities. Their relative thermal stabilities were measured and were consistent with the MD simulation data. Structures of the three ShK analogue proteins were determined by quasi-racemic X-ray crystallography and were similar to wild-type ShK. All three ShK analogues retained ion-channel blocking activity.

| S-EPMC5507063 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data