RESUMO
Eighty-six carbapenem non-susceptible Pseudomonas aeruginosa isolates collected in the National Institute of Respiratory Diseases of Mexico City were screened for the presence of metallo-beta-lactamase (MBL) activity using both E-test strips and a microbiological assay with EDTA-imipenem. Genomic comparisons and sequence analyses conducted with these isolates revealed the presence of bla(VIM-2) in two clonally related isolates, and bla(IMP-15) in a clonally unrelated isolate. Both genes were found to be carried by class 1 integrons, and bla(IMP-15) was additionally present on a broad host-range plasmid. This is the first report of co-existing P. aeruginosa strains producing different MBLs in a Mexican hospital, highlighting the necessity of appropriate surveillance to prevent dissemination of carbapenem resistance.
Assuntos
Proteínas de Bactérias/biossíntese , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/isolamento & purificação , beta-Lactamases/biossíntese , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Hospitais , Humanos , Integrons , México , Testes de Sensibilidade Microbiana/métodos , Plasmídeos , beta-Lactamases/genéticaRESUMO
Cu(A) is an electron-transfer copper center present in heme-copper oxidases and N2O reductases. The center is a binuclear unit, with two cysteine ligands bridging the metal ions and two terminal histidine residues. A Met residue and a peptide carbonyl group are located on opposite sides of the Cu2S2 plane; these weaker ligands are fully conserved in all known Cu(A) sites. The Met160Gln mutant of the soluble subunit II of Thermus thermophilus ba3 oxidase has been studied by NMR spectroscopy. In its oxidized form, the binuclear copper is a fully delocalized mixed-valence pair, as are all natural Cu(A) centers. The faster nuclear relaxation in this mutant suggests that a low-lying excited state has shifted to higher energies compared to that of the wild-type protein. The introduction of the Gln residue alters the coordination mode of His114 but does not affect His157, thereby confirming the proposal that the axial ligand-to-copper distances influence the copper-His interactions (Robinson, H.; Ang, M. C.; Gao, Y. G.; Hay, M. T.; Lu, Y.; Wang, A. H. Biochemistry 1999, 38, 5677). Changes in the hyperfine coupling constants of the Cys beta-CH2 groups are attributed to minor geometrical changes that affect the Cu-S-C(beta)-H(beta) dihedral angles. These changes, in addition, shift the thermally accessible excited states, thus influencing the spectral position of the Cys beta-CH2 resonances. The Cu-Cys bonds are not substantially altered by the Cu-Gln160 interaction, in contrast to the situation found in the evolutionarily related blue copper proteins. It is possible that regulatory subunits in the mitochondrial oxidases fix the relative positions of thermally accessible Cu(A) excited states by tuning axial ligand interactions.
Assuntos
Cobre/química , Grupo dos Citocromos b/química , Complexo IV da Cadeia de Transporte de Elétrons/química , Grupo dos Citocromos b/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Glutamina/química , Glutamina/genética , Metionina/química , Metionina/genética , Modelos Moleculares , Mutagênese , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação Proteica , Estrutura Terciária de Proteína , Prótons , Thermus thermophilus/enzimologia , Thermus thermophilus/genéticaRESUMO
The structural and functional features of class B b-lactamases, which are metal-dependent, are reviewed in this article. Enzymes from different bacterial strains exhibit a common fold and sequence similarity in their active sites. However, the protein scaffold fine tunes the metal binding affinity and substrate selectivity. In this way, some metallo-b-lactamases seem to be functional with only one Zn(II) equivalent per enzyme, whereas others require a binuclear active site. The sequence similarity leads to a subdivision of these enzymes into three subclasses. The substrate specificities are rather broad, except for enzymes belonging to subclass B2. Some inhibitors have been designed and tested, but none of them is able to exhibit a broad spectrum against these enzymes.
Assuntos
Metaloproteínas/antagonistas & inibidores , Metaloproteínas/química , Zinco/química , Inibidores de beta-Lactamases , beta-Lactamases/química , Antibacterianos/química , Antibacterianos/farmacologia , Sequência de Bases , Sítios de Ligação , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Hidrólise , Metaloproteínas/classificação , Modelos Moleculares , Dados de Sequência Molecular , beta-Lactamases/classificação , beta-LactamasRESUMO
The zinc metalloenzyme beta-lactamase II (betaLII) from Bacillus cereus has been overexpressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and the metal binding properties of recombinant betaLII toward Zn(II) and Co(II) have been studied by fluorescence and activity measurements. The apoenzyme is able to bind two metal ion equivalents, which confer on betaLII its maximum enzymatic efficiency. The enzyme is partially active with one metal ion equivalent. The diCo(II) and a mixed Zn(II)Co(II) derivative of betaLII were obtained and probed by electronic and paramagnetic NMR spectroscopy. In the high-affinity site, the metal is bound to three His residues and a solvent molecule, adopting a tetrahedral geometry. A Cys, a His, and an Asp residue are coordinated to the low-affinity metal site, together with two or three solvent molecules. This coordination polyhedron resembles the binuclear metal site of the Bacteroides fragilis beta-lactamase [Concha, N., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J. M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] but differs from that resulting from the X-ray study of betaLII [Carfi, A., Pares, S., Duée, E., Galleni, M., Duez, C., Frère, J. M., and Dideberg, O. (1995) EMBO J. 14, 4914-4921]. These results suggest that this binuclear metal site may be a general feature of metallo-beta-lactamases.
Assuntos
Bacillus cereus/enzimologia , Cefalosporinase/química , Zinco/química , Sequência de Aminoácidos , Sítios de Ligação , Cefalosporinase/biossíntese , Cefalosporinase/genética , Cobalto/química , Sequência Conservada , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Fluorescência , EspectrofotometriaRESUMO
Stellacyanin from Rhus vernificera is a blue copper protein in which the metal is coordinated to a Cys, two His, and a Gln residue. It displays a low redox potential, a fast electron exchange rate, and a reversible alkaline transition. We have studied this transition in Cu(II)- and Co(II)-stellacyanin by means of electronic and NMR spectroscopy. The data indicate that a conformational rearrangement of the metal site occurs at high pH. A drastic alteration in the Gln coordination mode, as initially proposed, is discarded. These results show that the metal site in stellacyanin is more flexible than the sites of other blue copper proteins. The present study demonstrates that the paramagnetic shifts of the bound Cys in the Co(II) derivative are sensitive indicators of the electron delocalization and conformational changes experienced by this residue.
Assuntos
Cobalto/química , Cobre/química , Metaloproteínas/química , Proteínas de Plantas/química , Plantas Tóxicas , Toxicodendron/química , Azurina/química , Proteínas de Bactérias/química , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Proteica , EspectrofotometriaRESUMO
The 1H NMR spectrum of Co(II) stellacyanin is reported, in which four signals not previously observed have been detected. NOE experiments were performed to assign the hyperfine shifted signals corresponding to a Cys and two His residues. Both His residues are solvent-accessible and are shown to bind the metal ion through their N delta 1 atoms. The beta-CH2 Cys proton shifts indicate the presence of a strong axial ligand.