Emergence of Pseudomonas aeruginosa strains producing metallo-beta-lactamases of the IMP-15 and VIM-2 types in Mexico.

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.

Protonation state and substrate binding to B2 metallo-beta-lactamase CphA from Aeromonas hydrofila.

The zinc enzymes metallo beta-lactamases counteract the beneficial action of beta-lactam antibiotics against bacterial infections, by hydrolyzing their beta-lactam rings. To understand structure/function relationships on a representative member of this class, the B2 M beta L CphA, we have investigated the H-bond pattern at the Zn enzymatic active site and substrate binding mode by molecular simulation methods. Extensive QM calculations at the DFT-BLYP level on eleven models of the protein active site, along with MD simulations of the protein in aqueous solution, allow us to propose two plausible protonation states for the unbound enzyme, which are probably in equilibrium. Docking procedures along with MD simulations and QM calculations suggest that in the complex between the enzyme and its substrate (biapenem), the latter is stable in only one of the two protonation states, in addition it exhibits two different binding modes, of which only one agrees with previous proposals. In both cases, the substrate is polarized as in aqueous solution. We conclude that addressing mechanistic issues on this class of enzymes requires a careful procedure to assign protonation states and substrate docking modes.

Axial ligand modulation of the electronic structures of binuclear copper sites: analysis of paramagnetic 1H NMR spectra of Met160Gln Cu(A).

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.

Backbone dynamics of plastocyanin in both oxidation states. Solution structure of the reduced form and comparison with the oxidized state.

A model-free analysis based on (15)N R(1), (15)N R(2), and (15)N-(1)H nuclear Overhauser effects was performed on reduced (diamagnetic) and oxidized (paramagnetic) forms of plastocyanin from Synechocystis sp. PCC6803. The protein backbone is rigid, displaying a small degree of mobility in the sub-nanosecond time scale. The loops surrounding the copper ion, involved in physiological electron transfer, feature a higher extent of flexibility in the longer time scale in both redox states, as measured from D(2)O exchange of amide protons and from NH-H(2)O saturation transfer experiments. In contrast to the situation for other electron transfer proteins, no significant difference in the dynamic properties is found between the two redox forms. A solution structure was also determined for the reduced plastocyanin and compared with the solution structure of the oxidized form in order to assess possible structural changes related to the copper ion redox state. Within the attained resolution, the structure of the reduced plastocyanin is indistinguishable from that of the oxidized form, even though small chemical shift differences are observed. The present characterization provides information on both the structural and dynamic behavior of blue copper proteins in solution that is useful to understand further the role(s) of protein dynamics in electron transfer processes.

The first solution structure of a paramagnetic copper(II) protein: the case of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803.

The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803 is here reported. The protein contains paramagnetic copper(II), whose electronic relaxation times are quite unfavorable for NMR solution studies. The structure has been solved on the basis of 1041 meaningful NOESY cross-peaks, 18 1D NOEs, 26 T(1) values, 96 dihedral angle constraints, and 18 H-bonds. The detection of broad hyperfine-shifted signals and their full assignment allowed the identification of the copper(II) ligands and the determination of the Cu-S-C-H dihedral angle for the coordinated cysteine. The global root-mean-square deviation from the mean structure for the solution structure family is 0.72 +/- 0.14 and 1.16 +/- 0.17 A for backbone and heavy atoms, respectively. The structure is overall quite satisfactory and represents a breakthrough, in that it includes paramagnetic copper proteins among the metalloproteins for which solution structures can be afforded. The comparison with the available X-ray structure of a triple mutant is also performed.

Class B beta-lactamases: the importance of being metallic.

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.

Spectroscopic characterization of a binuclear metal site in Bacillus cereus beta-lactamase II.

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.

Alkaline transition of Rhus vernicifera stellacyanin, an unusual blue copper protein.

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.

Paramagnetic NMR Spectroscopy of Cobalt(II) and Copper(II) Derivatives of Pseudomonas aeruginosa His46Asp Azurin.

NMR spectra of paramagnetic Co(II) and Cu(II) derivatives of Pseudomonas aeruginosa His46Asp azurin have been investigated. In each derivative, assignment of hyperfine-shifted resonances outside the diamagnetic envelope of spectra recorded at 200 and 500 MHz confirms that the Asp carboxylate is coordinated to the paramagnetic metal center. The reduced paramagnetic shifts of the Cys112 proton resonances in Cu(II) and Co(II) His46Asp azurins compared to those of the corresponding wild type proteins indicate that metal-S(Cys) bonding is weakened in this mutant. The downfield shifts of the gamma-CH(2) of Met121 suggest a stronger interaction between the metal and the Met thioether group than is present in the wild type protein. Molecular modeling of the metal site structure indicates a distorted tetrahedral geometry with Asp46 (monodentate carboxylate), Cys112, and His117 equatorial ligands. In this structure, the metal ion is shifted 0.3 Å out of the O(Asp)S(Cys)N(His) trigonal plane toward Met121.

A 1H NMR NOE study on Co(II) stellacyanin: some clues about the structure of the metal site.

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.

Strategies of signal assignments in paramagnetic metalloproteins. An NMR investigation of the thiocyanate adduct of the cobalt (II)-substituted human carbonic anhydrase II.

The title protein with MW 30,000 containing high-spin cobalt (II) has been thoroughly investigated through 1H NMR spectroscopy with the aid of selectively deuterated amino acids and 15N enrichment. The aim is that of showing the potentiality of the approach when local information by NMR is needed and the X-ray structure is available. The potential use of the pseudocontact shift is discussed; 90, 200, and 600 MHz spectrometers are used to investigate a spherical region at various distances from the metal ion. More than 35 signals of protons around cobalt (II) have been assigned.

NMR investigation of isotopically labeled cyanide derivatives of lignin peroxidase and manganese peroxidase.

The 1H NMR spectroscopy was used to study lignin peroxidase (LiP) and manganese peroxidase (MnP) containing deuterated histidines. LiP and MnP were obtained from a histidine auxotroph of the fungus Phanerochaete chrysosporium grown in the presence of deuterated histidines. The derivatives with deuterated histidines have allowed a firm assignment of the protons of the distal and proximal histidines. We have also found that the LiP from this strain exhibits different orientations of the 2-vinyl group compared to the LiP from the strain previously studied. Mobility of the group has also been detected, thus explaining the apparent inconsistency between X-ray solid-state and NMR solution data. The 15N shift values of 15N-enriched CN- in the cyanide derivatives of LiP and MnP have also been measured. The shift patterns, both for 15N and for the proximal histidine protons of several peroxidases, are consistent with predominant contact shift contributions which reflect the bond strength of the metal-axial ligand. Finally, our results confirm a correlation between shift values of 15N and those of proximal histidine protons and the Fe3+/Fe2+ redox potentials.

One- and two-dimensional NMR characterization of oxidized and reduced cytochrome c' from Rhodocyclus gelatinosus.

1D and 2D NMR spectra of both the reduced and oxidized forms of cytochrome c' from Rhodocyclus gelatinosus have been recorded. The analysis of the pH dependence of the 1H NMR spectrum of the ferric form has been performed, and two main ionizing groups have been identified. By comparison of the pH dependence of the available spectra of cytochromes c', an ambiguity remaining from previous studies on related cytochromes c' has been solved. By means of 2D spectra, an assignment of all the paramagnetically shifted signals is proposed both for the ferrous and for the ferric forms.

The interaction of acetate and formate with cobalt carbonic anhydrase. An NMR study.

The interaction of formate and acetate ions with cobalt-substituted carbonic anhydrase (CA) has been investigated through 13C-NMR and one-dimensional and two-dimensional 1H-NMR spectroscopy. 13C data on formate are consistent with a regularly coordinated ligand, as previously proposed for the acetate anion [Bertini, I., Luchinat, C. & Scozzafava, A. (1977) J. Chem. Soc. Dalton Trans., 1962-1965]. 1H-NOE experiments on both anions give evidence of through-space interactions between ligand protons and protein protons. The latter are assigned to specific residues in the active cavity through nuclear Overhauser effect spectroscopy (NOESY) experiments. The 13C-derived and 1H-derived constrains allow reliable docking of these ligands in the active-site cavity. The resulting geometries are similar to one another and consistent with five-coordinated structures around the metal ion, as previously proposed from electronic spectroscopy [Bertini, I., Canti, G., Luchinat, C. & Scozzafava, A. (1978) J. Am. Chem. Soc. 100, 4873-4877]. The results are discussed in light of the current debate on anion binding to metal ions in carbonic anhydrase [Lindahl, M., Svensson, A. & Liljas, A. (1992) Proteins, in the press]; Bertini, I., Luchinat, C., Pierattelli, R. & Vila, A. J. (1992) Inorg. Chem., in the press; Banci, L. & Merz, K. (1992) unpublished results] and, in particular, of the proposed long Zn-O distance found in the recent X-ray results on the formate adduct [Hakanson, K., Carlsson, M., Svensson, A. & Liljas, A. (1992) J. Mol. Biol., in the press].