The Experts below are selected from a list of 921 Experts worldwide ranked by ideXlab platform

Alessandro Desideri - One of the best experts on this subject based on the ideXlab platform.

  • Single mutations at the subunit interface modulate copper reactivity in Photobacterium leiognathi Cu,Zn superoxide dismutase.
    Journal of molecular biology, 2001
    Co-Authors: M.e. Stroppolo, Alessandra Pesce, Melania D'orazio, Peter O'neill, Domenico Bordo, Camillo Rosano, Mario Milani, Andrea Battistoni, Martino Bolognesi, Alessandro Desideri
    Abstract:

    The functional properties and X-ray structures of five mutant forms of Photobacterium leiognathi Cu,Zn superoxide dismutase carrying single mutations at residues located at the dimer association interface have been investigated. When compared to the wild-type enzyme, the three-dimensional structures of the mutants show structural perturbations limited to the proximity of the mutation sites and substantial identity of active site geometry. Nonetheless, the catalytic rates of all mutants, measured at neutral pH and low ionic strength by pulse radiolysis, are higher than that of the wild-type protein. Such enzymatic activity increase is paralleled by enhanced active site accessibility to external chelating agents, which, in the mutated enzyme, remove more readily the active site copper ion. It is concluded that mutations at the prokaryotic Cu,Zn superoxide dismutase subunit interface can transduce dynamical perturbation to the active site region, promoting substrate active site accessibility. Such long-range intramolecular communication effects have not been extensively described before within the Cu,Zn superoxide dismutase homology family.

  • Cu,Zn superoxide dismutase from Photobacterium leiognathi is an hyperefficient enzyme.
    Biochemistry, 1998
    Co-Authors: Maria Elena Stroppolo, Peter O'neill, Marco Sette, Francesca Polizio, Maria Teresa Cambria, Alessandro Desideri
    Abstract:

    The catalytic rate constant of recombinant Photobacterium leiognathi Cu,Zn superoxide dismutase has been determined as a function of pH by pulse radiolysis. At pH 7 and low ionic strength (I = 0.02 M) the catalytic rate constant is 8.5 x 10(9) M-1 s-1, more than two times the value found for all the native eukaryotic Cu,Zn superoxide dismutases investigated to date. Similarly, Brownian dynamics simulations indicate an enzyme-substrate association rate more than two times higher than that found for bovine Cu,Zn superoxide dismutase. Titration of the paramagnetic contribution to the water proton relaxation rate of the P. leiognathi with increasing concentration of halide ions with different radii indicates that the proteic channel delimiting the active site is wider than 4.4 A. This is at variance with that found on the eukariotic enzymes, and provides a rationale for the high catalytic rate of the bacterial enzyme. Evidence for solvent exposure of the active site different from that observed in the eukaryotic enzyme is suggested from the pH dependence of the water proton relaxation rate and of the EPR spectrum line shape, which indicate the occurrence of a prototropic equilibrium at pH 9.1 and 9.0, respectively. The pH dependence of the P. leiognathi catalytic rate has a trend different from that observed in the bovine enzyme, indicating that groups differently exposed to the solvent are involved in the modulation of the enzyme-substrate encounter.

  • Spectroscopic characterization of recombinant Cu,Zn superoxide dismutase from Photobacterium leiognathi expressed in Escherichia coli: evidence for a novel catalytic copper binding site.
    Biochemistry, 1997
    Co-Authors: D. Foti, M.e. Stroppolo, Francesca Polizio, B. Lo Curto, G. Cuzzocrea, Mariano Venanzi, Alessandro Desideri
    Abstract:

    Cu,Zn superoxide dismutase from Photobacterium leiognathi has been cloned and expressed in Escherichia coli. The circular dichroism spectrum in the UV region of the recombinant protein indicates an higher content of random coil structure with respect to the eukaryotic enzymes. Investigation of the active site by optical, CD, and EPR spectroscopy indicates a different coordination geometry around the catalytic copper site with respect to the eukaryotic enzymes. In particular a different orientation of the metal bridging histidine is suggested. The pH dependence of the copper EPR spectrum shows the presence of a single equilibrium which is at least one unit lower than the pK value observed for the bovine enzyme. Despite such structural differences the catalytic rate of this enzyme is identical to that observed for the eukaryotic Cu,Zn superoxide dismutase, suggesting that the overall electric field distribution is similar to that observed in the eukaryotic enzymes.

Maria Elena Stroppolo - One of the best experts on this subject based on the ideXlab platform.

  • Evidence of stable monomeric species in the unfolding of Cu,Zn superoxide dismutase from Photobacterium leiognathi.
    Archives of biochemistry and biophysics, 1999
    Co-Authors: Flaminia Malvezzi-campeggi, Maria Elena Stroppolo, Giampiero Mei, Nicola Rosato, A. Desideri
    Abstract:

    The equilibrium unfolding process of Photobacterium leiognathi Cu,Zn superoxide dismutase has been quantitatively monitored through circular dichroism (CD) and fluorescence spectroscopy, upon increasing the guanidinium hydrochloride concentration. The study has been undertaken for both the holo- and the copper-free derivative to work out the role of copper in protein stability. In both cases the unfolding was reversible. The denaturation curve derived from CD and fluorescence spectroscopy was not coincident, suggesting that the denaturation process occurs through a three-state model with formation of an intermediate monomeric species. The occurrence of an intermediate species has been unambiguously demonstrated following CD and steady-state fluorescence spectra of the enzyme at various concentrations in presence of a fixed amounts of guanidinium hydrochloride.

  • Cu,Zn superoxide dismutase from Photobacterium leiognathi is an hyperefficient enzyme.
    Biochemistry, 1998
    Co-Authors: Maria Elena Stroppolo, Peter O'neill, Marco Sette, Francesca Polizio, Maria Teresa Cambria, Alessandro Desideri
    Abstract:

    The catalytic rate constant of recombinant Photobacterium leiognathi Cu,Zn superoxide dismutase has been determined as a function of pH by pulse radiolysis. At pH 7 and low ionic strength (I = 0.02 M) the catalytic rate constant is 8.5 x 10(9) M-1 s-1, more than two times the value found for all the native eukaryotic Cu,Zn superoxide dismutases investigated to date. Similarly, Brownian dynamics simulations indicate an enzyme-substrate association rate more than two times higher than that found for bovine Cu,Zn superoxide dismutase. Titration of the paramagnetic contribution to the water proton relaxation rate of the P. leiognathi with increasing concentration of halide ions with different radii indicates that the proteic channel delimiting the active site is wider than 4.4 A. This is at variance with that found on the eukariotic enzymes, and provides a rationale for the high catalytic rate of the bacterial enzyme. Evidence for solvent exposure of the active site different from that observed in the eukaryotic enzyme is suggested from the pH dependence of the water proton relaxation rate and of the EPR spectrum line shape, which indicate the occurrence of a prototropic equilibrium at pH 9.1 and 9.0, respectively. The pH dependence of the P. leiognathi catalytic rate has a trend different from that observed in the bovine enzyme, indicating that groups differently exposed to the solvent are involved in the modulation of the enzyme-substrate encounter.

M.e. Stroppolo - One of the best experts on this subject based on the ideXlab platform.

  • Single mutations at the subunit interface modulate copper reactivity in Photobacterium leiognathi Cu,Zn superoxide dismutase.
    Journal of molecular biology, 2001
    Co-Authors: M.e. Stroppolo, Alessandra Pesce, Melania D'orazio, Peter O'neill, Domenico Bordo, Camillo Rosano, Mario Milani, Andrea Battistoni, Martino Bolognesi, Alessandro Desideri
    Abstract:

    The functional properties and X-ray structures of five mutant forms of Photobacterium leiognathi Cu,Zn superoxide dismutase carrying single mutations at residues located at the dimer association interface have been investigated. When compared to the wild-type enzyme, the three-dimensional structures of the mutants show structural perturbations limited to the proximity of the mutation sites and substantial identity of active site geometry. Nonetheless, the catalytic rates of all mutants, measured at neutral pH and low ionic strength by pulse radiolysis, are higher than that of the wild-type protein. Such enzymatic activity increase is paralleled by enhanced active site accessibility to external chelating agents, which, in the mutated enzyme, remove more readily the active site copper ion. It is concluded that mutations at the prokaryotic Cu,Zn superoxide dismutase subunit interface can transduce dynamical perturbation to the active site region, promoting substrate active site accessibility. Such long-range intramolecular communication effects have not been extensively described before within the Cu,Zn superoxide dismutase homology family.

  • Spectroscopic characterization of recombinant Cu,Zn superoxide dismutase from Photobacterium leiognathi expressed in Escherichia coli: evidence for a novel catalytic copper binding site.
    Biochemistry, 1997
    Co-Authors: D. Foti, M.e. Stroppolo, Francesca Polizio, B. Lo Curto, G. Cuzzocrea, Mariano Venanzi, Alessandro Desideri
    Abstract:

    Cu,Zn superoxide dismutase from Photobacterium leiognathi has been cloned and expressed in Escherichia coli. The circular dichroism spectrum in the UV region of the recombinant protein indicates an higher content of random coil structure with respect to the eukaryotic enzymes. Investigation of the active site by optical, CD, and EPR spectroscopy indicates a different coordination geometry around the catalytic copper site with respect to the eukaryotic enzymes. In particular a different orientation of the metal bridging histidine is suggested. The pH dependence of the copper EPR spectrum shows the presence of a single equilibrium which is at least one unit lower than the pK value observed for the bovine enzyme. Despite such structural differences the catalytic rate of this enzyme is identical to that observed for the eukaryotic Cu,Zn superoxide dismutase, suggesting that the overall electric field distribution is similar to that observed in the eukaryotic enzymes.

Melania D'orazio - One of the best experts on this subject based on the ideXlab platform.

  • The dimeric assembly of Photobacterium leiognathi and Salmonella typhimurium SodC1 Cu,Zn superoxide dismutases is affected differently by active site demetallation and pH: An analytical ultracentrifuge study
    Archives of biochemistry and biophysics, 2008
    Co-Authors: Bruno Catacchio, Melania D'orazio, Andrea Battistoni, Emilia Chiancone
    Abstract:

    Abstract To establish whether the species-specific variations at the subunit interface of bacterial Cu,Zn superoxide dismutases affect dimer assembly, the association state of the Photobacterium leiognathi (PlSOD) and Salmonella typhimurium (StSOD) enzymes, which differ in 11 out of 19 interface residues, was investigated by analytical ultracentrifugation. The same linkage pattern correlates quaternary assembly, active site metallation, and pH in the two enzymes albeit with quantitative differences. Both holo-enzymes are stable dimers at pH 6.8 and 8.0, although their shape is altered at alkaline pH. In contrast, dimer stability is affected differently by metal removal. Thus, apo-StSOD is a stable dimer at pH 6.8 whereas apo-PlSOD is in reversible monomer–dimer equilibrium. In both apoproteins a pH increase to 8.0 favors monomerization. These effects prove the existence of long-range communication between the active site and the subunit interface and provide a structural explanation for the known functional differences between the two enzymes.

  • Single mutations at the subunit interface modulate copper reactivity in Photobacterium leiognathi Cu,Zn superoxide dismutase.
    Journal of molecular biology, 2001
    Co-Authors: M.e. Stroppolo, Alessandra Pesce, Melania D'orazio, Peter O'neill, Domenico Bordo, Camillo Rosano, Mario Milani, Andrea Battistoni, Martino Bolognesi, Alessandro Desideri
    Abstract:

    The functional properties and X-ray structures of five mutant forms of Photobacterium leiognathi Cu,Zn superoxide dismutase carrying single mutations at residues located at the dimer association interface have been investigated. When compared to the wild-type enzyme, the three-dimensional structures of the mutants show structural perturbations limited to the proximity of the mutation sites and substantial identity of active site geometry. Nonetheless, the catalytic rates of all mutants, measured at neutral pH and low ionic strength by pulse radiolysis, are higher than that of the wild-type protein. Such enzymatic activity increase is paralleled by enhanced active site accessibility to external chelating agents, which, in the mutated enzyme, remove more readily the active site copper ion. It is concluded that mutations at the prokaryotic Cu,Zn superoxide dismutase subunit interface can transduce dynamical perturbation to the active site region, promoting substrate active site accessibility. Such long-range intramolecular communication effects have not been extensively described before within the Cu,Zn superoxide dismutase homology family.

Andrea Battistoni - One of the best experts on this subject based on the ideXlab platform.

  • The dimeric assembly of Photobacterium leiognathi and Salmonella typhimurium SodC1 Cu,Zn superoxide dismutases is affected differently by active site demetallation and pH: An analytical ultracentrifuge study
    Archives of biochemistry and biophysics, 2008
    Co-Authors: Bruno Catacchio, Melania D'orazio, Andrea Battistoni, Emilia Chiancone
    Abstract:

    Abstract To establish whether the species-specific variations at the subunit interface of bacterial Cu,Zn superoxide dismutases affect dimer assembly, the association state of the Photobacterium leiognathi (PlSOD) and Salmonella typhimurium (StSOD) enzymes, which differ in 11 out of 19 interface residues, was investigated by analytical ultracentrifugation. The same linkage pattern correlates quaternary assembly, active site metallation, and pH in the two enzymes albeit with quantitative differences. Both holo-enzymes are stable dimers at pH 6.8 and 8.0, although their shape is altered at alkaline pH. In contrast, dimer stability is affected differently by metal removal. Thus, apo-StSOD is a stable dimer at pH 6.8 whereas apo-PlSOD is in reversible monomer–dimer equilibrium. In both apoproteins a pH increase to 8.0 favors monomerization. These effects prove the existence of long-range communication between the active site and the subunit interface and provide a structural explanation for the known functional differences between the two enzymes.

  • Single mutations at the subunit interface modulate copper reactivity in Photobacterium leiognathi Cu,Zn superoxide dismutase.
    Journal of molecular biology, 2001
    Co-Authors: M.e. Stroppolo, Alessandra Pesce, Melania D'orazio, Peter O'neill, Domenico Bordo, Camillo Rosano, Mario Milani, Andrea Battistoni, Martino Bolognesi, Alessandro Desideri
    Abstract:

    The functional properties and X-ray structures of five mutant forms of Photobacterium leiognathi Cu,Zn superoxide dismutase carrying single mutations at residues located at the dimer association interface have been investigated. When compared to the wild-type enzyme, the three-dimensional structures of the mutants show structural perturbations limited to the proximity of the mutation sites and substantial identity of active site geometry. Nonetheless, the catalytic rates of all mutants, measured at neutral pH and low ionic strength by pulse radiolysis, are higher than that of the wild-type protein. Such enzymatic activity increase is paralleled by enhanced active site accessibility to external chelating agents, which, in the mutated enzyme, remove more readily the active site copper ion. It is concluded that mutations at the prokaryotic Cu,Zn superoxide dismutase subunit interface can transduce dynamical perturbation to the active site region, promoting substrate active site accessibility. Such long-range intramolecular communication effects have not been extensively described before within the Cu,Zn superoxide dismutase homology family.