JBIC Journal of Biological Inorganic Chemistry, 2002
The present study describes the cloning, isolation, and thorough biochemical characterization of ... more The present study describes the cloning, isolation, and thorough biochemical characterization of UreE from Bacillus pasteurii, a novel protein putatively involved in the transport of Ni in the urease assembly process. A DNA fragment of the B. pasteurii urease operon, containing all four accessory genes (ureE, ureF, ureG, and ureD) required for the incorporation of Ni ions into the active site of urease, was cloned, sequenced, and analyzed. B. pasteurii ureE was cloned, and the UreE protein (BpUreE) was over-expressed and purified to homogeneity. The identity of the recombinant protein was determined by N- and C-terminal sequencing and by mass spectrometry. BpUreE has a chain length of 147 amino acids, and features a p I value of 4.7. As isolated, BpUreE contains one Zn(II) ion per dimer, while no Ni(II) is present, as shown by mass spectrometry and atomic absorption spectroscopy. BpUreE behaves as a dimer independently of the presence of Zn(II), as shown by gel filtration and mass spectrometry. Paramagnetic NMR spectroscopy on concentrated (2 mM) UreE solutions reveals a one Ni atom per tetramer stoichiometry, with the Ni(II) ion bound to histidines in an octahedral coordination environment. BpUreE has a high sequence similarity with UreE proteins isolated from different biological sources, while no sequence homology is observed with proteins belonging to different classes. In particular, BpUreE is most similar to UreE from Bacillus halodurans (55% identity). A multiple sequence alignment reveals the presence of four strictly conserved residues (Leu55, Gly97, Asn98, His100; BpUreE numbering), in addition to position 115, conservatively occupied by an Asp or a Glu residue. Several secondary structure elements, including a betaalphabetabetaalphabeta "ferredoxin-like" motif, are highly conserved throughout the UreE sequences.
The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803... more 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.
The structure of β-mercaptoethanol-inhibited urease from Bacillus pasteurii, a highly ureolytic ... more The structure of β-mercaptoethanol-inhibited urease from Bacillus pasteurii, a highly ureolytic soil micro-organism, was solved at 1.65 Å using synchrotron X-ray cryogenic diffraction data. The structure clearly shows the unexpected binding mode of β-mercaptoethanol, which bridges the two nickel ions in the active site through the sulfur atom and chelates one Ni through the OH functionality. Another molecule of inhibitor forms a mixed disulfide with a Cys residue, thus sealing the entrance to the active site cavity by steric hindrance. The possible implications of the results on structure-based molecular design of new urease inhibitors are discussed.
The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined ... more The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined using synchrotron X-ray diffraction data from a vitrified crystal (1.85 Å resolution, 99.3% completeness, data redundancy 4.6, R-factor 17.3%, PDB code 6UBP). A distance of 3.5 Å separates the two Ni ions in the active site. The binding mode of the inhibitor involves the formation of four coordination bonds with the two Ni ions: one phosphate oxygen atom symmetrically bridges the two metal ions (1.9–2.0 Å), while two of the remaining phosphate oxygen atoms bind to the Ni atoms at 2.4 Å. The fourth phosphate oxygen is directed into the active site channel. Analysis of the H-bonding network around the bound inhibitor indicates that phosphate is bound as the H2PO4– anion, and that an additional proton is present on the Oδ2 atom of Aspα363, an active site residue involved in Ni coordination through Oδ1. The flexible flap flanking the active site cavity is in the open conformation. Analysis of the complex reveals why phosphate is a relatively weak inhibitor and why sulfate does not bind to the nickels in the active site. The implications of the results for the understanding of the urease catalytic mechanism are reviewed. A novel alternative for the proton donor is presented.
Interplay between Metal Ions and Nucleic …, Jan 1, 2012
The dramatic changes in the environmental conditions that organisms encountered during evolution ... more The dramatic changes in the environmental conditions that organisms encountered during evolution and adaptation to life in specific niches, have influenced intracellular and extracellular metal ion contents and, as a consequence, the cellular ability to sense and utilize different metal ions. This metal-driven differentiation is reflected in the specific panels of metal-responsive transcriptional regulators found in different organisms, which finely tune the intracellular metal ion content and all metal-dependent processes. In order to understand the processes underlying this complex metal homeostasis network, the study of the molecular processes that determine the protein-metal ion recognition, as well as how this event is transduced into a transcriptional output, is necessary. This chapter describes how metal ion binding to specific proteins influences protein interaction with DNA and how this event can influence the fate of genetic expression, leading to specific transcriptional outputs. The features of representative metal-responsive transcriptional regulators, as well as the molecular basis of metal-protein and protein-DNA interactions, are discussed on the basis of the structural information available. An overview of the recent advances in the understanding of how these proteins choose specific metal ions among the intracellular metal ion pool, as well as how they allosterically respond to their effector binding, is given.
The reaction of the sodium or potassium salts of H 2 acen [H 2 acen= NN-ethylenebis (acetylaceton... more The reaction of the sodium or potassium salts of H 2 acen [H 2 acen= NN-ethylenebis (acetylacetonylideneimine)] with NbCl 4· 2thf (thf= tetrahydrofuran) led to trans-[Nb (acen) Cl 2](1), while reaction with the lithium salt of H 2 L (H 2 L= 7, 16-dihydro-6, 8, 15, 17-...
Ion pair complexes,[M (tmtaa) Na (thf) 3](M= Fe, Co, or Ni; tmtaa= dibenzotetramethyltetra-aza [1... more Ion pair complexes,[M (tmtaa) Na (thf) 3](M= Fe, Co, or Ni; tmtaa= dibenzotetramethyltetra-aza [14] annulene dianion; thf= tetrahydrofuran), in which both metal ions are associated with the macrocyclic ligand have been obtained form single-electron reduction of the ...
JBIC Journal of Biological Inorganic Chemistry, 2002
The present study describes the cloning, isolation, and thorough biochemical characterization of ... more The present study describes the cloning, isolation, and thorough biochemical characterization of UreE from Bacillus pasteurii, a novel protein putatively involved in the transport of Ni in the urease assembly process. A DNA fragment of the B. pasteurii urease operon, containing all four accessory genes (ureE, ureF, ureG, and ureD) required for the incorporation of Ni ions into the active site of urease, was cloned, sequenced, and analyzed. B. pasteurii ureE was cloned, and the UreE protein (BpUreE) was over-expressed and purified to homogeneity. The identity of the recombinant protein was determined by N- and C-terminal sequencing and by mass spectrometry. BpUreE has a chain length of 147 amino acids, and features a p I value of 4.7. As isolated, BpUreE contains one Zn(II) ion per dimer, while no Ni(II) is present, as shown by mass spectrometry and atomic absorption spectroscopy. BpUreE behaves as a dimer independently of the presence of Zn(II), as shown by gel filtration and mass spectrometry. Paramagnetic NMR spectroscopy on concentrated (2 mM) UreE solutions reveals a one Ni atom per tetramer stoichiometry, with the Ni(II) ion bound to histidines in an octahedral coordination environment. BpUreE has a high sequence similarity with UreE proteins isolated from different biological sources, while no sequence homology is observed with proteins belonging to different classes. In particular, BpUreE is most similar to UreE from Bacillus halodurans (55% identity). A multiple sequence alignment reveals the presence of four strictly conserved residues (Leu55, Gly97, Asn98, His100; BpUreE numbering), in addition to position 115, conservatively occupied by an Asp or a Glu residue. Several secondary structure elements, including a betaalphabetabetaalphabeta "ferredoxin-like" motif, are highly conserved throughout the UreE sequences.
The NMR solution structure of oxidized plastocyanin from the cyanobacterium Synechocystis PCC6803... more 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.
The structure of β-mercaptoethanol-inhibited urease from Bacillus pasteurii, a highly ureolytic ... more The structure of β-mercaptoethanol-inhibited urease from Bacillus pasteurii, a highly ureolytic soil micro-organism, was solved at 1.65 Å using synchrotron X-ray cryogenic diffraction data. The structure clearly shows the unexpected binding mode of β-mercaptoethanol, which bridges the two nickel ions in the active site through the sulfur atom and chelates one Ni through the OH functionality. Another molecule of inhibitor forms a mixed disulfide with a Cys residue, thus sealing the entrance to the active site cavity by steric hindrance. The possible implications of the results on structure-based molecular design of new urease inhibitors are discussed.
The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined ... more The structure of Bacillus pasteurii urease (BPU) inhibited with phosphate was solved and refined using synchrotron X-ray diffraction data from a vitrified crystal (1.85 Å resolution, 99.3% completeness, data redundancy 4.6, R-factor 17.3%, PDB code 6UBP). A distance of 3.5 Å separates the two Ni ions in the active site. The binding mode of the inhibitor involves the formation of four coordination bonds with the two Ni ions: one phosphate oxygen atom symmetrically bridges the two metal ions (1.9–2.0 Å), while two of the remaining phosphate oxygen atoms bind to the Ni atoms at 2.4 Å. The fourth phosphate oxygen is directed into the active site channel. Analysis of the H-bonding network around the bound inhibitor indicates that phosphate is bound as the H2PO4– anion, and that an additional proton is present on the Oδ2 atom of Aspα363, an active site residue involved in Ni coordination through Oδ1. The flexible flap flanking the active site cavity is in the open conformation. Analysis of the complex reveals why phosphate is a relatively weak inhibitor and why sulfate does not bind to the nickels in the active site. The implications of the results for the understanding of the urease catalytic mechanism are reviewed. A novel alternative for the proton donor is presented.
Interplay between Metal Ions and Nucleic …, Jan 1, 2012
The dramatic changes in the environmental conditions that organisms encountered during evolution ... more The dramatic changes in the environmental conditions that organisms encountered during evolution and adaptation to life in specific niches, have influenced intracellular and extracellular metal ion contents and, as a consequence, the cellular ability to sense and utilize different metal ions. This metal-driven differentiation is reflected in the specific panels of metal-responsive transcriptional regulators found in different organisms, which finely tune the intracellular metal ion content and all metal-dependent processes. In order to understand the processes underlying this complex metal homeostasis network, the study of the molecular processes that determine the protein-metal ion recognition, as well as how this event is transduced into a transcriptional output, is necessary. This chapter describes how metal ion binding to specific proteins influences protein interaction with DNA and how this event can influence the fate of genetic expression, leading to specific transcriptional outputs. The features of representative metal-responsive transcriptional regulators, as well as the molecular basis of metal-protein and protein-DNA interactions, are discussed on the basis of the structural information available. An overview of the recent advances in the understanding of how these proteins choose specific metal ions among the intracellular metal ion pool, as well as how they allosterically respond to their effector binding, is given.
The reaction of the sodium or potassium salts of H 2 acen [H 2 acen= NN-ethylenebis (acetylaceton... more The reaction of the sodium or potassium salts of H 2 acen [H 2 acen= NN-ethylenebis (acetylacetonylideneimine)] with NbCl 4· 2thf (thf= tetrahydrofuran) led to trans-[Nb (acen) Cl 2](1), while reaction with the lithium salt of H 2 L (H 2 L= 7, 16-dihydro-6, 8, 15, 17-...
Ion pair complexes,[M (tmtaa) Na (thf) 3](M= Fe, Co, or Ni; tmtaa= dibenzotetramethyltetra-aza [1... more Ion pair complexes,[M (tmtaa) Na (thf) 3](M= Fe, Co, or Ni; tmtaa= dibenzotetramethyltetra-aza [14] annulene dianion; thf= tetrahydrofuran), in which both metal ions are associated with the macrocyclic ligand have been obtained form single-electron reduction of the ...
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Papers by Stefano Ciurli