S. Mangani - Academia.edu (original) (raw)
Papers by S. Mangani
The Journal of Physical Chemistry B, 2003
An analysis of the high resolution 3D structures of zinc enzymes shows that the Zn-His-carboxyl (... more An analysis of the high resolution 3D structures of zinc enzymes shows that the Zn-His-carboxyl (ate)-HX (X= OH or NH) H-bond motif is common. We investigate here the influence of this motif in the active site of horse liver alcohol dehydrogenase, which features the Zn-His-Asp-H2O motif. Density functional theory calculations are carried out on models of the active site complexed with the NAD+ cofactor, in which the metal ion binds either the alcohol substrate [Bahnson et al. Pro c. Nat l. Aca d. Sc i. USA 1997, 94, 12797-12802] 1 or a ...
Journal of Molecular Biology, 1999
The crystal structure of the engineered monomeric human Cu,ZnSOD triple mutant F50E/G51E/E133Q (Q... more The crystal structure of the engineered monomeric human Cu,ZnSOD triple mutant F50E/G51E/E133Q (Q133M2SOD) is reported at atomic resolution (1.02 A Ê ). This derivative has about 20 % of the wild-type activity. Crystals of Q133M2SOD have been obtained in the presence of CdCl 2 . The metal binding site is disordered, with both cadmium and copper ions simultaneously binding to the copper site. The cadmium (II) ions occupy about 45 % of the copper sites by binding the four histidine residues which ligate copper in the native enzyme, and two further water molecules to complete octahedral coordination. The copper ion is tri-coordinate, and the fourth histidine (His63) is detached from copper and bridges cadmium and zinc. X-ray absorption spectroscopy performed on the crystals suggests that the copper ion has undergone partial photoreduction upon exposure to the synchrotron light. The structure is also disordered in the disul®de bridge region of loop IV that is located at the subunit/subunit interface in the native SOD dimer. As a consequence, the catalytically relevant Arg143 residue is disordered. The present structure has been compared to other X-ray structures on various isoenzymes and to the solution structure of the same monomeric form. The structural results suggest that the low activity of monomeric SOD is due to the disorder in the conformation of the side-chain of Arg143 as well as of loop IV. It is proposed that the subunit-subunit interactions in the multimeric forms of the enzyme are needed to stabilize the correct geometry of the cavity and the optimal orientation of the charged residues in the active channel. Furthermore, the different coordination of cadmium and copper ions, contemporaneously present in the same site, are taken as models for the oxidized and reduced copper species, respectively. These properties of the structure have allowed us to revisit the enzymatic mechanism.
Journal of Molecular Biology, 1995
Journal of Inorganic Biochemistry, 1991
Journal of Biological Inorganic Chemistry, 2000
The structure of Bacillus pasteurii urease inhibited with acetohydroxamic acid was solved and ref... more The structure of Bacillus pasteurii urease inhibited with acetohydroxamic acid was solved and refined anisotropically using synchrotron X-ray cryogenic diffraction data (1.55 Å resolution, 99.5% completeness, data redundancy p 26, R-factor p 15.1%, PDB code 4UBP). The two Ni ions in the active site are separated by a distance of 3.53 Å. The structure clearly shows the binding mode of the inhibitor anion, symmetrically bridging the two Ni ions in the active site through the hydroxamate oxygen and chelating one Ni ion through the carbonyl oxygen. The flexible flap flanking the active site cavity is in the open conformation. The possible implications of the results on structure-based molecular design of new urease inhibitors are discussed.
Chemistry & Biology, 2009
Crystals of OXA-48 have been grown, by the sitting drop vapour diffusion technique, from a soluti... more Crystals of OXA-48 have been grown, by the sitting drop vapour diffusion technique, from a solution containing 0.1 M Hepes buffer at pH 7.5, 10% PEG 4000, 8% 1-butanol with and OXA-48 at a final concentration of 10 mg/ml. Several datasets from these crystals have been collected on the ID14-1 beamline at ESRF (Grenoble, France) and on the BW7A beamline at EMBL-DESY (Hamburg, Germany). All datasets were collected at 100 K and the crystals were cooled by using the precipitating solution with added 10-15 % (v/v) ethylene glycol as cryoprotectant. The OXA-48 crystals provided a complete dataset to 1.9 Å resolution with maximum diffraction up to 1.6 Å and belong to space group P2 1 with cell parameters a = 63.70 Å; b = 107.18 Å; c = 80.79 Å; β = 111.04°. The crystal asymmetric unit contains four molecules and 48.3 % solvent (Matthews coefficient, 2.38 Å 3 /Da). The data were processed by the program MOSFLM (Leslie, 1991) and scaled using the program SCALA with the TAILS and SECONDARY correction on to achieve an empirical absorption correction . The data collection and data reduction parameters are reported in of the manuscript. Despite trials with several crystals from different batches, in all cases, the observed diffraction was not of the highest possible quality, as shown by the indicators reported in . The relatively low quality of the crystals is probably due to the presence of highly disordered portions of many of the loops present in the structure (see refinement part) resulting in low symmetry and a high Z number. The structure was solved by molecular replacement methods (MOLREP program [Vagin and Teplyakov, 1997] from the CCP4 suite [Collaborative Computational Project N4, 2008]) using as model the structure of OXA-10 (PDB code 1K4F). Model rebuilding and manipulation was performed at all stages with the program COOT (Emsley and Cowtan, 2004). The refinement was carried out by the program REFMAC5 (Murshudov et al., 1997) (from the CCP4 suite). The presence of four independent subunits in the asymmetric unit organized in two dimers related by an approximate non-crystallographic two-fold axis suggested the use of a refinement protocol, similar to that described consisting of NCS restrained cycles followed by TLS refinement, before the final cycles of usual isotropic refinement. This resulted in final R cryst and R free factors of 0.211 and 0.285 respectively whit good geometry and Ramachandran distribution . The final 2Fo-Fc and ∆F maps are of good quality except for the loop regions consisting of the beginning of the Ω-loop in subunit A, where the density for the residue Ile-127 is weak and is totally missing for residue Ser-128. The presence of such disorder explains the relatively high values of the final R cryst and R free factors. The final model consists of two OXA-48 dimers (A-B and C-D), where each subunit spans the residues 3-243, four ethylene glycol molecules and 786 water molecules.
Antimicrobial Agents and Chemotherapy, 2010
Metallo--lactamases (MBLs) are important enzymatic factors in resistance to -lactam antibiotics... more Metallo--lactamases (MBLs) are important enzymatic factors in resistance to -lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes -lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for -lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.
Matrix Metalloproteinases (MMPs) are a family of zinc endopeptidases involved in essential physio... more Matrix Metalloproteinases (MMPs) are a family of zinc endopeptidases involved in essential physiological processes [1, 2]. To date 23 different enzymes and few isoforms have been identified in the human genome but only half of them have been structurally characterized. The proteolysis of the extracellular matrix (ECM) is a fundamental step in all processes involving the degradation and the remodelling of connective tissue such as wound healing, organ morphogenesis, embryonic development. Matrix metalloproteinases are released ...
The Journal of Physical Chemistry B, 2003
An analysis of the high resolution 3D structures of zinc enzymes shows that the Zn-His-carboxyl (... more An analysis of the high resolution 3D structures of zinc enzymes shows that the Zn-His-carboxyl (ate)-HX (X= OH or NH) H-bond motif is common. We investigate here the influence of this motif in the active site of horse liver alcohol dehydrogenase, which features the Zn-His-Asp-H2O motif. Density functional theory calculations are carried out on models of the active site complexed with the NAD+ cofactor, in which the metal ion binds either the alcohol substrate [Bahnson et al. Pro c. Nat l. Aca d. Sc i. USA 1997, 94, 12797-12802] 1 or a ...
Journal of Molecular Biology, 1999
The crystal structure of the engineered monomeric human Cu,ZnSOD triple mutant F50E/G51E/E133Q (Q... more The crystal structure of the engineered monomeric human Cu,ZnSOD triple mutant F50E/G51E/E133Q (Q133M2SOD) is reported at atomic resolution (1.02 A Ê ). This derivative has about 20 % of the wild-type activity. Crystals of Q133M2SOD have been obtained in the presence of CdCl 2 . The metal binding site is disordered, with both cadmium and copper ions simultaneously binding to the copper site. The cadmium (II) ions occupy about 45 % of the copper sites by binding the four histidine residues which ligate copper in the native enzyme, and two further water molecules to complete octahedral coordination. The copper ion is tri-coordinate, and the fourth histidine (His63) is detached from copper and bridges cadmium and zinc. X-ray absorption spectroscopy performed on the crystals suggests that the copper ion has undergone partial photoreduction upon exposure to the synchrotron light. The structure is also disordered in the disul®de bridge region of loop IV that is located at the subunit/subunit interface in the native SOD dimer. As a consequence, the catalytically relevant Arg143 residue is disordered. The present structure has been compared to other X-ray structures on various isoenzymes and to the solution structure of the same monomeric form. The structural results suggest that the low activity of monomeric SOD is due to the disorder in the conformation of the side-chain of Arg143 as well as of loop IV. It is proposed that the subunit-subunit interactions in the multimeric forms of the enzyme are needed to stabilize the correct geometry of the cavity and the optimal orientation of the charged residues in the active channel. Furthermore, the different coordination of cadmium and copper ions, contemporaneously present in the same site, are taken as models for the oxidized and reduced copper species, respectively. These properties of the structure have allowed us to revisit the enzymatic mechanism.
Journal of Molecular Biology, 1995
Journal of Inorganic Biochemistry, 1991
Journal of Biological Inorganic Chemistry, 2000
The structure of Bacillus pasteurii urease inhibited with acetohydroxamic acid was solved and ref... more The structure of Bacillus pasteurii urease inhibited with acetohydroxamic acid was solved and refined anisotropically using synchrotron X-ray cryogenic diffraction data (1.55 Å resolution, 99.5% completeness, data redundancy p 26, R-factor p 15.1%, PDB code 4UBP). The two Ni ions in the active site are separated by a distance of 3.53 Å. The structure clearly shows the binding mode of the inhibitor anion, symmetrically bridging the two Ni ions in the active site through the hydroxamate oxygen and chelating one Ni ion through the carbonyl oxygen. The flexible flap flanking the active site cavity is in the open conformation. The possible implications of the results on structure-based molecular design of new urease inhibitors are discussed.
Chemistry & Biology, 2009
Crystals of OXA-48 have been grown, by the sitting drop vapour diffusion technique, from a soluti... more Crystals of OXA-48 have been grown, by the sitting drop vapour diffusion technique, from a solution containing 0.1 M Hepes buffer at pH 7.5, 10% PEG 4000, 8% 1-butanol with and OXA-48 at a final concentration of 10 mg/ml. Several datasets from these crystals have been collected on the ID14-1 beamline at ESRF (Grenoble, France) and on the BW7A beamline at EMBL-DESY (Hamburg, Germany). All datasets were collected at 100 K and the crystals were cooled by using the precipitating solution with added 10-15 % (v/v) ethylene glycol as cryoprotectant. The OXA-48 crystals provided a complete dataset to 1.9 Å resolution with maximum diffraction up to 1.6 Å and belong to space group P2 1 with cell parameters a = 63.70 Å; b = 107.18 Å; c = 80.79 Å; β = 111.04°. The crystal asymmetric unit contains four molecules and 48.3 % solvent (Matthews coefficient, 2.38 Å 3 /Da). The data were processed by the program MOSFLM (Leslie, 1991) and scaled using the program SCALA with the TAILS and SECONDARY correction on to achieve an empirical absorption correction . The data collection and data reduction parameters are reported in of the manuscript. Despite trials with several crystals from different batches, in all cases, the observed diffraction was not of the highest possible quality, as shown by the indicators reported in . The relatively low quality of the crystals is probably due to the presence of highly disordered portions of many of the loops present in the structure (see refinement part) resulting in low symmetry and a high Z number. The structure was solved by molecular replacement methods (MOLREP program [Vagin and Teplyakov, 1997] from the CCP4 suite [Collaborative Computational Project N4, 2008]) using as model the structure of OXA-10 (PDB code 1K4F). Model rebuilding and manipulation was performed at all stages with the program COOT (Emsley and Cowtan, 2004). The refinement was carried out by the program REFMAC5 (Murshudov et al., 1997) (from the CCP4 suite). The presence of four independent subunits in the asymmetric unit organized in two dimers related by an approximate non-crystallographic two-fold axis suggested the use of a refinement protocol, similar to that described consisting of NCS restrained cycles followed by TLS refinement, before the final cycles of usual isotropic refinement. This resulted in final R cryst and R free factors of 0.211 and 0.285 respectively whit good geometry and Ramachandran distribution . The final 2Fo-Fc and ∆F maps are of good quality except for the loop regions consisting of the beginning of the Ω-loop in subunit A, where the density for the residue Ile-127 is weak and is totally missing for residue Ser-128. The presence of such disorder explains the relatively high values of the final R cryst and R free factors. The final model consists of two OXA-48 dimers (A-B and C-D), where each subunit spans the residues 3-243, four ethylene glycol molecules and 786 water molecules.
Antimicrobial Agents and Chemotherapy, 2010
Metallo--lactamases (MBLs) are important enzymatic factors in resistance to -lactam antibiotics... more Metallo--lactamases (MBLs) are important enzymatic factors in resistance to -lactam antibiotics that show important structural and functional heterogeneity. BJP-1 is a subclass B3 MBL determinant produced by Bradyrhizobium japonicum that exhibits interesting properties. BJP-1, like CAU-1 of Caulobacter vibrioides, overall poorly recognizes -lactam substrates and shows an unusual substrate profile compared to other MBLs. In order to understand the structural basis of these properties, the crystal structure of BJP-1 was obtained at 1.4-Å resolution. This revealed significant differences in the conformation and locations of the active-site loops, determining a rather narrow active site and the presence of a unique N-terminal helix bearing Phe-31, whose side chain binds in the active site and represents an obstacle for -lactam substrate binding. In order to probe the potential of sulfonamides (known to inhibit various zinc-dependent enzymes) to bind in the active sites of MBLs, the structure of BJP-1 in complex with 4-nitrobenzenesulfonamide was also obtained (at 1.33-Å resolution), thereby revealing the mode of interaction of these molecules in MBLs. Interestingly, sulfonamide binding resulted in the displacement of the side chain of Phe-31 from its hydrophobic binding pocket, where the benzene ring of the molecule is now found. These data further highlight the structural diversity shown by MBLs but also provide interesting insights in the structure-function relationships of these enzymes. More importantly, we provided the first structural observation of MBL interaction with sulfonamides, which might represent an interesting scaffold for the design of MBL inhibitors.
Matrix Metalloproteinases (MMPs) are a family of zinc endopeptidases involved in essential physio... more Matrix Metalloproteinases (MMPs) are a family of zinc endopeptidases involved in essential physiological processes [1, 2]. To date 23 different enzymes and few isoforms have been identified in the human genome but only half of them have been structurally characterized. The proteolysis of the extracellular matrix (ECM) is a fundamental step in all processes involving the degradation and the remodelling of connective tissue such as wound healing, organ morphogenesis, embryonic development. Matrix metalloproteinases are released ...