Stefano Ciurli

A new proposal for urease mechanism based on the crystal structures of the native and inhibited enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels

BACKGROUND Urease catalyzes the hydrolysis of urea, the final step of organic nitrogen mineralization, using a bimetallic nickel centre. The role of the active site metal ions and amino acid residues has not been elucidated to date. Many pathologies are associated with the activity of ureolytic bacteria, and the efficiency of soil nitrogen fertilization with urea is severely decreased by urease activity. Therefore, the development of urease inhibitors would lead to a reduction of environmental pollution, to enhanced efficiency of nitrogen uptake by plants, and to improved therapeutic strategies for treatment of infections due to ureolytic bacteria. Structure-based design of urease inhibitors would require knowledge of the enzyme mechanism at the molecular level. RESULTS The structures of native and inhibited urease from Bacillus pasteurii have been determined at a resolution of 2.0 A by synchrotron X-ray cryogenic crystallography. In the native enzyme, the coordination sphere of each of the two nickel ions is completed by a water molecule and a bridging hydroxide. A fourth water molecule completes a tetrahedral cluster of solvent molecules. The enzyme crystallized in the presence of phenylphosphorodiamidate contains the tetrahedral transition-state analogue diamidophosphoric acid, bound to the two nickel ions in an unprecedented mode. Comparison of the native and inhibited structures reveals two distinct conformations of the flap lining the active-site cavity. CONCLUSIONS The mode of binding of the inhibitor, and a comparison between the native and inhibited urease structures, indicate a novel mechanism for enzymatic urea hydrolysis which reconciles the available structural and biochemical data.

Chemistry of Ni2+ in urease: sensing, trafficking, and catalysis.

Transition metals are both essential to enzymatic catalysis and limited in environmental availability. These two biological facts have together driven organisms to evolve mechanisms for selective metal ion sensing and utilization. Changes in metal ion concentrations are perceived by metal-dependent transcription factors and transduced into appropriate cellular responses, which regulate the machineries of competitive metal ion homeostasis and metallo-enzyme activation. The intrinsic toxicity of the majority of metal ions further creates a need for regulated intracellular trafficking, which is carried out by specific chaperones. The Ni(2+)-dependent urease enzymatic system serves as a paradigm for studying the strategies that cells use to handle an essential, yet toxic, metal ion. Although the discovery of urease as the first biological system for which nickel is essential for activity dates to 1975, the rationale for Ni(2+) selection, as well as the cascade of events involving metal-dependent gene regulation and protein-protein interactions leading to enzyme activation, have yet to be fully unraveled. The past 14 years since the Account by Hausinger and co-workers (Karplus, P. A.; Pearson, M. A.; Hausinger, R. P. Acc. Chem. Res. 1997, 30, 330-337) have witnessed impressive achievements in the understanding of the biological chemistry of Ni(2+) in the urease system. In our Account, we discuss more recent advances in the comprehension of the specific role of Ni(2+) in the catalysis and the interplay between Ni(2+) and other metal ions, such as Zn(2+) and Fe(2+), in the metal-dependent enzyme activity. Our discussion focuses on work carried out in our laboratory. In particular, the structural features of the enzyme bound to inhibitors, substrate analogues, and transition state or intermediate analogues have shed light on the catalytic mechanism. Structural and functional information has been correlated to understand the Ni(2+) sensing effected by NikR, a nickel-dependent transcription factor. The urease activation process, involving insertion of Ni(2+) into the urease active site, has been in part dissected and analyzed through the investigation of the molecular properties of the accessory proteins UreD, UreF, and UreG. The intracellular trafficking of Ni(2+) has been rationalized through a deeper understanding of the structural and metal-binding properties of the metallo-chaperone UreE. All the while, a number of key general concepts have been revealed and developed. These include an understanding of (i) the overall ancillary role of Zn(2+) in nickel metabolism, (ii) the intrinsically disordered nature of the GTPase responsible for coupling the energy consumption to the carbon dioxide requirement for the urease activation process, and (iii) the role of the accessory proteins regulating this GTPase activity.

Structural properties of the nickel ions in urease: novel insights into the catalytic and inhibition mechanisms

Abstract This work provides a comprehensive critical summary of urease spectroscopy, crystallography, inhibitor binding, and site-directed mutagenesis, with special emphasis given to the relationships between the structural features of the Ni-containing active site and the physico–chemical and biochemical properties of this metallo-enzyme. In addition, the recently determined structure of a complex between urease and a transition state analogue is discussed as it leads to a novel, thought-provoking proposal for the enzyme mechanism.

The complex of Bacillus pasteurii urease with acetohydroxamate anion from X-ray data at 1.55 A resolution.

Abstract 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 = 26, R-factor = 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.

The complex of Bacillus pasteurii urease with beta-mercaptoethanol from X-ray data at 1.65-A resolution

Abstract 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.

Nonredox Nickel Enzymes

ion as well as other steps contributed to the mechanism Figure 11. Sequence alignments of selected class I and class II glyoxalase I enzymes created using Clustal W2. Amino acids are colored by property (hydrophobic (red), acidic (blue), basic (purple), other (green)). Metal binding residues are highlighted in yellow. Residues marked with an asterisk (∗) are invariant; thosemarked by other symbols represent low (:) andmoderate (.) variability. TheN-terminal extension and additional loops found in class I enzymes are highlighted in blue. The S. cerevisiae sequence was truncated after 226 of 326 residues. Chemical Reviews Review dx.doi.org/10.1021/cr4004488 | Chem. Rev. 2014, 114, 4206−4228 4218 of product formation. One possible explanation for the difference between the Ni(II)and Cd(II)-bound enzyme kinetics is that differences in the extent of polarization of the substrate result from different metals in the active site, with Cd(II) being less polarizing and therefore less efficient at lowering the energy barrier for proton abstraction. Differences in aqua ligand exchange rates may also play a role, particularly for the Ni(II) enzyme where a slower on-rate for the substrate might suppress the observation of the KIE. Once formed, the enediol(ate) can be protonated at the alternate C atom to complete the isomerization. Based in part on crystal structures of inhibitor complexes (vide supra), mechanisms involving coordination of the enediolate have been proposed (Figure 14), as have mechanisms that involve activatingmetal-bound water molecules to serve as catalytic bases without binding the substrate to the metal. The crystal structures reveal the apparent importance of two cis-aqua ligands in the active site, which could indicate the need for two ciscoordination positions, or a mechanism that employs proton transfers involving both aqua ligands. Coordination of the substrate with loss of a carboxylate ligand has the attractive feature that the Ni(II) site is always charge neutral, and consistent with model chemistry. Efforts to distinguish these mechanisms have employed kinetics and XAS studies of the E. coli Ni(II) enzyme in solution in the presence of various inhibitors, mutations, etc., and the crystal structure of human Glo I obtained in the presence of the transition state analogue, S(N-hydroxy-N-p-iodophenylcarbamoyl)glutathione. The latter structure reveals a five-coordinate Zn center in which both water molecules and Glu172 are displaced upon binding the inhibitor, which binds in a bidentate fashion. Glu172 was proposed to play a role as the catalytic base in the deprotonation and reprotonation of the substrate, a role that is consistent with computational models. Both Rand S-enantiomers of substrates are bound and stereospecifically reprotonated. The presence of two chemically equivalent Glu ligands suggests that one (Glu172) might be the catalytic base for the S-enantiomer, while the other (Glu99) serves that role for the Renantiomer. The corresponding residues in the E. coli enzyme are Glu122 and Glu56, and XAS studies of a hydroxamate inhibitor (L-γ-glutamyl-N-hydroxy-N-methyl-L-glutaminylglycine) complex are consistent with substitution of both aqua ligands, although evidence for the release of a carboxylate was not specifically observed. Although changes in metal ligands greatly affect metal binding affinities, mutation of Glu56 in E56A-E. coli Glo I resulted in the reduction of enzymatic activity to <4% of wild type under conditions of metal saturation, consistent with similar mutations of class I enzymes and the putative role for the carboxylate as a catalytic base. Figure 12. Ribbon diagram of the crystal structure of E. coliGlo I, (PDB code 1F9Z) showing the two subunits of the homo dimer in cyan and gray and the location of the two Ni sites (green spheres) at subunit interfaces. Figure 13.Comparison of the metal site structure of the Ni(II) complex (panel A, PDB code 1F9Z) and the Zn(II) complex (panel B, 1FA5) of E. coli Glo I showing the change in coordination number and geometry for the two metals. The nickel and zinc ions are represented in gray and dark blue, respectively, while CPK coloring is used for all other atoms. WAT = solvent molecules. Protein residues are distinguished by letters indicating the two different subunits of the enzyme. Chemical Reviews Review dx.doi.org/10.1021/cr4004488 | Chem. Rev. 2014, 114, 4206−4228 4219

Urease from the soil bacterium Bacillus pasteurii: immobilization on Ca-polygalacturonate.

Abstract Urease purified from the soil bacterium Bacillus pasteurii was adsorbed and immobilized on a preformed network of Ca-polygalacturonate, a substrate which has a similar composition and morphology to the mucigel present at the root-soil interface. The adsorption proceeded with an essentially quantitative yield, and the immobilized enzyme showed no decrease of specific activity with respect to the free enzyme. The dependence of urease adsorption on NaCl concentration suggested that the enzyme is bound to the carrier gel through electrostatic interactions. The immobilized enzyme showed increased stability, with respect to the free enzyme, with increasing time or temperature, and in the presence of proteolytic enzymes. The pH activity profile revealed that the adsorbed enzyme showed no change in the optimum pH (8.0), but it was more active than the free form in the pH range 5–8. The Michaelis-Menten kinetic pararneters V max and K m were measured for the free ( V max = 1960 ± 250 units ml −1 ; K m = 235 ± 20 mM) and immobilized ( V max = 1740 ± 185 units ml −1 ; K m = 315 ± 25 mM) urease. The substantial similarity of V max in the two cases suggests that there were no conformational changes involving the active site upon enzyme immobilization, while substrate partitioning effects between the bulk solution and the micro-environment surrounding the immobilized enzyme must be operating so as to partly increase its K m . These results suggest that bacterial urease present in plant root mucigel plays a large role in the mobilization of urea N. Its activity is in fact significantly mantained and protected by immobilization on hydrophilic gels such as those produced by root exudates.

Structure-based computational study of the catalytic and inhibition mechanisms of urease

Abstract. The viability of different mechanisms of catalysis and inhibition of the nickel-containing enzyme urease was explored using the available high-resolution structures of the enzyme isolated from Bacillus pasteurii in the native form and inhibited with several substrates. The structures and charge distribution of urea, its catalytic transition state, and three enzyme inhibitors were calculated using ab initio and density functional theory methods. The DOCK program suite was employed to determine families of structures of urease complexes characterized by docking energy scores indicative of their relative stability according to steric and electrostatic criteria. Adjustment of the parameters used by DOCK, in order to account for the presence of the metal ion in the active site, resulted in the calculation of best energy structures for the nickel-bound inhibitors β-mercaptoethanol, acetohydroxamic acid, and diamidophosphoric acid. These calculated structures are in good agreement with the experimentally determined structures, and provide hints on the reactivity and mobility of the inhibitors in the active site. The same docking protocol was applied to the substrate urea and its catalytic transition state, in order to shed light onto the possible catalytic steps occurring at the binuclear nickel active site. These calculations suggest that the most viable pathway for urea hydrolysis involve a nucleophilic attack by the bridging, and not the terminal, nickel-bound hydroxide onto a urea molecule, with active site residues playing important roles in orienting and activating the substrate, and stabilizing the catalytic transition state.

The electronic structure of FeS centers in proteins and models a contribution to the understanding of their electron transfer properties

Iron-sulfur polymetallic centers pose interesting questions on the oxidation state of each metal ion, on the possibility of electron delocalization over the metal ions versus the possibility of existence of equilibria among different localized-valence states, and on the factors determining if, and to what extent, electron delocalization is present. By interpreting the hyperfine coupling of unpaired electrons with 57Fe as well as with 1H, and by analyzing the EPR spectra, the valence distributions of Fe-S polymetallic centers in proteins and model systems are obtained. The systems analyzed here are: [Fe2S2]2+/+, [Fe3S4]+/0, [Fe4S4]3+/2+/+. The understanding of the parameters describing the exchange coupling within pairs of spin vectors, as well as the double exchange coupling, is attempted on the basis of the oxidation states of the various ions. Upon proper scaling of these parameters, we show that it is possible to transfer them from one polymetallic center to another. The electronic properties are tentatively related to the microscopic reduction potentials of each iron in the center.

Zn2+-linked dimerization of UreG from Helicobacter pylori, a chaperone involved in nickel trafficking and urease activation.

The biosynthesis of the active metal‐bound form of the nickel‐dependent enzyme urease involves the formation of a lysine‐carbamate functional group concomitantly with the delivery of two Ni2+ ions into the precast active site of the apoenzyme and with GTP hydrolysis. In the urease system, this role is performed by UreG, an accessory protein belonging to the group of homologous P‐loop GTPases, often required to complete the biosynthesis of nickel‐enzymes. This study is focused on UreG from Helicobacter pylori (HpUreG), a bacterium responsible for gastric ulcers and cancer, infecting large part of the human population, and for which urease is a fundamental virulence factor. The soluble HpUreG was expressed in E. coli and purified to homogeneity. On‐line size exclusion chromatography and light scattering indicated that apo‐HpUreG exists as a monomer in solution. Circular dichroism, which demonstrated the presence of a well‐defined secondary structure, and NMR spectroscopy, which revealed a large number of residues that appear structured on the basis of their backbone amide proton chemical shift dispersion, indicated that, at variance with other UreG proteins so far characterized, this protein is significantly folded in solution. The amino acid sequence of HpUreG is 29% identical to that of HypB from Methanocaldococcus jannaschii, a dimeric zinc‐binding GTPase involved in the in vivo assembly of [Ni,Fe]‐hydrogenase. A homology‐based molecular model of HpUreG was calculated, which allowed us to identify structural and functional features of the protein. Isothermal titration microcalorimetry demonstrated that HpUreG specifically binds 0.5 equivalents of Zn2+ per monomer (Kd = 0.33 ± 0.03 μM), whereas it has 20‐fold lower affinity for Ni2+ (Kd = 10 ± 1 μM). Zinc ion binding (but not Ni2+ binding) causes protein dimerization, as confirmed using light scattering measurements. The structural rearrangement occurring upon Zn2+‐binding and consequent dimerization was evaluated using circular dichroism and fluorescence spectroscopy. Fully conserved histidine and cysteine residues were identified and their role in zinc binding was verified by site‐directed mutagenesis and microcalorimetry. The results are analyzed and discussed with respect to analogous examples of GTPases in nickel metabolism. Proteins 2009.