Stefano Benini

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.

Structural basis for DNA recognition and loading into a viral packaging motor

Genome packaging into preformed viral procapsids is driven by powerful molecular motors. The small terminase protein is essential for the initial recognition of viral DNA and regulates the motor’s ATPase and nuclease activities during DNA translocation. The crystal structure of a full-length small terminase protein from the Siphoviridae bacteriophage SF6, comprising the N-terminal DNA binding, the oligomerization core, and the C-terminal β-barrel domains, reveals a nine-subunit circular assembly in which the DNA-binding domains are arranged around the oligomerization core in a highly flexible manner. Mass spectrometry analysis and four further crystal structures show that, although the full-length protein exclusively forms nine-subunit assemblies, protein constructs missing the C-terminal β-barrel form both nine-subunit and ten-subunit assemblies, indicating the importance of the C terminus for defining the oligomeric state. The mechanism by which a ring-shaped small terminase oligomer binds viral DNA has not previously been elucidated. Here, we probed binding in vitro by using EPR and surface plasmon resonance experiments, which indicated that interaction with DNA is mediated exclusively by the DNA-binding domains and suggested a nucleosome-like model in which DNA binds around the outside of the protein oligomer.

The crystal structure of Sporosarcina pasteurii urease in a complex with citrate provides new hints for inhibitor design

Urease, the enzyme that catalyses the hydrolysis of urea, is a virulence factor for a large number of ureolytic bacterial human pathogens. The increasing resistance of these pathogens to common antibiotics as well as the need to control urease activity to improve the yield of soil nitrogen fertilization in agricultural applications has stimulated the development of novel classes of molecules that target urease as enzyme inhibitors. We report on the crystal structure at 1.50-Å resolution of a complex formed between citrate and urease from Sporosarcina pasteurii, a widespread and highly ureolytic soil bacterium. The fit of the ligand to the active site involves stabilizing interactions, such as a carboxylate group that binds the nickel ions at the active site and several hydrogen bonds with the surrounding residues. The citrate ligand has a significantly extended structure compared with previously reported ligands co-crystallized with urease and thus represents a unique and promising scaffold for the design of new, highly active, stable, selective inhibitors.

Biomolecular Characterization of the Levansucrase of Erwinia amylovora, a Promising Biocatalyst for the Synthesis of Fructooligosaccharides

Erwinia amylovora is a plant pathogen that affects Rosaceae, such as apple and pear. In E. amylovora the fructans, produced by the action of a levansucrase (EaLsc), play a role in virulence and biofilm formation. Fructans are bioactive compounds, displaying health-promoting properties in their own right. Their use as food and feed supplements is increasing. In this study, we investigated the biomolecular properties of EaLsc using HPAEC-PAD, MALDI-TOF MS, and spectrophotometric assays. The enzyme, which was heterologously expressed in Escherichia coli in high yield, was shown to produce mainly fructooligosaccharides (FOSs) with a degree of polymerization between 3 and 6. The kinetic properties of EaLsc were similar to those of other phylogenetically related Gram-negative bacteria, but the good yield of FOSs, the product spectrum, and the straightforward production of the enzyme suggest that EaLsc is an interesting biocatalyst for future studies aimed at producing tailor-made fructans.

Fluoride inhibition of Sporosarcina pasteurii urease: structure and thermodynamics

Urease is a nickel-dependent enzyme and a virulence factor for ureolytic bacterial human pathogens, but it is also necessary to convert urea, the most worldwide used fertilizer, into forms of nitrogen that can be taken up by crop plants. A strategy to control the activity of urease for medical and agricultural applications is to use enzyme inhibitors. Fluoride is a known urease inhibitor, but the structural basis of its mode of inhibition is still undetermined. Here, kinetic studies on the fluoride-induced inhibition of urease from Sporosarcina pasteurii, a widespread and highly ureolytic soil bacterium, were performed using isothermal titration calorimetry and revealed a mixed competitive and uncompetitive mechanism. The pH dependence of the inhibition constants, investigated in the 6.5–8.0 range, reveals a predominant uncompetitive mechanism that increases by increasing the pH, and a lesser competitive inhibition that increases by lowering the pH. Ten crystal structures of the enzyme were independently determined using five crystals of the native form and five crystals of the protein crystallized in the presence of fluoride. The analysis of these structures revealed the presence of two fluoride anions coordinated to the Ni(II) ions in the active site, in terminal and bridging positions. The present study consistently supports an interaction of fluoride with the nickel centers in the urease active site in which one fluoride competitively binds to the Ni(II) ion proposed to coordinate urea in the initial step of the catalytic mechanism, while another fluoride uncompetitively substitutes the Ni(II)-bridging hydroxide, blocking its nucleophilic attack on urea.

The crystal structure of Erwinia amylovora levansucrase provides a snapshot of the products of sucrose hydrolysis trapped into the active site.

Levansucrases are members of the glycoside hydrolase family and catalyse both the hydrolysis of the substrate sucrose and the transfer of fructosyl units to acceptor molecules. In the presence of sufficient sucrose, this may either lead to the production of fructooligosaccharides or fructose polymers. Aim of this study is to rationalise the differences in the polymerisation properties of bacterial levansucrases and in particular to identify structural features that determine different product spectrum in the levansucrase of the Gram-negative bacterium Erwinia amylovora (Ea Lsc, EC 2.4.1.10) as compared to Gram-positive bacteria such as Bacillus subtilis levansucrase. Ea is an enterobacterial pathogen responsible for the Fire Blight disease in rosaceous plants (e.g., apple and pear) with considerable interest for the agricultural industry. The crystal structure of Ea Lsc was solved at 2.77 Å resolution and compared to those of other fructosyltransferases from Gram-positive and Gram-negative bacteria. We propose the structural features, determining the different reaction products, to reside in just a few loops at the rim of the active site funnel. Moreover we propose that loop 8 may have a role in product length determination in Gluconacetobacter diazotrophicus LsdA and Microbacterium saccharophilum FFase. The Ea Lsc structure shows for the first time the products of sucrose hydrolysis still bound in the active site.

Kinetic and Structural Studies Reveal a Unique Binding Mode of Sulfite to the Nickel Center in Urease.

Urease is the most efficient enzyme known to date, and catalyzes the hydrolysis of urea using two Ni(II) ions in the active site. Urease is a virulence factor in several human pathogens, while causing severe environmental and agronomic problems. Sporosarcina pasteurii urease has been used extensively in the structural characterization of the enzyme. Sodium sulfite has been widely used as a preservative in urease solutions to prevent oxygen-induced oxidation, but its role as an inhibitor has also been suggested. In the present study, isothermal titration microcalorimetry was used to establish sulfite as a competitive inhibitor for S. pasteurii urease, with an inhibition constant of 0.19mM at pH7. The structure of the urease-sulfite complex, determined at 1.65Å resolution, shows the inhibitor bound to the dinuclear Ni(II) center of urease in a tridentate mode involving bonds between the two Ni(II) ions in the active site and all three oxygen atoms of the inhibitor, supporting the observed competitive inhibition kinetics. This coordination mode of sulfite has never been observed, either in proteins or in small molecule complexes, and could inspire synthetic coordination chemists as well as biochemists to develop urease inhibitors based on this chemical moiety.