No abstract

The persistence of Helicobacter pylori in the hostile environment of the human stomach is ensured by the activity of urease. The essentiality of Ni(2+) for this enzyme demands proper intracellular trafficking of this metal ion. The metallo-chaperone UreE promotes Ni(2+) insertion into the apo-enzyme in the last step of urease maturation while facilitating concomitant GTP hydrolysis. The present study focuses on the metal-binding properties of HpUreE (Helicobacter pylori UreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. ITC (isothermal titration calorimetry) showed that HpUreE binds one equivalent of Ni(2+) (Kd=0.15 microM) or Zn(2+) (Kd=0.49 microM) per dimer, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn(2+) and Ni(2+), which involve crucial histidine residues, were revealed by site-directed mutagenesis, suggesting a mechanism for discriminating metal-ion-specific binding. The formation of a HpUreE-HpUreG protein complex was revealed by NMR spectroscopy, and the thermodynamics of this interaction were established using ITC. A role for Zn(2+), and not for Ni(2+), in the stabilization of this complex was demonstrated using size-exclusion chromatography, light scattering, and ITC experiments. A calculated viable structure for the complex suggested the presence of a novel binding site for Zn(2+), actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidence of a UreE-UreG functional interaction in vivo. A possible role for Zn(2+) in the Ni(2+)-dependent urease system is envisaged.

The binding constants between Ni2+ and Helicobacterpylori NikR have been determined using isothermal titration microcalorimetry in order to rationalize the role of this protein as a nickel-dependent biological sensor.

The survival and growth of the pathogen Helicobacter pylori in the gastric acidic environment is ensured by the activity of urease, an enzyme containing two essential Ni2+ ions in the active site. The metallo-chaperone UreE facilitates in vivo Ni2+ insertion into the apo-enzyme. Crystals of apo-HpUreE and its Ni2+ and Zn2+ bound forms were obtained from protein solutions in the absence and presence of the metal ions. The crystal structures of the homodimeric protein, determined at 2.00 Å (apo), 1.59 Å (Ni) and 2.52 Å (Zn) resolution, show the conserved proximal and solvent-exposed His102 residues from two adjacent monomers invariably involved in metal binding. The C-terminal regions of the apo-protein are disordered in the crystal, but acquire significant ordering in the presence of the metal ions due to the binding of His152. The analysis of X-ray absorption spectral data obtained on solutions of Ni2+- and Zn2+-HpUreE provided accurate information of the metal ion environment in the absence of solid-state effects. These results reveal the role of the histidine residues at the protein C-terminus in metal ion binding, and the mutual influence of protein framework and metal ion stereo-electronic properties in establishing coordination number and geometry leading to metal selectivity.

The pathogenicity of Helicobacter pylori depends on the activity of urease for pH modification. Urease activity requires assembly of a dinickel active site that is facilitated in part by GTP hydrolysis by UreG. The proper functioning of Helicobacter pylori UreG (HpUreG) is dependent on Zn(II) binding and dimerization. X-ray absorption spectroscopy and structural modeling were used to elucidate the structure of the Zn(II) site in HpUreG. These studies independently indicated a site at the dimer interface that has trigonal bipyramidal geometry and is composed of two axial cysteines at 2.29(2)Å, two equatorial histidines at 1.99(1)Å, and a solvent-accessible coordination site. The final model for the Zn(II) site structure was determined by refining multiple-scattering extended X-ray absorption fine structure fits using the geometry predicted by homology modeling and ab initio calculations.

Cyanogenic glycosides form part of a binary plant defense system that, upon catabolism, detonates a toxic hydrogen cyanide bomb. In seed plants, the initial step of cyanogenic glycoside biosynthesis—the conversion of an amino acid to the corresponding aldoxime—is catalyzed by a cytochrome P450 from the CYP79 family. An evolutionary conundrum arises, as no CYP79s have been identified in ferns, despite cyanogenic glycoside occurrence in several fern species. Here, we report that a flavin-dependent monooxygenase (fern oxime synthase; FOS1), catalyzes the first step of cyanogenic glycoside biosynthesis in two fern species (Phlebodium aureum and Pteridium aquilinum), demonstrating convergent evolution of biosynthesis across the plant kingdom. The FOS1 sequence from the two species is near identical (98%), despite diversifying 140 MYA. Recombinant FOS1 was isolated as a catalytic active dimer, and in planta, catalyzes formation of an N-hydroxylated primary amino acid; a class of metabolite not previously observed in plants.

Ppdfn1 is a defensin gene previously identified in peach (Prunus persica). The biological role of Ppdfn1 was investigated by analysing its expression profile in leaves, flowers and fruits, either inoculated with the Monilinia laxa fungal pathogen or mock‐inoculated. Ppdfn1 expression was highest in flowers and, in fruits, did not vary upon M. laxa inoculation. To characterize the PpDFN1 antifungal activity, the recombinant mature peptide was expressed in Escherichia coli and purified; recombinant PpDFN1 displays antifungal activity against Botrytis cinerea, M. laxa and Penicillium expansum, with IC50 values of 15·1, 9·9 and 1·1 μg mL−1, respectively. Treatment of fungal hyphae with FITC‐labelled PpDFN1 indicated that the peptide is not internalized by fungal hyphae, but localizes on their external cell surface. At this site, PpDFN1 is capable of membrane destabilization and permeabilization, as demonstrated by SYTOX Green fluorescence uptake by the treated mycelia. Using artificial lipid monolayers, it was shown that PpDFN1 interacts with sphingolipid‐containing membranes; however the strongest interaction occurs with monolayers composed of lipids extracted from sensitive fungi, such as P. expansum. These data suggest that the lipid composition of fungal membranes is of key relevance for defensin specificity.

The analysis of the sequence of Helicobacter pylori UreD(H), an accessory protein involved in the activation of urease through the assembly of the Ni(2+)-containing active site, revealed the presence of two domains. The structure of these domains was calculated using threading and modeling algorithms. A search for putative binding sites on the protein surface was carried out using dedicated algorithms sensitive to either sequence conservation or structural similarity based on geometry and physicochemical properties. The results suggest that UreD(H) acts as a multifunctional molecular recognition platform facilitating the interaction between apo-urease and the ancillary proteins UreG, UreF, and UreE, responsible for nickel trafficking and delivering.