Jaburetox is a polypeptide derived from jack bean (Canavalia ensiformis) urease and toxic to a broad spectrum of insects, phytopathogenic filamentous fungi and yeasts of medical importance. The elucidation of the structural basis for the mode of action of Jaburetox is the focus of this multifaceted study. Jaburetox in solution is a monomer of 11.0 kDa featuring a large hydrodynamic radius, suggestive of a disordered polypeptide. The intrinsically disordered nature of Jaburetox was theoretically predicted by a comprehensive bioinformatics analysis and experimentally confirmed by light scattering as well as by circular dichroism and NMR spectroscopy. NMR signal assignment provided backbone secondary chemical shifts that indicated that Jaburetox has a low propensity to assume a stable secondary structure. 15N relaxation studies revealed significant backbone mobility, especially in the N-terminal portion of the polypeptide. The solution structure of Jaburetox shows the presence of an a-helical motif close to the N terminus, together with two turn-like structures situated in the central portion of the protein and close to the C terminus. Similar regions were predicted as potential protein-protein interaction sites using computational tools. The knowledge of the structural properties of Jaburetox in solution is a key step to correlate its structural and biological activities.Abbreviations HSQC, heteronuclear single quantum coherence; IDP, intrinsically disordered protein; JBU, jack bean urease; LUV, large unilamellar vesicle;

BackgroundAlthough the entomotoxicity of plant ureases has been reported almost 20 years ago, their insecticidal mechanism of action is still not well understood. Jaburetox is a recombinant peptide derived from one of the isoforms of Canavalia ensiformis (Jack Bean) urease that presents biotechnological interest since it is toxic to insects of different orders. Previous studies of our group using the Chagas disease vector and model insect Rhodnius prolixus showed that the treatment with Jack Bean Urease (JBU) led to hemocyte aggregation and hemolymph darkening, among other effects. In this work, we employed cell biology and biochemical approaches to investigate whether Jaburetox would induce not only cellular but also humoral immune responses in this species.ResultsThe findings indicated that nanomolar doses of Jaburetox triggered cation-dependent, in vitro aggregation of hemocytes of fifth-instar nymphs and adults. The use of specific eicosanoid synthesis inhibitors revealed that the cellular immune response required cyclooxygenase products since indomethacin prevented the Jaburetox-dependent aggregation whereas baicalein and esculetin (inhibitors of the lipoxygenases pathway) did not. Cultured hemocytes incubated with Jaburetox for 24 h showed cytoskeleton disorganization, chromatin condensation and were positive for activated caspase 3, an apoptosis marker, although their phagocytic activity remained unchanged. Finally, in vivo treatments by injection of Jaburetox induced both a cellular response, as observed by hemocyte aggregation, and a humoral response, as seen by the increase of spontaneous phenoloxidase activity, a key enzyme involved in melanization and defense. On the other hand, the humoral response elicited by Jaburetox injections did not lead to an increment of antibacterial or lysozyme activities. Jaburetox injections also impaired the clearance of the pathogenic bacteria Staphylococcus aureus from the hemolymph leading to increased mortality, indicating a possible immunosuppression induced by treatment with the peptide.ConclusionsIn our experimental conditions and as part of its toxic action, Jaburetox activates some responses of the immune system of R. prolixus both in vivo and in vitro, although this induction does not protect the insects against posterior bacterial infections. Taken together, these findings contribute to the general knowledge of insect immunity and shed light on Jaburetox’s mechanism of action.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1710-3) contains supplementary material, which is available to authorized users.

The novel ternary Zn­(II)-thiourea–urea ionic cocrystal [Zn­(thiourea)­(urea)­Cl2], (ZnTU) has been prepared by both solution and mechanochemical processes and structurally characterized by solid-state methods. ZnTU exhibited improved response properties to water as relative humidity as inherited from thiourea. The results of enzymatic activity measurements provide evidence that ZnTU is effective in modulating urea hydrolysis both in vitro (negatively impacting on the activity of isolated urease) and in vivo (decreasing the ureolytic activity of Sporosarcina pasteurii, a widespread soil bacterium), and that Zn­(II) is the component of the cocrystal acting as the actual urease inhibitor. Concomitantly, the analysis of the ammonia monooxygenase (AMO) enzymatic activity in Nitrosomonas europaea, taken as a representative of soil ammonia-oxidizing bacteria, in the presence of ZnTU reveals that thiourea is the only component of ZnTU able to inhibit ammonia conversion to nitrite. It has also been shown that ZnTU maintains these capabilities when applied to bacterial cultures containing both S. pasteurii and N. europaea working in tandem. The compound can thus act both as a fertilizer via urea and via the Zn­(II) and thiourea components, as a dual action inhibitor of the activities of the enzymes urease and AMO, which are responsible for the negative environmental and economic impact of the agricultural use of urea as soil fertilizer. These results indicate that ZnTU should be considered a novel material to improve N fertilization efficiency, toward a more environment-friendly agricultural practice.

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.

Ammonia monooxygenase is a copper-dependent membrane-bound enzyme that catalyzes the first step of nitrification in ammonia-oxidizing bacteria to convert ammonia to hydroxylamine, through the reductive insertion of a dioxygen-derived O atom in an N–H bond. This reaction is analogous to that carried out by particulate methane monooxygenase, which catalyzes the conversion of methane to methanol. The enzymatic activity of ammonia monooxygenase must be modulated to reduce the release of nitrogen-based soil nutrients for crop production into the atmosphere or underground waters, a phenomenon known to significantly decrease the efficiency of primary production as well as increase air and water pollution. The structure of ammonia monooxygenase is not available, rendering the rational design of enzyme inhibitors impossible. This study describes a successful attempt to build a structural model of ammonia monooxygenase, and its accessory proteins AmoD and AmoE, from Nitrosomonas europaea, taking advantage of the high sequence similarity with particulate methane monooxygenase and the homologous PmoD protein, for which crystal structures are instead available. The results obtained not only provide the structural details of the proteins ternary and quaternary structures, but also suggest a location for the copper-containing active site for both ammonia and methane monooxygenases, as well as support a proposed structure of a CuA-analogue dinuclear copper site in AmoD and PmoD. Graphic abstract

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Jaburetox is a recombinant peptide derived from a Canavalia ensiformis urease that presents toxic effects upon several species of insects, phytopathogenic fungi and yeasts of medical importance. So far, no toxicity of Jaburetox to mammals has been shown. Previous reports have identified biochemical targets of this toxic peptide in insect models, although its mechanism of action is not completely understood. In this work, we aimed to characterize the effects of Jaburetox in hemolymphatic insect cells. For this purpose, the model insect and Chagas' disease vector Rhodnius prolixus was used. In vivo and in vitro experiments indicated that Jaburetox interacts with a subset of hemocytes and it can be found in various subcellular compartments. In insects injected with Jaburetox there was an increase in the gene expression of the enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP), chitin synthase and nitric oxide synthase (NOS). Nevertheless, the expression of NOS protein, the enzyme activities of UAP and acid phosphatase (a possible link between UAP and NOS) as well as the phosphorylation state of proteins remained unchanged upon the in vivo Jaburetox treatment. Nitric oxide (NO) imaging using fluorescent probes showed that Jaburetox augmented NO production in the hemocyte aggregates when compared to controls. Even though Jaburetox activated the hemocytes, as demonstrated by wheat germ agglutinin binding assays, the peptide did not lead to an increase of their phagocytic behavior. Taken together, these findings contribute to our understanding of toxic effects of Jaburetox, a peptide with biotechnological applications and a prospective tool for rational insect control.