One enzyme, many faces: urease is also canatoxin.

Ureases catalyze the hydrolysis of urea into carbamate and ammonia. Well-conserved proteins, most plant ureases are hexamers of a single chain subunit, like the most abundant isoform of the jack bean (Canavalia ensiformis) urease (JBU). Canatoxin (CNTX) was originally isolated from these seeds as a neurotoxic protein, and later characterized as an isoform of JBU with lower molecular mass and enzyme activity. Inactive CNTX oligomers form upon storage and stabilization of CNTX was achieved by treatment with low concentration of formaldehyde, avoiding its oligomerization. Here, nano-LC-MS/MS-based peptide analysis of CNTX revealed 804 amino acids identical to those of JBU's sequence (840 amino acids). De novo sequencing of CNTX revealed 15 different peptides containing substitution of amino acid residues, denoting CNTX as a product of a paralog gene of JBU. The MS/MS analysis of formaldehyde-treated CNTX showed that amino acid residues located at the trimer-trimer interface of JBU's hexamer were modified. The data confirmed that CNTX is an isoform of JBU and elucidated that stabilization by formaldehyde treatment occurs by modification of amino acids at the protein's surface that prevents the formation of the hexamer and of higher molecular mass inactive aggregates. HIGHLIGHTSCanatoxin (CNTX) is an isoform of jack bean urease (JBU, hexamer of 90 kDa chains)MS/MS sequencing of CNTX showed 804 amino acids identical in JBU (840 residues)Formaldehyde treatment of CNTX stabilizes its toxicity and avoids oligomerizationModified amino acid residues in CNTX are at the trimer-trimer interface of JBUCommunicated by Ramaswamy H. Sarma.

Risk assessment of the antifungal and insecticidal peptide Jaburetox and its parental protein the Jack bean (Canavalia ensiformis) urease.

Jaburetox (JBTX) is an insecticidal and antifungal peptide derived from jack bean (Canavalia ensiformis) urease that has been considered a candidate for developing genetically modified crops. This study aimed to perform the risk assessment of the peptide JBTX following the general recommendations of the two-tiered, weight-of-evidence approach proposed by International Life Sciences Institute. The urease of C. ensiformis (JBU) and its isoform JBURE IIb (the JBTX parental protein) were assessed. The history of safe use revealed no hazard reports for the studied proteins. The available information shows that JBTX possesses selective activity against insects and fungi. JBTX and JBU primary amino acids sequences showed no relevant similarity to toxic, antinutritional or allergenic proteins. Additionally, JBTX and JBU were susceptible to in vitro digestibility, and JBU was also susceptible to heat treatment. The results did not identify potential risks of adverse effects and reactions associated to JBTX. However, further allergen (e.g. serum IgE binding test) and toxicity (e.g. rodent toxicity tests) experimentation can be done to gather additional safety information on JBTX, and to meet regulatory inquiries for commercial approval of transgenic cultivars expressing this peptide.

Jaburetox affects gene expression and enzyme activities in Rhodnius prolixus, a Chagas' disease vector.

Jaburetox, a recombinant peptide of ∼11kDa derived from one of the Canavalia ensiformis (Jack Bean) urease isoforms, is toxic and lethal to insects belonging to different orders when administered orally or via injection. Previous findings indicated that Jaburetox acts on insects in a complex fashion, inhibiting diuresis and the transmembrane potential of Malpighian tubules, interfering with muscle contractility and affecting the immune system. In vitro, Jaburetox forms ionic channels and alters permeability of artificial lipid membranes. Moreover, recent data suggested that the central nervous system (CNS) is a target organ for ureases and Jaburetox. In this work, we employed biochemical, molecular and cellular approaches to explore the mode of action of Jaburetox using Rhodnius prolixus, one of the main Chagas' disease vectors, as experimental model. In vitro incubations with fluorescently labeled Jaburetox indicated a high affinity of the peptide for the CNS but not for salivary glands (SG). The in vitro treatment of CNS or SG homogenates with Jaburetox partially inhibited the activity of nitric oxide synthase (NOS), thus disrupting nitrinergic signaling. This inhibitory effect was also observed in vivo (by feeding) for CNS but not for SG, implying differential modulation of NOS in these organs. The inhibition of NOS activity did not correlate to a decrease in expression of its mRNA, as assessed by qPCR. UDP-N-acetylglucosamine pyrophosphorylase (UAP), a key enzyme in chitin synthesis and glycosylation pathways and a known target of Jaburetox in insect CNS, was also affected in SG, with activation of the enzyme seen after both in vivo or in vitro treatments with the peptide. Unexpectedly, incubation of Jaburetox with a recombinant R. prolixus UAP had no effect on its activity, implying that the enzyme's modulation by the peptide requires the participation of other factor(s) present in CNS or SG homogenates. Feeding Jaburetox to R. prolixus decreased the mRNA levels of UAP and chitin synthase, indicating a complex regulation exerted by the peptide on these enzymes. No changes were observed upon Jaburetox treatment in vivo and in vitro on the activity of the enzyme acid phosphatase, a possible link between UAP and NOS. Here we have demonstrated for the first time that the Jaburetox induces changes in gene expression and that SG are another target for the toxic action of the peptide. Taken together, these findings contribute to a better understanding of the mechanism of action of Jaburetox as well as to the knowledge on basic aspects of the biochemistry and neurophysiology of insects, and might help in the development of optimized strategies for insect control.

Pliable natural biocide: Jaburetox is an intrinsically disordered insecticidal and fungicidal polypeptide derived from jack bean urease.

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. (15)N 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 α-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.

Expression analysis and molecular characterization of aquaporins in Rhodnius prolixus.

Aquaporins (AQPs) are water channels responsible for transport of water and, in some cases, transport of small solutes such as urea and glycerol across lipid bilayer membranes. Hematophagous insects, such as Rhodnius prolixus, ingest large volumes of fluid and must rapidly eliminate the excess of water and salts from the blood meal within the gut. In order to deal with this increase in body fluid volume, a hormone-controlled diuresis is activated, during which a high rate of water and salt absorption occurs across the anterior midgut, followed by secretion of water and salts by the Malpighian tubules (MTs). Previously, one member of the MIP family (major intrinsic protein that includes the AQP family) was identified in the MTs of R. prolixus, and named RpMIP. We have described here that the RpMIP gene has different variants, and is present in tissues other than MTs. In addition, we have characterized a new AQP (RhoprAQP1) found in different tissues of R. prolixus. The expression of these transcripts in unfed insects as well as blood fed insects was evaluated using real-time quantitative PCR. Molecular models of the predicted proteins were constructed and the characteristics of their pores evaluated. A yeast complementation assay was used to validate that the products of these transcripts were bona fide AQPs. Both RhoprAQP1 and RhoprMIP-A were capable of transporting water whereas RhoprMIP-A was also capable of transporting H2O2. Taken together, these analyses suggest that RhoprMIP is probably an aquaglyceroporin, while RhoprAQP1 appears to be a strict aquaporin that transports only water.

Role of lysine and acidic amino acid residues on the insecticidal activity of Jackbean urease.

Canavalia ensiformis has three isoforms of urease: Jackbean urease (JBU), Jackbean urease II and canatoxin. These isoforms present several biological activities, independent from the enzymatic property, such as entomotoxicity and antifungal properties. The entomotoxic activity is a property of the whole protein, as well as of a 10 kDa peptide released by insect digestive enzymes. Here we have used chemical modification to observe the influence of lysines and acidic residues on JBU enzymatic and insecticidal activities. Chemical modification of lysine residues was performed with dimethylamine-borane complex and formaldehyde, and acidic residues were modified by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and ethylenediamine. Derivatized ureases, called JBU-Lys (lysine-modified) and JBU-Ac (acidic residues-modified), were assayed for their biochemical and insecticidal properties. Neither modification altered significantly the kinetic parameters analyzed, indicating that no residue critical for the enzyme activity was affected and that the modifications did not incur in any significant structural alteration. On the other hand, both modifications reduced the toxic activity of the native protein fed to Dysdercus peruvianus. The changes observed in the entomotoxic property of the derivatized proteins reflect alterations in different steps of JBU's toxicity towards insects. JBU-Ac is not susceptible to hydrolysis by insect digestive enzymes, hence impairing the release of toxic peptide(s), while JBU-Lys is processed as the native protein. On the other hand, the antidiuretic effect of JBU on Rhodnius prolixus is altered in JBU-Lys, but not in JBU-Ac. Altogether, these data emphasize the role of lysine and acidic residues on the insecticidal properties of ureases.

Evidence-based docking of the urease activation complex.

Ureases require accessory proteins for their activation and proper function. In Klebsiella aerogenes, UreD, UreF, UreG, and UreE are sequentially complexed to UreABC as required for its activation. Until now, only low-resolution structures are available for this activation complex. To circumvent such limitation, our work intends to provide an atomic-level model for the (UreABC-UreDFG)3 complex from K. aerogenes, by employing comparative modeling associated to sequential macromolecular dockings, validated through small-angle X-ray scattering profiles and comparison with results from cross-linking, mutagenesis, and pull-down experiments. Additionally, normal mode analyses of the obtained complex supported the characterization of the elevated flexibility of both UreD-UreF dimer and (UreABC-UreDFG)3 oligomer, explaining the previously observed diffuse binding of UreD to the apoenzyme. The model shown here is the first atomic-level depiction of this complex, a required step for the unraveling of the urease activation process. (1)Both authors share senior authorship. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:6.

Biochemical and structural studies on native and recombinant Glycine max UreG: a detailed characterization of a plant urease accessory protein.

Urea is the nitrogen fertilizer most utilized in crop production worldwide. Understanding all factors involved in urea metabolism in plants is an essential step towards assessing and possibly improving the use of urea by plants. Urease, the enzyme responsible for urea hydrolysis, and its accessory proteins, necessary for nickel incorporation into the enzyme active site and concomitant activation, have been extensively characterized in bacteria. In contrast, little is known about their plant counterparts. This work reports a detailed characterization of Glycine max UreG (GmUreG), a urease accessory protein. Two forms of native GmUreG, purified from seeds, were separated by metal affinity chromatography, and their properties (GTPase activity in absence and presence of Ni(2+) or Zn(2+), secondary structure and metal content) were compared with the recombinant protein produced in Escherichia coli. The binding affinity of recombinant GmUreG (rGmUreG) for Ni(2+) and Zn(2+) was determined by isothermal titration calorimetry. rGmUreG binds Zn(2+) or Ni(2+) differently, presenting a very tight binding site for Zn(2+) (K (d) = 0.02 ± 0.01 µM) but not for Ni(2+), thus suggesting that Zn(2+) may play a role on the plant urease assembly process, as suggested for bacteria. Size exclusion chromatography showed that Zn(2+) stabilizes a dimeric form of the rGmUreG, while NMR measurements indicate that rGmUreG belongs to the class of intrinsically disordered proteins. A homology model for the fully folded GmUreG was built and compared to bacterial UreG models, and the possible sites of interaction with other accessory proteins were investigated.

Stage-specific gut proteinases of the cotton stainer bug Dysdercus peruvianus: role in the release of entomotoxic peptides from Canavalia ensiformis urease.

Canavalia ensiformis ureases are toxic to insects of different orders. The entomotoxicity of urease is due to a 10 kDa internal peptide released by proteinases in the insect digestive tract. We previously observed that, given orally, urease is toxic to nymphs of Dysdercus peruvianus, but does not affect adults. Here we characterized the major proteolytic activities of D. peruvianus midgut homogenates and investigated their in vitro-catalyzed release of the 10 kDa entomotoxic peptide from urease. Cysteine, aspartic and metalloproteinases are present in both homogenates. Variations in optimal pH and susceptibility to inhibitors indicated differences in the enzyme profiles in the two developmental stages. Only nymph homogenates released approximately 10 kDa fragment(s) from urease, recognized by antibodies against the entomotoxic peptide. Fluorogenic substrates containing urease partial sequences flanking the N-terminal or the C-terminal portion of the entomotoxic peptide were efficiently cleaved by homogenates from nymphs, but much more slowly by the adult homogenate. Different classes of enzymes in the homogenates cleaved both substrates suggesting that in vivo the release of the entomotoxic peptide results from the concerted action of at least two different proteinases. Our findings support the view that a differential processing of ingested urease by the insects explains at least in part the lack of toxicity in adults.

Jackbean, soybean and Bacillus pasteurii ureases: biological effects unrelated to ureolytic activity.

In this work we compared two plant ureases, jackbean urease (JBU) and embryo-specific soybean urease (SBU) and a bacterial (Bacillus pasteurii) urease, for kinetic parameters and other biological properties described recently for ureases that are independent of the ureolytic activity. The insecticidal effect of ureases was investigated in feeding trials with the cotton sucker bug, Dysdercus peruvianus (Hemiptera) as an insect model. Contrasting with B. pasteurii urease (PBU), both plant ureases presented potent insecticidal activity, with LD(50) values of 0.017% (w/w) and 0.052% (w/w) for JBU and SBU, respectively. The insecticidal property of JBU or SBU was not affected by treatment with p-hydroxymercuribenzoate, an irreversible inhibitor of ureolytic activity of both proteins. Also, contrasting with canatoxin - a urease isoform from jackbean seeds that displays a toxic effect in mice (LD(50) = 2 mg x kg(-1)) - no lethality was seen in mice injected intraperitoneally with JBU or SBU (20 mg x kg(-1)). Similarly to canatoxin, the three enzymes promoted aggregation of blood platelets (EC(50) = 400.0 micro g x mL(-1), 22.2 micro g x mL(-1), 15.8 micro g x mL(-1) for BPU, SBU and JBU, respectively). This platelet activating property was also independent of urease activity. Comparison of the kinetic properties indicated that SBU is fivefold less susceptible than JBU to inhibition by acetohydroxamic acid, a chelator of Ni(+2) and Zn(+2) ions. The ureases also showed different susceptibility to agents that modify cysteine residues, such as p-hydroxymercuribenzoate and p-benzoquinone. Altogether, these data emphasize that biological properties that are independent of ureolytic activity are not restricted to jackbean ureases and that these proteins may have a role in plant defense against insect predators.