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Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO(2). These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli. The antifungal activity of ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H(+) and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (~10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to ureases. Plant ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10(-6)-10(-7)M.

Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea to produce ammonia and carbon dioxide These enzymes, which are found in fungi, bacteria, and plants, show very similar structures. Despite an abundance of urease in vegetal tissues, the physiological role of this enzyme in plants is still poorly understood. It has been previously described that ureases from the legumes jackbean ( Canavalia ensiformis) and soybean ( Glycine max) have insecticidal activity and antifungal properties. This work presents the physicochemical purification and characterization of a urease from cotton ( Gossypium hirsutum) seeds, the first description of this enzyme in Malvaceae. The urease content varied among different cotton cultivars. Cotton seed urease (98.3 kDa) displayed low ureolytic activity but exhibited potent antifungal properties at sub-micromolar concentrations against different phytopathogenic fungi. As described for other ureases, the antifungal effect of cotton urease persisted after treatment with an irreversible inhibitor of its enzyme activity. The data suggest an important role of these proteins in plant defense.

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Soybean (Glycine max (L) Merril) seeds are known to contain different proteins displaying antinutritional and/or toxic effects, such as soybean agglutinin (an N-acetylgalactosamine-specific lectin), proteinase inhibitors (Kunitz-and Bowman-Birk-type trypsin and chymotrypsin inhibitors) and urease (seed and tissue isoforms). Two other toxic proteins were previously isolated from soybeans, soyatoxin (21 kDa) and soybean toxin (18.4 kDa), which are immunologically related to canatoxin, a toxic protein from Canavalia ensiformis (jackbean) seeds. In this work we have screened crude extracts from seeds of six different soybean cultivars, which together represent most of the crop harvested in the southern region of Brazil, for the presence of urease, trypsin inhibitory and haemagglutination activities, intraperitoneal toxicity in mice and immunoreactivity against anti-canatoxin antibodies. Significant differences were found in the contents of proteinase inhibitors, lectin, urease activity and lethality in mice. The relevance of these findings to the agronomic qualities and to the choice of soybean cultivars to be used as food or feed is discussed.

The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-012-9894-1) contains supplementary material, which is available to authorized users.

According to the World Bank and FAO, the population grows worldwide and the poorest countries are expected to double their population within the next decades, reaching approximately 7.2 billion in 2015. Moreover, the food and financial crisis together with the global economic recession pushed the number of hungry and undernourished people in the world to unprecedented levels. The substitution of animal proteins by plant proteins in food and feed is a general trend because of the lower cost and better production efficiency. Pathogens and pests can reduce the crop yields up to 30%. In some places, the losses can reach 80% due to climate conditions, proliferation of insects, and fungal diseases. All together, the harvest and postharvest losses vary from 5% to 20% and depending on the commodity can be as high as 50%. Plants have a complex chemical armory for defense composed of low and high molecular mass compounds that can act over a variety of pests and pathogens, from micro-organisms to phytophagous insects or grazing animals. Among them, plant fungitoxic and insecticidal polypeptides represent promising alternatives to increase the supply of plant-derived proteins and tackle the hunger in a global scale.

Ureases, nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and bicarbonate, are widespread in plants, bacteria, and fungi. Previously, we cloned a cDNA encoding a Canavalia ensiformis urease isoform named JBURE-II, corresponding to a putative smaller urease protein (78kDa) when compared to other plant ureases. Aiming to produce the recombinant protein, we obtained jbure-IIb, with different 3' and 5' ends, encoding a 90kDa urease. Three peptides unique to the JBURE-II/-IIb protein were detected by mass spectrometry in seed extracts, indicating that jbure-II/-IIb is a functional gene. Comparative modeling indicates that JBURE-IIb urease has an overall shape almost identical to C. ensiformis major urease JBURE-I with all residues critical for urease activity. The cDNA was cloned into the pET101 vector and the recombinant protein was produced in Escherichia coli. The JBURE-IIb protein, although enzymatically inactive presumably due to the absence of Ni atoms in its active site, impaired the growth of a phytopathogenic fungus and showed entomotoxic properties, inhibiting diuresis of Rhodnius prolixus isolated Malpighian tubules, in concentrations similar to those reported for JBURE-I and canatoxin. The antifungal and entomotoxic properties of the recombinant JBURE-IIb apourease are consistent with a protective role of ureases in plants.