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Ureases são enzimas que realizam hidrólise da uréia em amônia e dióxido de carbono e são isoladas de plantas, fungos e bactérias. A urease de C. ensiformis (JBU) tem algumas atividades biológicas independentes da sua atividade enzimática, como por exemplo: agregação plaquetária e efeito inseticida. O efeito inseticida é devido à liberação de um peptídeo interno por enzimas digestivas específicas dos insetos. Este peptídeo foi isolado, caracterizado e um análogo recombinante foi obtido e denominado Jaburetox-2Ec. Somente insetos com enzimas digestivas acídicas, como catepsinas, são capazes de liberar o peptídeo tóxico a partir da hidrólise de JBU. Insetos com enzimas básicas do tipo tripsina não são suscetíveis pois não há formação do peptídeo tóxico. Ninfas de D. peruvianus são sensíveis aos efeitos da JBU enquanto adultos não são. Este trabalho teve como objetivo estudar as diferenças enzimáticas entre os dois estágios do inseto para elucidar o processamento diferencial da JBU. Realizando a hidrólise in vitro da JBU com o homogeneizado de intestino de ninfas e adultos foi visto que tanto adultos como ninfas hidrolisam a JBU, mas somente ninfas liberam o peptídeo tóxico identificado pelo anticorpo específico anti-Jaburetox-2Ec. Ainda, através de ensaios enzimáticos utilizando substratos fluorogênicos e inibidores específicos, foi observada uma diferença no pH ótimo de atividade e na susceptibilidade a inibidores das enzimas digestivas presentes nos dois estágios. Substratos fluorogênicos correspondentes às regiões flanqueadoras do peptídeo dentro da JBU intacta foram desenhados e testados com os dois homogeneizados, o que também revelou que os homogeneizados dos dois estágios têm ação diferencial sobre estes substratos, sendo que ninfas liberariam mais eficientemente a extremidade Nterminal do peptídeo quando comparado aos adultos. Verificamos ainda que, em estudos eletrofisiológicos utilizando a técnica de “Planar Lipid Bilayer”, tanto a JBU como o Jaburetox-2Ec são capazes de se inserir em membrana lipídica planar, formando canais iônicos. Os canais formados pela JBU apresentaram quatro níveis de condutância majoritárias e seletividade para íons cloreto. / Ureases are enzymes that hydrolyze urea in ammonium and carbon dioxide and they have been isolated from plants, fungi and bacteria. Jackbean urease, from Canavalia ensiformis (JBU) displays biological activities unrelated from its enzymatic activity, as platelet aggregation and insecticide effect. This insecticide effect is due to the release of an internal peptide by insect specific digestive enzymes. This peptide was isolated, characterized and the recombinant peptide obtained was called Jaburetox- 2Ec. Only insects that rely on cathepsin-like digestive enzymes are able to hydrolyze JBU and release the toxic peptide. Insects with alkaline enzymes like tripsins are not susceptible because they don’t release the toxic peptide. Nymphs of D. peruvianus are susceptible to JBU effects while adults are not. The goal of this work was to study the enzymatic differences between both insect stages to elucidate JBU’s differential processing. In vitro hydrolysis were performed with nymphs and adults midgut homogenates and we observed that both adults and nymphs hydrolyze JBU but only nymphs are able to release the toxic peptide identified by Jaburetox-2Ec antibodies. Furthermore, in enzymatic assays using different fluorogenic substrates and specific inhibitors a difference in optimum pH and susceptibility to inhibitors of the digestive enzymes in both stages was observed. Fluorogenic substrates corresponding to the flanking regions of the peptide inside the intact JBU were produced and tested with both homogenates. Homogenates from both stages have differential action upon these substrates, considering that nymphs hydrolyses more efficiently the N-terminal of the peptide compared to adults. In electrophysiological studies using the Planar Lipid Bilayer technique we verify that JBU and Jaburetox-2Ec are able to insert in planar lipid membrane forming ionic channels. JBU’s channels display four major conductance levels and selectivity for chloride íons.

Ureases são enzimas níquel-dependentes que catalisam a hidrólise da ureia em amônia e dióxido de carbono. Além disso, apresentam diversas propriedades independentes da catálise, sendo consideradas proteínas moonlighting. São amplamente distribuídas na natureza, sendo encontradas em bactérias, arqueas, plantas e fungos, podendo se organizar em unidades funcionais compostas por uma, duas ou três subunidades. Sua ativação, que envolve a passagem da enzima de sua forma apo-urease a sua forma holourease, requer pelo menos três proteínas acessórias. Parte de suas propriedades não-catalíticas é associada à liberação de peptídeos internos da proteína nativa. Nesse contexto, a presente tese se dedicou ao estudo de diferentes aspectos da biologia estrutural de ureases. Ao elaborar uma narrativa filogenética, envolvendo varredura de bancos de dados em larga escala e diferentes algoritmos de reconstrução de árvores, foi possível propor uma rota evolutiva indicando a transição de três subunidades para uma única unidade funcional, sem passar por intermediários de duas cadeias. Quanto ao seu processo de ativação, por meio de múltiplos cálculos de atracamento baseados em dados experimentais prévios (especialmente SAXS), foram propostas estruturas para suas diferentes etapas, em resolução atomística. Finalmente, o comportamento dinâmico de diferentes peptídeos derivados de urease foram analisados computacionalmente por meio de simulações de longa duração (500 ns) e associados a outros dados obtidos in vitro, permitindo justificar efeitos diferenciais obtidos na aplicação destes peptídeos. De maneira geral, o trabalho empregou técnicas computacionais à análise de ureases, fornecendo bases para futuros estudos de suas propriedades, sejam catalíticas ou não, incluindo sua aplicação biotecnológica. / Ureases are nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and carbon dioxide. They have many catalysis-independent properties, being considered moonlighting proteins. Ureases are found in bacteria, archaea, plants, and fungi, and may be organized in functional units composed by one, two, or three subunits. Their activation, involving the transition from apo to holourease, requires at least three accessory proteins. Some of their non-catalytic properties are related to the release of internal peptides from the native protein. In this context, this thesis was developed upon the study of different aspects of urease structural biology. By phylogenetical reconstruction, employing large-scale databank scans and different tree-building algorithms, we were able to propose an evolutionary route by which the transition from three to one functional subunits was possible, with no need for a two-chained intermediate. Regarding the activation mechanism, we have proposed structural models for the oligomeric intermediates of the process by multiple docking calculations, at atomistic resolution, based on previous experimental data (especially SAXS). Additionally, the dynamical behavior of different ureasederived peptides was analyzed by computational simulations at large time scales (500 ns) and correlated to in vitro results, allowing us to explain the variable effects observed after their application on test systems. In short, in this work we have employed computational techniques to the analysis of urease, providing working grounds for further studies of this enzyme and its properties (catalytical or not), including its biotechnological applicability.

A ampla distribuição das ureases na natureza é um indício da grande importância desta enzima para os mais diferentes organismos, fato que levou diversos pesquisadores ao redor do globo a dedicarem-se exclusivamente à caracterização destas enzimas. Apesar de ser uma proteína estudada ha quase um século, até o início deste trabalho apenas três ureases possuíam suas estruturas cristalográficas elucidadas e todas de origem bacteriana. A urease de Canavalia ensiformis (JBU) foi a primeira enzima desta família a ser descrita, em 1926 e ainda assim, apesar dos incansáveis esforços de diferentes grupos na caracterização estrutural e biológica desta proteína, sua estrutura cristalográfica só foi obtida no primeiro semestre de 2010. Tentativas anteriores de obtenção da estrutura cristalográfica de JBU falharam devido a baixa qualidade dos cristais formados por esta proteína através de técnicas convencionais de cristalização. Desta forma, na primeira parte deste trabalho relatamos o uso de técnicas alternativas de cristalização como o uso de ligantes ou aditivos, proteólise in situ e modificação química de resíduos de amino ácidos na obtenção de cristais de JBU com qualidade superior aos anteriormente obtidos, validando o uso destas metodologias na cristalização de proteínas recalcitrantes. Os cristais aqui obtidos, apesar de apresentarem qualidade superior aos previamente descritos na literatura, não foram otimizados ao ponto de obtenção da estrutura cristalográfica da enzima A atividade enzimática das ureases é dependente da presença de dois íons de níquel precisamente incorporados em seus sítios ativos. A biossíntese deste sítio ativo, bem como a incorporação de níquel na enzima consiste de um processo altamente regulado, cuja ocorrência depende da participação de diversas chaperonas (proteínas acessórias) agindo como reguladores pós traducionais. Apesar dos esforços já realizados, o mecanismo de ativação de ureases ainda permanece obscuro. Até hoje, os estudos concentraram-se em ureases de origem microbiana, sendo a informação disponível ainda extremamente limitada. Menos ainda é conhecido para o sistema de ativação de ureases de origem vegetal. Na segunda parte deste trabalho relatamos pioneiramente as primeiras tentativas de produção e caracterização de proteínas acessórias de origem vegetal. As proteínas UreD, UreF e UreG de soja (Glycine max) foram clonadas e expressas em sistema bacteriano. A proteína UreG de soja, purificada diretamente da planta e também produzida de forma recombinante em E.coli, foi caracterizada quanto a sua estrutura, capacidade de ligação a metais e atividade GTPásica, descrevendo algumas características nunca antes observadas para outras proteínas da mesma família. Também descrevemos o primeiro sucesso na obtenção da proteína acessória UreF tipo 5 selvagem na sua forma solúvel e a caracterização estrutural desta proteína foi realizada. As proteínas acessórias de soja também foram caracterizadas em relação ao seu perfil de expressão em diferentes tecidos, ao longo do desenvolvimento da planta, utilizando PCR em tempo real. Observamos uma grande variação dos níveis de expressão dos diferentes mRNAs nos tecidos avaliados. Uma possível relação entre estes níveis de expressão e a atividade ureásica de cada tecido foi investigada, visando uma melhor compreensão da dinâmica entre a expressão e atividade das proteínas responsáveis pela ativação das ureases vegetais. / The wide distribution of ureases in nature is an indication of the importance of this enzyme for the most different organisms, fact that lead researchers all over the world to dedicate their careers exclusively to the study of these enzymes. Despite of being studied for almost a century, until last year only three crystallographic structures of ureases had been solved, all from bacterial source. The urease from Canavalia ensiformis (JBU) was the first member of this family to be described, in 1926 and still, despite the restless efforts from different research groups on the structural and biological characterization of this protein, its structure was not solved until the beginning of 2010. Early trials to obtain the crystallographic structure of JBU have failed due to the poor quality crystals formed by this protein using conventional crystallization techniques. In this panorama, the first part of this work describes the use of alternative crystallization techniques, such as the use of ligands, in situ proteolisis and chemical modification of amino acid residues, for the obtention of JBU crystals with superior quality than the ones previously described in the literature, validating the use of such techniques on the crystallization of recalcitrant proteins. The crystals obtained here, despite of their superior characteristics, were not refined to the point of generating the crystallographic structure of JBU. The enzymatic activity of ureases is dependent of the presence of nickel ions precisely incorporated in their active sites. The bioassembly of these active sites, including the nickel incorporation, consist of a tightly regulated process, whose occurence depends on the participation of several chaperones (accessory proteins) that work as post-translational regulators. A great deal of effort has already been done on the study of the urease’s activation process; however its details remain unclear. To date, all work done concentrates on the study of microbial ureases, but the available information is very limited and even less information is available for the plant ureases activation process. The second part this work presents the first trials on the production and characterization of plant urease accessory proteins. The proteins UreD, UreF and UreG from soybean (Glycine max) were cloned and expressed in bacterial system. The soybean UreG protein, purified directly from the plant seeds and also produced in recombinant fashion in Escherichia coli, was characterized regarding its structure, metal biding capacity and GTPasic activity, describing a few characteristics never before observed for other proteins of the same family. It is also described the first success on the obtention of the wild type ureF accessory protein in its soluble form and a structural characterization was performed. All accessory proteins from soybean were also characterized for its expression profile in several tissues during its development utilizing real-time PCR and a correlation with the levels of urease activity were investigated. Altogether, these data improve the understanding of multiple factors involved on the urease activation process.

The hydrolysis of urea by the widely distributed enzyme urease is special in that it is one of the few biologically occurring reactions that can generate carbonate ions without an associated production of protons. When this hydrolysis occurs in a calcium-rich environment, calcite (calcium carbonate) precipitates from solution forming a solid-crystalline material. The binding strength of the precipitated crystals is highly dependent on the rate of carbonate formation and under suitable conditions it is possible to control the reaction to generate hard binding calcite cement (or Biocement). The objective of this thesis was to develop an industrially suitable cost-effective microbial process for the production of urease active cells and investigate the potential for urease active cells to act as a catalyst for the production of Biocement. The biocementation capability of two suitable strains was compared. Sporosarcina pasteurii (formally Bacillus pasteurii) produced significantly higher levels of urease activity compared to Proteus vulgaris, however the level of urease activity was variable with respect to biomass suggesting that the enzyme was not constitutive as indicated by the literature, but subject to regulation. The environmental and physiological conditions for maximum urease activity in S. pasteurii were investigated and it was found that the potential urease capacity of the organism was very high (29 mM urea.min-1.OD-1) and sufficient for biocementation without additional processing (e.g. concentration, cell lysis). The regulation mechanism for S. pasteurii urease was not fully elucidated in this study, however it was shown that low specific urease activity was not due to depletion of urea nor due to the high concentrations of the main reaction product, ammonium. pH conditions were shown to have a regulatory effect on urease but it was evident that another co-regulating mechanism existed. Despite not fully exploiting the urease capability of S. pasteurii, sufficient urease activity to allow direct application of the enzyme without additional processing could still be achieved and the organism was considered suitable for biocementation. Urease was the most expensive component of the cementation process and cost-efficient production was desired, thus an economic growth procedure was developed for large-scale cultivation of S. pasteurii. The organism is a moderate alkaliphile (growth optimum pH 9.25) and it was shown that sufficient activity for biocementation could be cultivated in non-sterile conditions with a minimum of upstream and downstream processing. The cultivation medium was economised and expensive components were replace with a food-grade protein source and acetate, which lowered production costs by 95%. A high level of urease activity (21 mM urea hydrolysed.min-1) was produced in the new medium at a low cost ($0.20 (AUD) per L). The performance of urease in whole S. pasteurii cells was evaluated under biocementation conditions (i.e. presence of high concentrations of urea, Ca2+, NH4 +/NH3, NO3 - and Cl- ions). It was established that the rate of urea hydrolysis was not constant during cementation, but largely controlled by the external concentrations of urea and calcium, which constantly changed during cementation due to precipitation of solid calcium carbonate from the system. A simple model was generated that predicted the change in urea hydrolysis rate over the course of cementation. It was shown that whole cell S. pasteurii urease was tolerant to concentrations of up to 3 M urea and 2 M calcium, and the rate of urea hydrolysis was unaffected up to by 3 M ammonium. This allowed the controlled precipitation of up to 1.5 M CaCO3 within one treatment, and indicated that the enzyme was very stable inspite of extreme chemical conditions. A cost-efficient cementation procedure for the production of high cementation strength was developed. Several biocementation trials were conducted into order to optimise the imparted cementation strength by determining the effect of urea hydrolysis rate on the development of strength. It was shown that high cementation strength was produced at low urea hydrolysis rates and that the development of cementation strength was not linear over the course of the reaction but mostly occurred in the first few hours of the reaction. In addition, the whole cell bacterial enzyme had capacity to be immobilised in the cementation material and re-used to subsequent applications, offering a significant cost-saving to the process. An industry-sponsored trial was undertaken to investigate the effectiveness of Biocement for increasing in-situ strength and stiffness of two different sandy soils; (a) Koolschijn sand and (b) 90% Koolschijn sand mixed with 10% peat (Holland Veen). After biocementation treatment, Koolschijn sand indicated a shear strength of 1.8 MPa and a stiffness of 250 MPa, which represents an 8-fold and 3-fold respective improvement in strength compared to unconsolidated sand. Significantly lower strength improvements were observed in sand mixed with peat. In combination, trials of producing bacteria under economically acceptable conditions and cementation trials support the possibility of on-site production and in-situ application of large field applications.

The hydrolysis of urea by the widely distributed enzyme urease is special in that it is one of the few biologically occurring reactions that can generate carbonate ions without an associated production of protons. When this hydrolysis occurs in a calcium-rich environment, calcite (calcium carbonate) precipitates from solution forming a solid-crystalline material. The binding strength of the precipitated crystals is highly dependent on the rate of carbonate formation and under suitable conditions it is possible to control the reaction to generate hard binding calcite cement (or Biocement). The objective of this thesis was to develop an industrially suitable cost-effective microbial process for the production of urease active cells and investigate the potential for urease active cells to act as a catalyst for the production of Biocement. The biocementation capability of two suitable strains was compared. Sporosarcina pasteurii (formally Bacillus pasteurii) produced significantly higher levels of urease activity compared to Proteus vulgaris, however the level of urease activity was variable with respect to biomass suggesting that the enzyme was not constitutive as indicated by the literature, but subject to regulation. The environmental and physiological conditions for maximum urease activity in S. pasteurii were investigated and it was found that the potential urease capacity of the organism was very high (29 mM urea.min-1.OD-1) and sufficient for biocementation without additional processing (e.g. concentration, cell lysis). The regulation mechanism for S. pasteurii urease was not fully elucidated in this study, however it was shown that low specific urease activity was not due to depletion of urea nor due to the high concentrations of the main reaction product, ammonium. pH conditions were shown to have a regulatory effect on urease but it was evident that another co-regulating mechanism existed. Despite not fully exploiting the urease capability of S. pasteurii, sufficient urease activity to allow direct application of the enzyme without additional processing could still be achieved and the organism was considered suitable for biocementation. Urease was the most expensive component of the cementation process and cost-efficient production was desired, thus an economic growth procedure was developed for large-scale cultivation of S. pasteurii. The organism is a moderate alkaliphile (growth optimum pH 9.25) and it was shown that sufficient activity for biocementation could be cultivated in non-sterile conditions with a minimum of upstream and downstream processing. The cultivation medium was economised and expensive components were replace with a food-grade protein source and acetate, which lowered production costs by 95%. A high level of urease activity (21 mM urea hydrolysed.min-1) was produced in the new medium at a low cost ($0.20 (AUD) per L). The performance of urease in whole S. pasteurii cells was evaluated under biocementation conditions (i.e. presence of high concentrations of urea, Ca2+, NH4 +/NH3, NO3 - and Cl- ions). It was established that the rate of urea hydrolysis was not constant during cementation, but largely controlled by the external concentrations of urea and calcium, which constantly changed during cementation due to precipitation of solid calcium carbonate from the system. A simple model was generated that predicted the change in urea hydrolysis rate over the course of cementation. It was shown that whole cell S. pasteurii urease was tolerant to concentrations of up to 3 M urea and 2 M calcium, and the rate of urea hydrolysis was unaffected up to by 3 M ammonium. This allowed the controlled precipitation of up to 1.5 M CaCO3 within one treatment, and indicated that the enzyme was very stable inspite of extreme chemical conditions. A cost-efficient cementation procedure for the production of high cementation strength was developed. Several biocementation trials were conducted into order to optimise the imparted cementation strength by determining the effect of urea hydrolysis rate on the development of strength. It was shown that high cementation strength was produced at low urea hydrolysis rates and that the development of cementation strength was not linear over the course of the reaction but mostly occurred in the first few hours of the reaction. In addition, the whole cell bacterial enzyme had capacity to be immobilised in the cementation material and re-used to subsequent applications, offering a significant cost-saving to the process. An industry-sponsored trial was undertaken to investigate the effectiveness of Biocement for increasing in-situ strength and stiffness of two different sandy soils; (a) Koolschijn sand and (b) 90% Koolschijn sand mixed with 10% peat (Holland Veen). After biocementation treatment, Koolschijn sand indicated a shear strength of 1.8 MPa and a stiffness of 250 MPa, which represents an 8-fold and 3-fold respective improvement in strength compared to unconsolidated sand. Significantly lower strength improvements were observed in sand mixed with peat. In combination, trials of producing bacteria under economically acceptable conditions and cementation trials support the possibility of on-site production and in-situ application of large field applications.

Thesis (Ph.D.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on Mar. 30, 2009) Includes bibliographical references. Also issued in print.

Ureases (3.5.1.5), enzimas níquel dependentes que catalisam a reação de hidrólise da uréia em amônia e dióxido de carbono, apresentam ampla distribuição em plantas, fungos e bactérias. A espiroqueta Helicobacter pylori causa úlceras pépticas e câncer gástrico por mecanismos ainda não totalmente conhecidos. H. pylori produz grande quantidade de urease, que neutraliza o meio ácido e permite sua sobrevivência no estômago. Nosso grupo demonstrou que as ureases de Canavalia ensiformis, soja e Bacillus pasteurii induzem agregação plaquetária independentemente de sua atividade ureolítica, por uma rota que requer ativação de canais de cálcio. ativação da rota do ácido araquidônico e secreção plaquetária. Estudos prévios mostraram ainda que a canatoxina, uma isoforma da urease de C.ensiformis, possui atividade pró-inflamatória, induzindo edema de pata em ratos. Neste trabalho, caracterizamos as propriedades ativadora de plaquetas e pró-inflamatória da urease recombinante de H. pylori (HPU). Em plaquetas, estudamos as vias recrutadas pela proteína na agregação plaquetária e comparamos com dados prévios para a canatoxina e a urease de Bacillus pasteurii. Em neutrófilos, demonstramos que a HPU, em doses nanomolares, induz quimiotaxia e produção de espécie reativas de oxigênio. A taxa de apoptose de neutrófilos ativados por HPU foi inibida, acompanhando alterações dos níveis de proteínas pró- e antiapoptóticas. Por último, mostramos que a resposta dos neutrófilos a HPU envolve aumento dos níveis de lipoxigenase(s), sem, contudo, haver alterações das ciclooxigenase( s). Concluímos que as propriedades não enzimáticas aqui descritas para a HPU podem potencialmente contribuir para o processo inflamatório promovido por H. pylori. / Ureases (EC 3.5.1.5), nickel-dependent enzymes that hydrolyze urea into ammonia and carbon dioxide, are widespread among plants, bacteria and fungi. The spirochete Helicobacter pylori is the etiological agent of gastric ulcers and gastric adenocarcinoma by mechanisms not yet fully understood. H. pylori produces high amounts of urease, which enables the bacterium to survive in the acidic medium of the stomach. We have previously reported that ureases from jackbean, soybean or Bacillus pasteurii induce blood platelet aggregation independently of their enzyme activity by a pathway requiring activation of calcium channels, lipoxigenase-derived eicosanoids and platelet secretion. We also showed that canatoxin, an isoform of C. ensiformis urease, presents pro-inflammatory property demonstrated by rat paw oedema. In this work we characterized the platelet aggregating and pro-inflammatory properties of the recombinant H. pylori urease (HPU). In platelets we studied the pathways recruited by the protein to induce platelet aggregation and compared the data to those previously reported for the plant urease canatoxin and for Bacillus pasteurii urease. Using neutrophils we demonstrated that nanomolar doses of HPU induce chemotaxis and production of oxygen reactive species in human neutrophils. The rate of apoptosis was decreased in HPU-treated neutrophils, accompanied by alterations in the levels of proand anti-apoptotic proteins. Moreover, we showed that the response of neutrophils to HPU requires increased levels of lipoxygenase(s) with no alterations of cyclooxygenase( s). We concluded that the non-enzymatic properties of HPU here described potentially contribute to the inflammatory process that underlies H. pylori infection.

Ureases (EC 3.5.1.5) são metaloenzimas que hidrolisam uréia para produzir amônia e dióxido de carbono. Essas proteínas têm atividade inseticida e fungicida, efeitos independentes da sua atividade ureolítica. A atividade inseticida de ureases depende, em parte, da liberação de peptídeos internos através da hidrólise por catepsinas digestivas do inseto. Um desses peptídeos foi isolado e um recombinante chamado Jaburetox –V5 foi produzido em E. coli a partir da sequência da urease. Outra propriedade relevante de ureases é sua atividade antifúngica, que ocorre em concentrações de 10-7 M para certos fungos filamentosos, causando danos à membrana celular, visualizados por microscopia eletrônica de varredura. Moléculas antifúngicas de origem vegetal representam uma alternativa estratégica para o surgimento de espécies resistentes de fungos. Sabendo que a atividade antifúngica das ureases é cerca de 3-4 de magnitude mais ativa do que a maioria das proteínas antifúngicas já descritas, neste trabalho, avaliamos o efeito tóxico da urease de C. ensiformis (JBU) sobre diferentes espécies de leveduras. Além disso, buscamos identificar as regiões responsáveis por essa atividade através da fragmentação da JBU por hidrólise enzimática. Os efeitos tóxicos da JBU também ocorrem em espécies de leveduras, indicando que atividade antifúngica não afeta somente fungos filamentosos. Os efeitos da JBU nas leveduras variam conforme o gênero e a espécie, tanto em termos qualitativos como quantitativos, indicando seletividade espécie-específica. Os efeitos fungitóxicos consistem de inibição da multiplicação, indução de alterações morfológicas com formação de pseudo-hifas, alterações do transporte de H+ e no metabolismo energético, permeabilização de membranas, podendo ocorrer morte celular. Nas condições testadas, não houve produção de espécies reativas de oxigênio associada ao efeito fungitóxico da JBU. A hidrólise da JBU com papaína produziu fragmentos tóxicos com massa molecular ~10 kD. Esses hidrolisados foram analisados por espectrometria de massas e um fragmento contendo parte da sequência N-terminal do peptídeo entomotóxico Jaburetox foi encontrado. A atividade fungitóxica do peptídeo recombinante Jaburetox – V5 foi testada, sendo observada atividade tóxica sobre leveduras e fungos filamentosos. A atividade antifúngica do Jaburetox-V5 requer concentrações 2-3 vezes maiores do que aquela observada para a holoproteína JBU, indicando a possibilidade de que outros domínios da proteína estejam envolvidos nessa atividade. A descoberta de novos agentes antifúngicos é urgente e imperativa, devido ao crescente número de casos de micoses invasivas. Ureases de plantas, como a JBU, e peptídeos derivados, podem representar uma nova alternativa para controle de fungos de importância clínica e fitopatogênicos, principalmente em se considerando a potente atividade, na faixa de 10-6 a 10-7 M . Estudos estrutura versus atividade adicionais, aprofundando a identificação de domínios antifúngicos, e a construção de recombinantes contendo esses domínios, são etapas futuras para avaliar o real potencial fungicida/fungistático das ureases e peptídeos derivados. / Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea to produce ammonia and carbon dioxide. These proteins have insecticidal and fungicidal effects not related to their enzyme activity. The insecticidal activity of urease is mostly dependent on the release of internal peptides consequent to hydrolysis of the ingested protein by insect digestive cathepsins. One of these peptides was isolated and its recombinant version, named Jaburetox-V5, was produced in E. coli. Another important property of ureases is their antifungal activity, which occurs at concentrations of 10-7 M for certain filamentous fungi, causing damage to the cell membranes, as visualized by scanning electron microscopy. Antifungal molecules from plants represent an alternative strategy to the emergence of resistant fungal species. Considering that the antifungal activity of urease is about 3-4 orders of magnitude more potent than most of the antifungal proteins already described, in this study, we evaluated the toxic effect of Canavalia ensiformis urease (JBU) on different species of yeast. Furthermore, studies aiming to identify antifungal domain(s) of JBU by enzymatic hydrolysis were carried out. The results showed that JBU exerts toxic effects on yeast species, indicating that antifungal activity is not restricted to filamentous fungi. The effects of JBU in yeast varied according to the genus and species of yeasts, both in qualitative and quantitative terms, indicating a species-specific selectivity. The fungitoxic effects consisted in inhibition of proliferation, induction of morphological alterations with formation of pseudo hyphae, changes in the transport of H+ and carbohydrate metabolism, permeabilization of membranes, eventually leading to cell death. Under the conditions tested, there was no production of reactive oxygen species associated with the antifungal effect of JBU. Hydrolysis of JBU with papain resulted in fungitoxic fragments with molecular mass ~ 10 kD. These peptides were analyzed by mass spectrometry, revealing the presence of a fragment containing the Nterminal sequence of the entomotoxic peptide Jaburetox. We tested the recombinant peptide Jaburetox-V5 for antifungal effects and observed fungitoxic activity on yeast and filamentous fungi. The antifungal activity of Jaburetox-V5 requires 2-3 times larger concentrations than those observed for the holoprotein JBU, indicating the possibility that other protein domains are involved in this activity. The discovery of new antifungal agents is imperative to face the increasing number of cases of invasive mycoses. Plant ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important and phytopathogenic fungi, especially considering their potent activity in the range of 10-6 to 10-7 M. More studies are necessary to clarify the structure versus activity relationships of ureases. Construction of mutants containing ureasederived antifungal domains is one of the necessary steps to assess the real fungicidal/ fungistatic potential of ureases and derived peptides.