RESUMO
The urease enzyme has been an important target for the discovery of effective pharmacological and agricultural products. Thirteen regio-selectively alkylated benzimidazole-2-thione derivatives have been designed to carry the essential features of urease inhibitors. The urease enzyme was isolated from Helicobacter pylori as a recombinant urease utilizing the His-tag method. The isolated enzyme was purified and characterized using chromatographic and FPLC techniques showing a maximal activity of 200 mg/mL. Additionally, the commercial Jack bean urease was purchased and included in this study for comparative and mechanistic investigations. The designed compounds were synthesized and screened for their inhibitory activity against the two ureases. Compound 2 inhibited H. pylori and Jack bean ureases with IC50 values of 0.11; and 0.26 mM; respectively. While compound 5 showed IC50 values of 0.01; and 0.29 mM; respectively. Compounds 2 and 5 were docked against Helicobacter pylori urease (PDB ID: 1E9Y; resolution: 3.00 Å) and exhibited correct binding modes with free energy (ΔG) values of -9.74 and -13.82 kcal mol-1; respectively. Further; the in silico ADMET and toxicity properties of 2 and 5 indicated their general safeties and likeness to be used as drugs. Finally, the compounds' safety was authenticated by an in vitro cytotoxicity assay against fibroblast cells.
Assuntos
Benzimidazóis/química , Inibidores Enzimáticos/química , Helicobacter pylori/enzimologia , Simulação de Acoplamento Molecular , Urease , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Helicobacter pylori/genética , Urease/antagonistas & inibidores , Urease/biossíntese , Urease/genética , Urease/isolamento & purificaçãoRESUMO
Ureases are microbial virulence factors either because of the enzymatic release of ammonia or due to many other non-enzymatic effects. Here we studied two neurotoxic urease isoforms, Canatoxin (CNTX) and Jack Bean Urease (JBU), produced by the plant Canavalia ensiformis, whose mechanisms of action remain elusive. The neurotoxins provoke convulsions in rodents (LD50 â¼2 mg/kg) and stimulate exocytosis in cell models, affecting intracellular calcium levels. Here, electrophysiological and brain imaging techniques were applied to elucidate their mode of action. While systemic administration of the toxins causes tonic-clonic seizures in rodents, JBU injected into rat hippocampus induced spike-wave discharges similar to absence-like seizures. JBU reduced the amplitude of compound action potential from mouse sciatic nerve in a tetrodotoxin-insensitive manner. Hippocampal slices from CNTX-injected animals or slices treated in vitro with JBU failed to induce long term potentiation upon tetanic stimulation. Rat cortical synaptosomes treated with JBU released L-glutamate. JBU increased the intracellular calcium levels and spontaneous firing rate in rat hippocampus neurons. MicroPET scans of CNTX-injected rats revealed increased [18]Fluoro-deoxyglucose uptake in epileptogenesis-related areas like hippocampus and thalamus. Curiously, CNTX did not affect voltage-gated sodium, calcium or potassium channels currents, neither did it interfere on cholinergic receptors, suggesting an indirect mode of action that could be related to the ureases' membrane-disturbing properties. Understanding the neurotoxic mode of action of C. ensiformis ureases could help to unveil the so far underappreciated relevance of these toxins in diseases caused by urease-producing microorganisms, in which the human central nervous system is affected.
Assuntos
Canavalia/química , Síndromes Neurotóxicas/etiologia , Proteínas de Plantas/toxicidade , Toxinas Biológicas/toxicidade , Urease/toxicidade , Animais , Convulsivantes/isolamento & purificação , Convulsivantes/toxicidade , Feminino , Masculino , Camundongos , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/patologia , Síndromes Neurotóxicas/fisiopatologia , Proteínas de Plantas/isolamento & purificação , Ratos , Ratos Wistar , Toxinas Biológicas/isolamento & purificação , Urease/isolamento & purificação , Xenopus laevisRESUMO
Helicobacter pylori causes one of the most common infections in human populations. The role of this bacterium in chronic gastritis, gastric ulcer, gastric cancer, as well as extra-digestive diseases such as ischemic heart disease and chronic obstructive pulmonary diseases, is well known. Prevention and control of these diseases can occur by early diagnosis and eradication of H. pylori infection. At present, different methods have been established to detect H. pylori infection. The biopsy-based tests, which are known as invasive methods, such as rapid urease test and histology, have the highest specificity among the others. Similarly, culture of biopsy samples is used for diagnosis of H. pylori infection. It has a high specificity value, and also allows us to perform antibiotic sensitivity testing. On the contrary, polymerase chain reaction and other molecular methods have good sensitivity and specificity, and can be used for detection of H. pylori infection, its virulence factors, and eradication success after treatment. While serological tests are more appropriate for epidemiological studies, their main weakness for clinical use is low specificity. Overall, specificity and sensitivity, cost, usefulness, and limitation of tests should be considered for selection of detection methods of H. pylori in each country.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Testes Diagnósticos de Rotina/métodos , Infecções por Helicobacter/diagnóstico , Helicobacter pylori/isolamento & purificação , Antígenos de Bactérias/genética , Biópsia , Análise Custo-Benefício , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/química , Humanos , Sensibilidade e Especificidade , Urease/química , Urease/isolamento & purificaçãoRESUMO
Urease is a potent metalloenzyme with diverse applications. This paper describes the scale up and purification of an extracellular urease from Arthrobacter creatinolyticus MTCC 5604. The urease production was scaled-up in 3.7 L and 20 L fermentor. A maximum activity of 27 and 27.8 U/mL and a productivity of 0.90 and 0.99 U/mL/h were obtained at 30 h and 28 h in 3.7 and 20 L fermentor, respectively. Urease was purified to homogeneity with 49.85-fold purification by gel filtration and anion exchange chromatography with a yield of 36% and a specific activity of 1044.37 U/mg protein. The enzyme showed three protein bands with molecular mass of 72.6, 11.2 and 6.1 kDa on SDS-PAGE and ~ 270 kDa on native PAGE. The cytotoxic effect of urease was assessed in vitro using cancer cell lines (A549 and MG-63) and normal cell line (HEK 293). Urease showed its inhibitory effects on cancer cell lines through the generation of toxic ammonia, which in turn increased the pH of the surrounding medium. This increase in extracellular pH, enhanced the cytotoxic effect of weak base chemotherapeutic drugs, doxorubicin (50 µM) and vinblastine (100 µM) in the presence of urease (5 U/mL) and urea (0-4 mM) significantly.
Assuntos
Arthrobacter/enzimologia , Urease/isolamento & purificação , Urease/farmacologia , Células A549/efeitos dos fármacos , Amônia/metabolismo , Arthrobacter/metabolismo , Arthrobacter/fisiologia , Linhagem Celular , Cromatografia em Gel/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio/efeitos dos fármacos , Peso Molecular , Ureia/metabolismo , Urease/fisiologiaRESUMO
Cementation of salt-containing soils can be achieved by salt-tolerant or halophilic calcite precipitation bacteria. Therefore, the isolation of calcite-producing bacteria in the presence of salt is the first step in the microbial cementation of saline soils. Urease producing bacteria can cause calcite nano-crystals to precipitate by producing urease in the presence of urea and calcium. The purpose of this study was to isolate urease producing halophilic bacteria in order to make calcite precipitate in saline soil. The calcite and the properties of the strains were further analyzed by X-ray diffraction (XRD) and scanning electron microscope equipped with an energy dispersive X-ray detector. In this study, a total of 110 halophilic strains were isolated, from which 58 isolates proved to have the ability of urease production. Four strains were identified to produce nano-calcite using urease activity in the precipitation medium. The XRD studies showed that the size of these particles was in the range of 40-60 nm. Strain H3 revealed that calcite is mostly produced in the precipitation medium containing 5% salt in comparison with other strains. This strain also produced calcite precipitates in the precipitation medium containing 15% salt. Phylogenetic analysis indicated that these isolates are about 99-100% similar to Staphylococcus saprophyticus.
Assuntos
Carbonato de Cálcio/metabolismo , Microscopia Eletrônica de Varredura/métodos , Nanopartículas/metabolismo , Staphylococcus saprophyticus/enzimologia , Urease/metabolismo , Difração de Raios X/métodos , Carbonato de Cálcio/química , Carbonato de Cálcio/isolamento & purificação , Microbiologia Ambiental , Irã (Geográfico) , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Filogenia , RNA Ribossômico 16S/genética , Staphylococcus saprophyticus/classificação , Staphylococcus saprophyticus/isolamento & purificação , Staphylococcus saprophyticus/metabolismo , Ureia/metabolismo , Urease/isolamento & purificaçãoRESUMO
Two series of ω-phenoxy contained acylhydroxamic acids as novel urease inhibitors were designed and synthesized. Biological activity evaluations revealed that ω-phenoxypropinoylhydroxamic acids were more active than phenoxyacetohydroxamic acids. Out of these compounds, 3-(3,4-dichlorophenoxy)propionylhydroxamic acid c24 showed significant potency against urease in both cell free extract (IC50â¯=â¯0.061⯱â¯0.003⯵M) and intact cell (IC50â¯=â¯0.89⯱â¯0.05⯵M), being over 450- and 120-fold more potent than the clinically prescribed urease inhibitor AHA, repectively. Non-linear fitting of experimental data (V-[S]) suggested a mixed-type inhibition mechanism and a dual site binding mode of these compounds.
Assuntos
Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Urease/antagonistas & inibidores , Antibacterianos/síntese química , Antibacterianos/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Infecções por Helicobacter/metabolismo , Helicobacter pylori/citologia , Helicobacter pylori/enzimologia , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Cinética , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Estrutura Molecular , Relação Estrutura-Atividade , Urease/isolamento & purificação , Urease/metabolismoRESUMO
Urease is a ubiquitous nickel metalloenzyme. In plants, its activation requires three urease accessory proteins (UAPs), UreD, UreF, and UreG. In bacteria, the UAPs interact with urease and facilitate activation, which involves the channeling of two nickel ions into the active site. So far this process has not been investigated in eukaryotes. Using affinity pulldowns of Strep-tagged UAPs from Arabidopsis and rice transiently expressed in planta, we demonstrate that a urease-UreD-UreF-UreG complex exists in plants and show its stepwise assembly. UreG is crucial for nickel delivery because UreG-dependent urease activation in vitro was observed only with UreG obtained from nickel-sufficient plants. This activation competence could not be generated in vitro by incubation of UreG with nickel, bicarbonate, and GTP. Compared with their bacterial orthologs, plant UreGs possess an N-terminal extension containing a His- and Asp/Glu-rich hypervariable region followed by a highly conserved sequence comprising two potential HXH metal-binding sites. Complementing the ureG-1 mutant of Arabidopsis with N-terminal deletion variants of UreG demonstrated that the hypervariable region has a minor impact on activation efficiency, whereas the conserved region up to the first HXH motif is highly beneficial and up to the second HXH motif strictly required for activation. We also show that urease reaches its full activity several days after nickel becomes available in the leaves, indicating that urease activation is limited by nickel accessibility in vivo Our data uncover the crucial role of UreG for nickel delivery during eukaryotic urease activation, inciting further investigations of the details of this process.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Modelos Moleculares , Níquel/metabolismo , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Urease/metabolismo , Sequência de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/isolamento & purificação , Apoenzimas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Células Cultivadas , Células Clonais , Sequência Conservada , Ativação Enzimática , Deleção de Genes , Hidroponia , Mutação , Oryza/enzimologia , Oryza/metabolismo , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/citologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Nicotiana/citologia , Nicotiana/genética , Nicotiana/crescimento & desenvolvimento , Urease/química , Urease/genética , Urease/isolamento & purificaçãoRESUMO
Here we present the development of a 13 week project-oriented biochemistry laboratory designed to introduce students to foundational biochemical techniques and then enable students to perform original research projects once they have mastered these techniques. In particular, we describe a semester-long laboratory that focuses on a biomedically relevant enzyme--Helicobacter pylori (Hp) urease--the activity of which is absolutely required for the gastric pathogen Hp to colonize the human stomach. Over the course of the semester, students undertake a biochemical purification of Hp urease, assess the success of their purification, and investigate the activity of their purified enzyme. In the final weeks of the semester, students design and implement their own experiments to study Hp urease. This laboratory provides students with an understanding of the importance of biochemistry in human health while empowering them to engage in an active area of research.
Assuntos
Bioquímica/educação , Educação/métodos , Helicobacter pylori/enzimologia , Aprendizagem Baseada em Problemas/métodos , Pesquisa/educação , Urease/isolamento & purificação , Bioquímica/métodos , Currículo , Humanos , Laboratórios , Estudantes , Urease/metabolismoRESUMO
BACKGROUND: Urease, one of the highly efficient known enzymes, catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The present study aimed to extract urease from pea seeds (Pisum Sativum L). The enzyme was then purified in three consequence steps: acetone precipitation, DEAE-cellulose ion-exchange chromatography, and gel filtration chromatography (Sephacryl S-200 column). RESULTS: The purification fold was 12.85 with a yield of 40%. The molecular weight of the isolated urease was estimated by chromatography to be 269,000 Daltons. Maximum urease activity (190 U/g) was achieved at the optimum conditions of 40°C and pH of 7.5 after 5 min of incubation. The kinetic parameters, Km and Vmax, were estimated by Lineweaver-Burk fits and found to be 500 mM and 333.3 U/g, respectively. The thermodynamic constants of activation, ΔH, Ea, and ΔS, were determined using Arrhenius plot and found to be 21.20 kJ/mol, 23.7 kJ/mol, and 1.18 kJ/mol/K, respectively. CONCLUSIONS: Urease was purified from germinating Pisum Sativum L. seeds. The purification fold, yield, and molecular weight were determined. The effects of pH, concentration of enzyme, temperature, concentration of substrate, and storage period on urease activity were examined. This may provide an insight on the various aspects of the property of the enzyme. The significance of extracting urease from different sources could play a good role in understanding the metabolism of urea in plants.
Assuntos
Bioquímica/métodos , Pisum sativum/enzimologia , Proteínas de Plantas/isolamento & purificação , Ureia/metabolismo , Urease/isolamento & purificação , Resinas Acrílicas , Precipitação Química , Cromatografia DEAE-Celulose , Cromatografia em Gel , Germinação , Hidrólise , Peso Molecular , Extratos Vegetais , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Sementes , Termodinâmica , Urease/química , Urease/metabolismoRESUMO
This paper describes the isolation of a potent extracellular urease producing microorganism, identified by 16S rRNA as Arthrobacter creatinolyticus MTCC 5604 and its medium optimization by classical one-factor-at-a-time method and central composite rotatable design (CCRD), a tool of response surface methodology (RSM). An optimal activity of 9.0 U ml(-1) was obtained by classical method and statistical optimization of the medium resulted in an activity of 17.35 U ml(-1) at 48 h and 30 °C. This activity was 4.91 times greater than the initial activity (3.53 U ml(-1) ) from the basal medium and the enzyme showed maximum activity at pH 8.0 and 60 °C and was stable at pH 7.0-9.0 and temperatures up to 50 °C. Furthermore, the enzyme was assessed for its activity reduction by determining the inhibitory concentration (IC50 ) of heavy metal ions and the inhibition of urease was in the order of Cu(II) > Cd(II) > Zn(II) > Ni(II). Urease was highly sensitive to Cu(II) and its inhibition was 94% and 100% in model solutions containing a mixture of Cu(II) with heavy metal ions Cd(II) and Zn(II), respectively. The results of these studies suggested that the enzyme could be utilized as sensors to determine the levels of Cu(II) ions in industrial effluents, contaminated soil and ground water.
Assuntos
Arthrobacter/enzimologia , Metais Pesados/análise , Urease/isolamento & purificação , Cátions Bivalentes , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Temperatura , Urease/biossínteseRESUMO
Jack bean (Canavalia ensiformis) is the source of interesting proteins that contribute to modern biochemistry, and urease is the primary of these proteins. Owing to its role and occurrence in nature, urease has become a part of extensive studies. In this study, jack bean urease (JBU) was purified by immobilized metal affinity chromatography using Cu(2+) chelated poly(hydroxyethyl methacrylate-N-methacryloyl-(L)-histidine methyl ester) [PHEMAH-Cu(2+)]-based cryogels. PHEMAH-Cu(2+) cryogel was synthesized and characterized for swelling degree, morphology (by SEM), N-methacryloyl-(L)-histidine methyl ester and Cu(2+) incorporation (by elemental analysis and atomic absorption spectrophotometry). The binding of JBU to PHEMAH-Cu(2+) cryogel was optimized by examining the effect of pH, flow rate and JBU concentration on binding. The maximal binding of JBU was 23.2 mg/dry gram of adsorbent. The maximal binding of JBU extracted from jack bean meal was 67.8 mg/dry gram of adsorbent. The elution of JBU from cryogel column was accomplished by 1.0 M NaCl in 20 mM phosphate buffer (pH 8.0). Molecular weight and purity of JBU from jack bean meal was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was observed that JBU could be repeatedly bound and eluted from (PHEMAH)-Cu(2+) cryogel with less than 10% loss in column capacity.
Assuntos
Canavalia/química , Quelantes/química , Cromatografia de Afinidade/métodos , Cobre/química , Histidina/análogos & derivados , Ácidos Polimetacrílicos/química , Urease/isolamento & purificação , Soluções Tampão , Criogéis , Eletroforese em Gel de Poliacrilamida , Histidina/química , Concentração de Íons de Hidrogênio , Cinética , Microscopia Eletrônica de Varredura , Peso Molecular , Espectrofotometria AtômicaRESUMO
This study aims to characterise biochemically urease from an atypical Campylobacter lari, namely urease-positive thermophilic Campylobacter (UPTC). Urease was purified from cells of a Japanese UPTC isolate (CF89-12) using phenyl-Sepharose chromatography. Two protein components (estimates molecular masses 24 kDa and 61 kDa) were obtained that appeared to be structural proteins of urease (subunits A and B), and these were fractionated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (PAGE). The native molecular weight for the final purified UPTC urease was estimated to be approximately 186,000 Da which is close to the calculated molecular weight (182,738 Da) based on all six open reading frames of UPTC CF89-12 urease genes (ureA, B, E, F, G and H), as described previously. Moreover, an active band was observed on phenol red staining after a nondenaturing native PAGE of the crude extract from the UPTC cells. In addition, the purified urease of UPTC CF8912 showed enzyme activity over a broad pH range (pH 6-10), with maximal activity at pH 8.0. The urease was also stable against heat treatment, with almost no loss of enzyme activity seen following 60-min incubation at temperatures of 20-60 degrees C. Urease subunits A and B were identified immunologically by Western blot analysis with rabbit anti-urease alpha (A) and beta (B) raised against Helicobacter pylori.
Assuntos
Campylobacter lari/enzimologia , Urease/química , Urease/isolamento & purificação , Western Blotting , Campylobacter lari/metabolismo , Catálise/efeitos dos fármacos , Cromatografia em Agarose , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacologia , Etanolaminas/química , Etanolaminas/farmacologia , Etilmaleimida/farmacologia , Hidroxiureia/farmacologia , Sefarose/análogos & derivados , Sefarose/química , Sefarose/farmacologia , Temperatura , Tioureia/farmacologia , Urease/antagonistas & inibidores , Urease/metabolismoRESUMO
All living systems depend on metalloproteins. Yet, while tools for the separation and identification of apo-proteins are well developed, those enabling identification and quantitation of individual metalloproteins within complex mixtures are still nascent. Here, we describe the electrophoretic separation of a mixture of carbonic anhydrase, ceruloplasmin, urease, and hemoglobin using native 2D gel electrophoresis and X-ray fluorescence mapping-an approach we have developed to be broadly applicable, not require specialized equipment for sample preparation, and likely to be extensible in the future.
Assuntos
Anidrases Carbônicas/isolamento & purificação , Ceruloplasmina/isolamento & purificação , Hemoglobinas/isolamento & purificação , Espectrometria por Raios X/métodos , Urease/isolamento & purificação , Animais , Anidrases Carbônicas/química , Bovinos , Ceruloplasmina/química , Eletroforese em Gel Bidimensional/métodos , Hemoglobinas/química , Focalização Isoelétrica , Metaloproteínas/química , Metaloproteínas/isolamento & purificação , Metais/química , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Conformação Proteica , Síncrotrons , Urease/química , Espectroscopia por Absorção de Raios X/métodosRESUMO
Small unextended molecules based on the diamidophosphate structure with a covalent carbon-to-phosphorus bond to improve hydrolytic stability were developed as a novel group of inhibitors to control microbial urea decomposition. Applying a structure-based inhibitor design approach using available crystal structures of bacterial urease, N-substituted derivatives of aminomethylphosphonic and P-methyl-aminomethylphosphinic acids were designed and synthesized. In inhibition studies using urease from Bacillus pasteurii and Canavalia ensiformis, the N,N-dimethyl derivatives of both lead structures were most effective with dissociation constants in the low micromolar range (Ki=13±0.8 and 0.62±0.09 µM, respectively). Whole-cell studies on a ureolytic strain of Proteus mirabilis showed the high efficiency of N,N-dimethyl and N-methyl derivatives of aminomethane-P-methylphosphinic acids for urease inhibition in pathogenic bacteria. The high hydrolytic stability of selected inhibitors was confirmed over a period of 30 days using NMR technique.
Assuntos
Domínio Catalítico , Inibidores Enzimáticos/síntese química , Ácidos Fosfínicos/síntese química , Proteus mirabilis/efeitos dos fármacos , Urease , Bacillus/efeitos dos fármacos , Canavalia/efeitos dos fármacos , Cinética , Modelos Moleculares , Ácidos Fosfínicos/química , Ácidos Fosfínicos/farmacologia , Compostos de Fósforo/química , Conformação Proteica , Relação Estrutura-Atividade , Urease/antagonistas & inibidores , Urease/química , Urease/isolamento & purificaçãoRESUMO
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.
Assuntos
Canavalia/enzimologia , Canavalia/genética , Urease/genética , Sequência de Aminoácidos , Antifúngicos/química , Antifúngicos/isolamento & purificação , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Sequência de Bases , Canavalia/química , Clonagem Molecular , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Homologia de Sequência do Ácido Nucleico , Urease/isolamento & purificação , Urease/metabolismo , Urease/farmacologiaRESUMO
Urease from Klebsiella aerogenes is composed of three subunits (UreA-UreB-UreC) that assemble into a (UreABC)(3) quaternary structure. UreC harbors the dinuclear nickel active site, whereas the functions of UreA and UreB remain unknown. UreD and UreF accessory proteins previously were suggested to reposition UreB and increase the level of exposure of the nascent urease active site, thus facilitating metallocenter assembly. In this study, cells were engineered to separately produce (UreAC)(3) or UreB, and the purified proteins were characterized. Monomeric UreB spontaneously binds to the trimeric heterodimer of UreA and UreC to form (UreABC*)(3) apoprotein, as shown by gel filtration chromatography, integration of electrophoretic gel band intensities, and mass spectrometry. Similar to the authentic urease apoprotein, the active enzyme is produced by incubation of (UreABC*)(3) with Ni(2+) and bicarbonate. Conversely, UreBΔ1-19, lacking the 19-residue potential hinge and tether to UreC, does not form a complex with (UreAC)(3) and yields negligible levels of the active enzyme when incubated under activation conditions with (UreAC)(3). Comparison of activities and nickel contents for (UreAC)(3), (UreABC*)(3), and (UreABC)(3) samples treated with Ni(2+) and bicarbonate and then desalted indicates that UreB facilitates efficient incorporation of the metal into the active site and protects the bound metal from chelation. Amylose resin pull-down studies reveal that MBP-UreD (a fusion of maltose binding protein with UreD) forms complexes with (UreABC)(3), (UreAC)(3), and UreB in vivo, but not in vitro. By contrast, MBP-UreD does not form an in vivo complex with UreBΔ1-19. The soluble MBP-UreD-UreF-UreG complex binds in vitro to (UreABC)(3), but not to (UreAC)(3) or UreB. Together, these data demonstrate that UreB facilitates the interaction of urease with accessory proteins during metallocenter assembly, with the N-terminal hinge and tether region being specifically required for this process. In addition to its role in urease activation, UreB enhances the stability of UreC against proteolytic cleavage.
Assuntos
Enterobacter aerogenes/enzimologia , Subunidades Proteicas/metabolismo , Urease/metabolismo , Enterobacter aerogenes/química , Enterobacter aerogenes/genética , Ativação Enzimática , Proteínas Ligantes de Maltose/metabolismo , Modelos Moleculares , Níquel/metabolismo , Peptídeo Hidrolases/metabolismo , Engenharia de Proteínas , Multimerização Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Urease/química , Urease/genética , Urease/isolamento & purificaçãoRESUMO
Helicobacter mustelae, a gastric pathogen of ferrets, synthesizes a distinct iron-dependent urease in addition to its archetypical nickel-containing enzyme. The iron-urease is oxygen-labile, with the inactive protein exhibiting a methemerythrin-like electronic spectrum. Significantly, incubation of the oxidized protein with dithionite under anaerobic conditions leads to restoration of activity and bleaching of the spectrum. Structural analysis of the oxidized species reveals a dinuclear iron metallocenter bridged by a lysine carbamate, closely resembling the traditional nickel-urease active site. Although the iron-urease is less active than the nickel-enzyme, its activity allows H. mustelae to survive the carnivore's low-nickel gastric environment.
Assuntos
Helicobacter mustelae/enzimologia , Ferro/metabolismo , Urease/metabolismo , Absorção/efeitos dos fármacos , Cristalografia por Raios X , Meios de Cultura/farmacologia , Elétrons , Helicobacter mustelae/efeitos dos fármacos , Íons , Cinética , Modelos Moleculares , Níquel/metabolismo , Oxigênio/metabolismo , Análise Espectral , Urease/química , Urease/isolamento & purificaçãoRESUMO
Jackbean (Canavalia ensiformis) ureases are entomotoxic upon the release of internal peptides by insect's digestive enzymes. Here we studied the digestive peptidases of Oncopeltus fasciatus (milkweed bug) and its susceptibility to jackbean urease (JBU). O. fasciatus nymphs fed urease showed a mortality rate higher than 80% after two weeks. Homogenates of midguts dissected from fourth instars were used to perform proteolytic activity assays. The homogenates hydrolyzed JBU in vitro, yielding a fragment similar in size to known entomotoxic peptides. The major proteolytic activity at pH 4.0 upon protein substrates was blocked by specific inhibitors of aspartic and cysteine peptidases, but not significantly affected by inhibitors of metallopeptidases or serine peptidases. The optimal activity upon N-Cbz-Phe-Arg-MCA was at pH 5.0, with complete blockage by E-64 in all pH tested. Optimal activity upon Abz-AIAFFSRQ-EDDnp (a substrate for aspartic peptidases) was detected at pH 5.0, with partial inhibition by Pepstatin A in the pH range 2-8. Fluorogenic substrates corresponding to the N- and C-terminal regions flanking a known entomotoxic peptide within urease sequence were also tested. While the midgut homogenate did not hydrolyze the N-terminal peptide, it cleaved the C-terminal peptide maximally at pH 4.0-5.0, and this activity was inhibited by E-64 (10 µM). The midgut homogenate was submitted to ion-exchange chromatography followed by gel filtration. A 22 kDa active fraction was obtained, resolved in SDS-PAGE (12%), the corresponding band was in-gel digested by trypsin, the peptides were analyzed by mass spectrometry, retrieving a cathepsin L protein. The purified cathepsin L was shown to have at least two possible cleavage sites within the urease sequence, and might be able to release a known insecticidal peptide in a single or cascade event. The results suggest that susceptibility of O. fasciatus nymphs to jackbean urease is, like in other insect models, due mostly to limited proteolysis of ingested protein and subsequent release of entomotoxic peptide(s) by cathepsin-like digestive enzymes.
Assuntos
Canavalia/toxicidade , Inseticidas/metabolismo , Ninfa/efeitos dos fármacos , Proteínas de Plantas/metabolismo , Precursores de Proteínas/metabolismo , Urease/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Canavalia/enzimologia , Catepsina L/química , Catepsina L/metabolismo , Cromatografia por Troca Iônica , Cumarínicos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Digestão , Dipeptídeos/farmacologia , Eletroforese em Gel de Poliacrilamida , Heterópteros/efeitos dos fármacos , Heterópteros/enzimologia , Concentração de Íons de Hidrogênio , Hidrólise/efeitos dos fármacos , Inseticidas/química , Inseticidas/isolamento & purificação , Inseticidas/toxicidade , Leucina/análogos & derivados , Leucina/farmacologia , Dados de Sequência Molecular , Ninfa/enzimologia , Fragmentos de Peptídeos/análise , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Proteínas de Plantas/toxicidade , Precursores de Proteínas/química , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/toxicidade , Urease/química , Urease/isolamento & purificação , Urease/toxicidadeRESUMO
BACKGROUND AND AIMS: Mongolian gerbils are frequently used to study Helicobacter pylori-induced gastritis and its consequences. The presence of some gastric flora with a suppressive effect on H. pylori suggests inhibitory microflora against H. pylori infection. The aim of the present study was to analyze the microflora in the stomach of Mongolian gerbils with H. pylori infection. METHODS: H. pylori ureA was detected by polymerase chain reaction (PCR) in the fecal samples of infected Mongolian gerbils. H. pylori was isolated from the gastric mucosa of the gerbils by microaerophilic cultivation. Gastric microflora were isolated by aerobic and anaerobic culture, and the identification of gastric bacterial species was performed by API20E and API20A. RESULTS: Oral administration of H. pylori TK1402 induced colonization and gastric inflammation of the stomach of the Mongolian gerbils. According to the frequency of detection of H. pylori ureA in fecal samples, the gerbils were divided into three groups (frequently detected, moderately detected and infrequently detected). According to the analysis of the gastric microflora in the frequently and infrequently detected groups, Lactobacillus spp. and Eubacterium limosum were isolated from the former and latter group, respectively. CONCLUSION: Some gastric flora, such as Lactobacillus spp., may inhibit colonization by H. pylori.
Assuntos
Mucosa Gástrica/microbiologia , Gastrite/microbiologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/isolamento & purificação , Animais , Proteínas de Bactérias/isolamento & purificação , Modelos Animais de Doenças , Eubacterium/isolamento & purificação , Fezes/microbiologia , Feminino , Gerbillinae , Helicobacter pylori/enzimologia , Helicobacter pylori/crescimento & desenvolvimento , Humanos , Lactobacillus/isolamento & purificação , Urease/isolamento & purificaçãoRESUMO
It has been demonstrated that acid urease is capable of decomposing urea in fermented beverage and foods. As urea is a precursor of ethylcarbamate, a potential carcinogenic compound, measures must be taken to control the level of urea. We herein describe the purification and characterization of a novel acid urease from Enterobacter sp. R-SYB082 and its application to the removal of urea in Chinese rice wine. The enzyme was purified to electrophoretic homogeneity using ethanol precipitation, Superdex 200 and Mono Q with a fold purification of 21.1 and a recovery of 49%. The molecular weight of the enzyme was 430,000 Da by gel filtration and 72,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that it was a hexamer. The activity of this purified enzyme was optimal at pH 4.5 and 35 degrees C. The temperature stability was under 55 degrees C, and the pH stability was 4.0~5.0. The enzyme exhibited an apparent K (m) of 19.5 micromol/l and a V (max) of 109 micromol urea/mg.min at 35 degrees C and pH 4.5. When incubating two different kinds of Chinese rice wine with the enzyme (0.08 U/ml) at 35 degrees C for 7 days, over 85% of urea was decomposed, and at 20 degrees C, above 78% was removed. The result showed that the enzyme is applicable to elimination of urea in Chinese rice wine.