Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations

Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 angstrom resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis, for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting beta-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.

Singlet oxygen reacts with 2 `,7 `-dichlorodihydrofluorescein and contributes to the formation of 2 `,7 `-dichlorofluorescein

There are controversial reports in the literature concerning the reactivity of singlet oxygen ((1)O(2)) with the redox probe 2`,7`-dichlorodihydrofluorescein (DCFH). By carefully preparing solutions in which (1)O(2) is quantitatively generated in the presence of DCFH, we were able to show that the formation rate of the fluorescent molecule derived from DCFH oxidation, which is 2`,7`-dichlorofluorescein (DCF), increases in D(2)O and decreases in sodium azide, proving the direct role of (1)O(2) in this process. We have also prepared solutions in which either (1)O(2) or dication (MB(center dot 2+)) and semi-reduced (MB(center dot)) radicals of the sensitizer and subsequently super-oxide radical (O(2)(center dot-)) are generated. The absence of any effect of SOD and catalase ruled out the DCFH oxidation by O(2)(center dot-), indicating that both (1)O(2) and MB(center dot 2+) react with DCFH. Although the formation of DCF was 1 order of magnitude larger in the presence of MB(center dot 2+) than in the presence of (1)O(2), considering the rate of spontaneous decays of these species in aqueous solution, we were able to conclude that the reactivity of (1)O(2) with DCFH is actually larger than that of MB(center dot 2+). We conclude that DCFH can continue to be used as a probe to monitor general redox misbalance induced in biologic systems by oxidizing radicals and (1)O(2).

The role in the substrate specificity and catalysis of residues forming the substrate aglycone-binding site of a beta-glycosidase

The relative contributions to the specificity and catalysis of aglycone, of residues E190, E194, K201 and M453 that form the aglycone-binding site of a beta-glycosidase from Spodoptera frugiperda (EC 3.2.1.21), were investigated through site-directed mutagenesis and enzyme kinetic experiments. The results showed that E190 favors the binding of the initial portion of alkyl-type aglycones (up to the sixth methylene group) and also the first glucose unit of oligosaccharidic aglycones, whereas a balance between interactions with E194 and K201 determines the preference for glucose units versus alkyl moieties. E194 favors the binding of alkyl moieties, whereas K201 is more relevant for the binding of glucose units, in spite of its favorable interaction with alkyl moieties. The three residues E190, E194 and K201 reduce the affinity for phenyl moieties. In addition, M453 favors the binding of the second glucose unit of oligosaccharidic aglycones and also of the initial portion of alkyl-type aglycones. None of the residues investigated interacted with the terminal portion of alkyl-type aglycones. It was also demonstrated that E190, E194, K201 and M453 similarly contribute to stabilize ES double dagger. Their interactions with aglycone are individually weaker than those formed by residues interacting with glycone, but their joint catalytic effects are similar. Finally, these interactions with aglycone do not influence glycone binding.

Characterization of a beta-1,3-glucanase active in the alkaline midgut of Spodoptera frugiperda larvae and its relation to beta-glucan-binding proteins

Spodoptera frugiperda beta-1,3-glucanase (SLam) was purified from larval midgut. It has a molecular mass of 37.5 kDa, an alkaline optimum pH of 9.0, is active against beta-1,3-glucan (laminarin), but cannot hydrolyze yeast beta-1,3-1,6-glucan or other polysaccharides. The enzyme is an endoglucanase with low processivity (0.4), and is not inhibited by high concentrations of substrate. In contrast to other digestive beta-1,3-glucanases from insects, SLam is unable to lyse Saccharomyces cerevisae cells. The cDNA encoding SLam was cloned and sequenced, showing that the protein belongs to glycosyl hydrolase family 16 as other insect glucanases and glucan-binding proteins. Multiple sequence alignment of beta-1,3-glucanases and beta-glucan-binding protein supports the assumption that the beta-1,3-glucanase gene duplicated in the ancestor of mollusks and arthropods. One copy originated the derived beta-1,3-glucanases by the loss of an extended N-terminal region and the beta-glucan-binding proteins by the loss of the catalytic residues. SLam homology modeling suggests that E228 may affect the ionization of the catalytic residues, thus displacing the enzyme pH optimum. SLam antiserum reacts with a single protein in the insect midgut. Immunocytolocalization shows that the enzyme is present in secretory vesicles and glycocalyx from columnar cells. (C) 2010 Elsevier Ltd. All rights reserved.

The catalytic and other residues essential for the activity of the midgut trehalase from Spodoptera frugiperda

Trehalase (EC 3.2.1.28) hydrolyzes only alpha, alpha`- trehalose and is present in a variety of organisms, but is most important in insects and fungi. Crystallographic data showed that bacterial trehalase has 0312 and E496 as the catalytical residues and three Arg residues in the active site. Those residues have homologous in all family 37 trehalases including Spodoptera frugiperda trehalase (0322, E520, R169, R227, R287). To test the role of these residues, mutants of trehalase were produced. All mutants were at least four orders of magnitude less active than wild type trehalase and no structural difference between these mutants and wild type enzyme were discernible by circular dichroism. D322A and E520 pH-activity profile lacked the alkaline arm and the acid arm, respectively, suggesting that D322 is the acid and E520 the basic catalyst. Azide increases E520A activity three times, confirming its action as the basic catalyst. Taking into account the decrease in activity after substitution for alanine residue, the three arginine residues are as important as the catalytical ones to trehalase activity. This clarifies the previous misidentification of an Arg residue as the acid catalyst. As far as we know, this is the first report on the functional identification residues important for trehalase activity. (C) 2010 Elsevier Ltd. All rights reserved.

Sistema purinérgico em plaquetas de ratos adultos e interações com o sistema renina-angiotensina

As plaquetas sangüíneas são fragmentos citoplasmáticos, oriundos da ruptura dos megacariócitos, cuja principal função está relacionada à manutenção da integridade vascular. Os nucleotídeos extracelulares, ATP e ADP, bem como a adenosina, têm sido implicados em um grande número de funções fisiológicas: o ADP é o principal fator recrutador de plaquetas, enquanto que o ATP é um inibidor competitivo da agregação induzida por ADP. A adenosina é uma molécula capaz de induzir vasodilatação e inibir a agregação plaquetária. Desta maneira, a manutenção da sinalização purinérgica normal tem se mostrado importante para o tratamento de doenças cardiovasculares. Os nucleosídeos di e trifosfatos circulantes podem ser hidrolisados por membros de várias famílias de ectonucleotidases de membrana e solúveis, incluindo as ecto-nucleosídeo trifosfato difosfoidrolases (E-NTPDases) e ecto-nucleotídeo pirofosfatase/fosfodiesterases (E-NPPs), que em conjunto com a ecto-5’-nucleotidase, levam à formação de adenosina. Na superfície das plaquetas, ambas enzimas, E-NTPDase e ecto-5’-nucleotidase, estão descritas. O sistema renina-angiotensina é o principal regulador da função renal e cardiovascular, desenvolvendo um papel fundamental na homeostasia da pressão arterial e do balanço eletrolítico. A angiotensina II (ANGII) induz fisiologicamente a ativação das plaquetas, possivelmente devido às suas propriedades vasoconstritoras. Os objetivos deste trabalho foram, portanto: 1) caracterizar cineticamente a enzima E-NPP em plaquetas de ratos, utilizando o substrato marcador p-Nph-5’TMP e 2) esclarecer, mesmo que em parte, os possíveis efeitos da ANGII sobre a hidrólise extracelular de nucleotídeos por plaquetas de ratos. No primeiro capítulo deste trabalho, descrevemos uma atividade enzimática em plaquetas de ratos que compartilha as principais características bioquímicas já descritas para as E-NPPs: pH ótimo alcalino; valores de KM e Vmax calculados de aproximadamente 106.22 ± 17.83 μM e 3.44 ± 0.18 nmol p-nitrophenol/min/mg, respectivamente; e dependência de cátions divalentes. Além disso, o AMP inibiu somente a hidrólise do p-Nph-5’TMP. Por outro lado, a azida de sódio, em altas concentrações, a angiotensina II e o cloreto de gadolínio alteraram apenas as hidrólises de ATP ou ADP ou de ambos. No segundo capítulo, mostramos que a ANGII foi capaz de aumentar as hidrólises de ATP, ADP e AMP em plaquetas em todas as doses testadas (5, 50, 500 e 5000 picomóis). Entretanto, nenhuma alteração foi observada com relação à hidrólise do p-Nph-5'TMP. Em adição, observamos um aumento na hidrólise de AMP e uma diminuição na hidrólise de p-Nph-5'TMP em plaquetas de ratos espontaneamente hipertensos (SHR) quando comparados a ratos Wistar normotensos. De maneira geral, esta dissertação traz a caracterização bioquímica da enzima E-NPP na superfície de plaquetas intactas de ratos como sendo parte de um complexo sistema para a hidrólise de nucleotídeos nestes fragmentos citoplasmáticos, podendo, assim, contribuir para o desenvolvimento de terapias antiplaquetárias e para o tratamento de doenças vasculares. Adicionalmente, apresentamos alguns resultados demonstrando interações entre os sistemas angiotensinérgico e adenosinérgico de plaquetas de ratos, o que poderá contribuir para o entendimento e o tratamento de doenças cardiovasculares como hipertensão e arteriosclerose.

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MWr of about 120 kDa and specific PNPP activity of 1200 U/tng. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/ mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 Wing), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and (3glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function. (c) 2006 Elsevier B.V. All rights reserved.

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper. we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4 h of incubation at 25 degreesC. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395 nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degreesC.Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate.The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Comparative Studies of the (Anti) Mutagenicity of Baccharis dracunculifolia and Artepillin C by the Bacterial Reverse Mutation Test

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Mutagenicity and antimutagenicity of (-)-hinokinin a trypanosomicidal compound measured by Salmonella microsome and comet assays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Síntese e atividade citotóxica de alguns azido-ciclopaladados estabilizados com ligantes bifosfínicos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The role of matrix effects on the quantification of abscisic acid and its metabolites in the leaves of Bauhinia variegata L. using liquid chromatography combined with tandem mass spectrometry

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Flavonóides, norisoprenóides e outros terpenos das folhas de Tapirira guianensis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Semi-selective culture medium for Curtobacterium flaccumfaciens pv. flaccumfaciens isolation from bean seeds

Curtobacterium wilt has become an important disease of beans in several localities in the country. Its causal agent, Curtobacterium flaccumfacciens pv. flaccumfaciens (Cff), survives and is disseminated through seeds. To date, few studies have been conducted with the objective of developing an effective and low-cost culture medium to isolate this bacterium from bean seeds, for health analysis purposes. Usually, the culture media employed for coryneform bacteria isolation contain specific carbon sources and antimicrobial products not available in the Brazilian market. A culture medium known as MSCFF was developed (peptone - 5 g, meat extract - 3 g, sucrose - 5 g, agar 15 g, skim milk powder* - 5 g. Congo red* - 0.05 g-, chlorothalonil* - 0.01 g, thiophanate methyl* - 0.01 g, nalidixic acid* - 0.01 g, nitrofurantoin* - 0.01 g. oxacillin* 0.001 g, sodium azide* - 0.001 g and distilled water q.s. 1L; *added after autoclaving the basal medium), which has the ability to inhibit growth of a large amount of saprophytic bacteria, but with low supressivity to Cff isolates. The MSCFF medium was highly effective for Cff isolation from naturally infected bean seeds and could be used for routine detection of this bacterium in bean seeds.