Crystallization and preliminary X-ray crystallographic analysis of chorismate synthase from Mycobacterium tuberculosis

The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria but absent from mammals. The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. Optimization of crystallization trials allowed the crystallization of homogeneous recombinant CS from Mycobacterium tuberculosis (MtCS). The crystals of MtCS belong to space group P6(4)22 (or P6(2)22) and diffract to 2.8 Angstrom resolution, with unit-cell parameters a = b = 129.7, c = 156.8 Angstrom. There are two molecules in the asymmetric unit. Molecular-replacement trials were not sucessful. Heavy-atom derivative screening is in progress.

Crystallization and preliminary diffraction data of BaP1, a haemorrhagic metalloproteinase from Bothrops asper snake venom

BaP1 is a metalloproteinase isolated from the venom of the Central American snake Bothrops asper (terciopelo). It is a 24 kDa protein consisting of a single chain which includes the metalloproteinase domain only, therefore being classified as a class P-I snake-venom metalloproteinase. BaP1 induces prominent local tissue damage, such as haemorrhage, myonecrosis, blistering, dermonecrosis and oedema. In order to elucidate its structure, BaP1 was crystallized by the hanging-drop vapour-diffusion technique in 0.1 M bicine pH 9.0, 10% PEG 20 000 and 2%(v/v) dioxane. Diffraction data were observed to a resolution of 2.7 Angstrom. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 38.22, b = 60.17, c = 86.09 Angstrom.

Crystallization of bothrombin, a fibrinogen-converting serine protease isolated from the venom of Bothrops jararaca

Bothrombin, a snake-venom serine protease, specifically cleaves fibrinogen, releasing fibrinopeptide A to form non-crosslinked soft clots, aggregates platelets in the presence of exogeneous fibrinogen and activates blood coagulation factor VIII. Bothrombin shares high sequence homology with other snake-venom proteases such as batroxobin (94% identity), but only 30 and 34% identity with human alpha-thrombin and trypsin, respectively. Single crystals of bothrombin have been obtained and X-ray diffraction data have been collected at the Laboratorio Nacional de Luz Sincrotron to a resolution of 2.8 Angstrom. The crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 94.81, b = 115.68, c = 155.97 Angstrom.

Initial structural analysis of an alpha(4)beta(4) C-type lectin from the venom of Crotalus durissus terrificus

Convulxin, an alphabeta C-type lectin, is a potent platelet activator isolated from the venom of the South American rattlesnake Crotalus durissus terrificus. It is a 26.5 kDa alphabeta heterodimer consisting of two homologous disulfide-linked chains. The crystals belong to space group I4, with unit-cell parameters a = b = 131.61, c = 121.85 Angstrom, and diffraction data were collected to 2.7 Angstrom. The structure was solved by molecular replacement and the asymmetric unit contains two alphabeta heterodimers, each of which forms a disulfide-linked cyclic alpha(4)beta(4) tetramer in the unit cell. These alpha(4)beta(4) tetramers are stacked to form a large solvent channel.

Crystallization and preliminary diffraction data of a platelet-aggregation inhibitor from the venom of Agkistrodon piscivorus piscivorus (North American water moccasin)

Applaggin (Agkistrodon piscivorus piscivorus platelet-aggregation inhibitor) is a potent inhibitor of blood platelet aggregation derived from the venom of the North American water moccasin, the protein consists of 71 amino acids, is rich in cysteines, contains the sequence-recognition site of adhesion proteins at positions 50-52 (Arg-Gly-Asp) and shares high sequence homology with other snake-venom disintegrins such as echistatin, kistrin and trigramin, Single crystals of applaggin have been grown and X-ray diffraction data have been collected to a resolution of 3.2 Angstrom. The crystals belong to space group P4(1)2(1)2 (or its enantiomorph), with unit-cell dimensions a = b = 63.35, c = 74.18 Angstrom and two molecules per asymmetric unit. Molecular replacement using models constructed from the NMR structures of echistatin and kistrin has not been successful in producing a trial structure for applaggin.

Structure of 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida

2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase from Pseudomonas putida is a key enzyme in the Entner-Doudoroff pathway which catalyses the cleavage of KDPG via a class I Schiff-base mechanism. The crystal structure of this enzyme has been refined to a crystallographic residual R = 17.1% (R-free = 21.4%). The N-terminal helix caps one side of the torus of the (betaalpha)(8)-barrel and the active site is located on the opposite, carboxylic side of the barrel. The Schiff-base-forming Lys145 is coordinated by a sulfate (or phosphate) ion and two solvent water molecules. The interactions that stabilize the trimer are predominantly hydrophobic, with the exception of the cyclically permuted bonds formed between Glu132 OE1 of one molecule and Thr129 OG1 of a symmetry-equivalent molecule. Except for the N-terminal helix, the structure of KDPG aldolase from P. putida closely resembles the structure of the homologous enzyme from Escherichia coli.

Crystallization and preliminary X-ray diffraction analysis of the catalytic subunit of ADP-glucose pyrophosphorylase from potato tuber

ADP-glucose pyrophosphorylase is the key regulatory enzyme in the biosynthesis of starch in plants and glycogen in bacteria. The enzyme from potato tuber is comprised of a regulatory subunit and a catalytic subunit and is present as a heterotetramer (alpha(2)beta(2)) the catalytic subunit from potato tuber (50 kDa) was crystallized in four different forms, two of which are suitable for structural studies. A tetragonal crystal form obtained in the presence of the substrate analog Cr-ATP diffracted to 2.2 Angstrom and belongs to space group P4(1) (or its enantiomorph), with unit-cell parameters a = b = 110.57, c = 190.14 Angstrom. A second crystal form obtained diffracted to 2.8 Angstrom and belongs to space group PZ, with unit-eel parameters a = 80.06, b = 138.84, c = 92.20 Angstrom, beta = 112.40 degrees. As this protein displays no significant homology to any currently known protein structure, a search for heavy-atom derivatives has been initiated.

Crystallization and preliminary crystallographic analysis of SMase I, a sphingomyelinase from Loxosceles laeta spider venom

SMase I, a 32 kDa sphingomyelinase found in Loxosceles laeta venom, is responsible for the major pathological effects of spider envenomation. This toxin has been cloned and functionally expressed as a fusion protein containing a 6 x His tag at its N-terminus to yield a 33 kDa protein [Fernandes-Pedrosa et al. (2002), Biochem. Biophys. Res. Commun. 298, 638 - 645]. The recombinant protein possesses all the biological properties ascribed to the whole L. laeta venom, including dermonecrotic and complement-dependent haemolytic activities. Dynamic light-scattering experiments conducted at 291 K demonstrate that the sample possesses a monomodal distribution, with a hydrodynamic radius of 3.57 nm. L. laeta SMase I was crystallized by the hanging-drop vapour-diffusion technique using the sparse-matrix method. Single crystals were obtained using a buffer solution consisting of 0.08 M HEPES and 0.9 M trisodium citrate, which was titrated to pH 7.5 using 0.25 M sodium hydroxide. Complete three-dimensional diffraction data were collected to 1.8 Angstrom at the Laboratorio Nacional de Luz Sincrotron (LNLS, Campinas, Brazil). The crystals belong to the hexagonal system ( space group P6(1) or P6(5)), with unit-cell parameters a = b = 140.6, c = 113.6 Angstrom. A search for heavy-atom derivatives has been initiated and elucidation of the crystal structure is currently in progress.

Crystallization, preliminary X-ray diffraction analysis and Patterson search of oxyhaemoglobin I from the wolf (Chrysocyon brachiurus)

Oxyhaemoglobin I isolated from the Brazilian wolf Chrysocyon brachiurus has been crystallized and X-ray diffraction data has been collected to 2.06 A resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P2(1)2(1)2(1) and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit. The structure was determined using standard molecular-replacement techniques and is currently being refined using maximum-likelihood protocols. This is the first haemoglobin isolated from a member of the Canidae family to be crystallized and it will provide further insights in the comparative biochemistry of vertebrate haemoglobins.

Diversidade e sazonalidade de ácaros (Acari) em seringal (Hevea brasiliensis, Muell. Arg.) no Noroeste do Estado de São Paulo

A Região Sudeste do Brasil apresenta aspectos edafo-climáticos favoráveis para o plantio e desenvolvimento da seringueira, sendo o estado de São Paulo o maior produtor nacional de látex. Este trabalho teve como objetivos determinar, através de coletas mensais durante três anos de estudo, a diversidade, a riqueza de espécies e a sua sazonalidade, bem como realizar estimativas de densidade populacional das principais espécies de ácaros presentes nas folhas. Todos os ácaros foram montados em lâminas de microscopia, totalizando 74.407 indivíduos, de 26 espécies pertencentes a 10 famílias. Os fitófagos representaram 95,4% do total de indivíduos coletados e os predadores 3,9%. Doze espécies foram consideradas acidentais, seis foram acessórias e oito constantes. Apresentaram maior número de espécies as famílias Phytoseiidae (cinco) e Tydeidae (quatro). A espécie mais abundante foi Calacarus heveae Feres (50.573), com maior abundância nos meses correspondentes ao término da estação chuvosa e início da estação seca na região. Dentre os predadores, a mais abundante foi Zetzellia quasagistemas Hernandes & Feres (1.345), seguida por Pronematus sp. (455), Zetzellia agistzellia Hernandes & Feres (409) e Euseius citrifolius Denmark & Muma (243). C. heveae apresentou maior densidade populacional em março e abril de 2003, e Lorryia formosa Cooreman e Tenuipalpus heveae Baker em março e maio de 2001, respectivamente. Muitos estigmeídeos foram observados associados a agrupamentos de L. formosa predando seus ovos e estágios imaturos.

Diversidade e flutuação populacional de ácaros (Acari) em Mabea fistulifera Mart. (Euphorbiaceae) de dois fragmentos de mata estacional semidecídua em São José do Rio Preto, SP

Amostrou-se mensalmente, durante 2001, ácaros de Mabea fistulifera Mart. em dois fragmentos de mata, um urbano e outro rural. em cada local e coleta, foram coletadas 20 folhas de cada um de três exemplares de M. fistulifera. As folhas foram examinadas sob estereomicroscópio e todos os ácaros encontrados, montados em lâminas e identificados e contados sob microscópio com contraste de fase. A diversidade e a uniformidade de espécies foram analisadas através dos índices de Shannon-Wiener e Pielou, respectivamente. O índice de similaridade de Morisita-Horn foi utilizado para determinar o grau de semelhança entre as áreas. A diversidade máxima teórica e a constância também foram determinadas. O índice de correlação de Pearson foi utilizado para verificar possíveis relações entre a abundância de ácaros com a pluviosidade. No fragmento urbano foram coletados 6326 ácaros pertencentes a 31 espécies, e no rural, 2330 ácaros de 25 espécies. Nos dois fragmentos, a diversidade excedeu 50% da diversidade máxima teórica, entretanto, no rural verificou-se maior diversidade. Registrou-se também, grande abundância de fitófagos, que foi maior no urbano, provavelmente como resultado do estresse das plantas favorecendo o desenvolvimento dos fitófagos. Alguns predadores apresentaram grande abundância durante a floração de M. fistulifera, provavelmente por utilizarem o pólen como alimento. A maior abundância de Euseius citrifolius Denmark & Muma, na estação seca, pode estar correlacionada com a maior disponibilidade de pólen, e não com a menor pluviosidade do período.

Ácaros (Acari) de dois sistemas de cultivo da seringueira no noroeste do estado de São Paulo

A cultura da seringueira (Hevea brasiliensis Muell. Agr.) vem se expandindo consideravelmente no noroeste do estado de São Paulo. O objetivo deste trabalho foi avaliar a fauna de ácaros em dois sistemas comuns de cultivo nessa região, um em monocultivo e outro em consórcio com gariroba (Syagrus oleracea (Mart.) Becc. O estudo foi conduzido entre maio de 2002 e abril de 2003, tomando-se amostras mensais de 150 folíolos de seringueira de cada plantação e 150 folíolos de gariroba. As faunas dos dois sistemas foram semelhantes; os números de espécies, gêneros e famílias, assim como as famílias mais diversas e mais abundantes foram semelhantes entre eles. Dezoito espécies ocorreram exclusivamente em plantas de gariroba. O número de ácaros sobre estas foi consideravelmente menor, mas a riqueza de espécies foi maior que em seringueiras. Dentre as espécies mais freqüentes e abundantes em seringueira, Eutetranychus banksi (McGregor) e Pronematus sp. foram as únicas também freqüentes em gariroba, porém bem menos abundantes. Nenhum dos predadores abundantes em seringueira (Zetzellia aff. yusti, Euseius citrifolius Denmark & Muma, Pronematus sp. e Spinibdella sp.) foi abundante em gariroba. O fungo Hirsutella thompsonii Fisher foi o único patógeno de ácaros encontrado neste estudo, atacando Calacarus heveae Feres. Aparentemente, na forma como cultivada na região, a gariroba não constitui um reservatório adequado dos ácaros predadores mais importantes em seringueira.

Diversidade de ácaros (Acari, Arachnida) em seringueiras (Hevea brasiliensis Muell. Arg., Euphorbiaceae) na região noroeste do Estado de São Paulo, Brasil

The mites of three rubber tree cultures (Cedral, Pindorama and Taquaritinga) in order to determine the abundance of populations, the richness, the diversity and the degree of similarity among the communities was studied. Twenty one species were found, five of which were common to the three cultures. The richness and the abundance were greatest at the beginning of the dry season. The composition of communities differed probably as consequence of the kind of neighboring vegetation to each area, and because of the acaricid pulverization on the culture of Taquaritinga, reductng the richness of mite species in that area. The influence of neighboring vegetation can be shown by the occurrence of Iphiseiodes zuluagui Denmark & Muma, 1972, a common species to citrus trees, on neighboring rubber trees in Taquaritinga, and Tenuipalpus heveae Baker, 1945, a common species on rubber trees, on a coffee culture neighbor to the rubber trees of Pindorama. This data suggests that mites move among neighbor cultures, and can be an importam factor towards pest management and control. The diversity was small on the three cultures, as a result of the occurrence of one dominam species on each area, Calacarus heveae Feres, 1992 or T. heveae, which are considered pests of the rubber tree. The small diversity and the occurrence of dominant species are patterns expected in monocultures, systems with small environmental heterogeneity.

Ácaros (Acari) de seringueira (Hevea brasiliensis Muell. Arg., Euphorbiaceae) e de euforbiáceas espontâneas no interior dos cultivos

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

Plant inhabiting mites (Acari) from the Cerrado biome of Mato Grosso State, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)