Crystallization and preliminary X-ray diffraction analysis of prephenate dehydratase from Mycobacterium tuberculosis H37Rv

Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen ( Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 angstrom, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 angstrom resolution using a synchrotron-radiation source.

Crystallization and preliminary X-ray diffraction analysis of XAC1151, a small heat-shock protein from Xanthomonas axonopodis pv. citri belonging to the alpha-crystallin family

The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein ( sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 angstrom resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 angstrom. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.

Purification, crystallization and Patterson search of haemoglobin IV from the armoured catfish Liposarcus anisitsi

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

Crystallization and preliminary X-ray diffraction analysis of an anti-H(O) lectin from Lotus tetragonolobus seeds

The seed lectin from Lotus tetragonolobus (LTA) has been crystallized. The best crystals grew over several days and were obtained using the vapour-diffusion method at a constant temperature of 293 K. A complete structural data set was collected at 2.00 angstrom resolution using a synchrotron-radiation source. LTA crystals were found to be monoclinic, belonging to space group P2(1), with unit-cell parameters a = 68.89, b = 65.83, c = 102.53 angstrom, alpha = gamma = 90, beta = 92 degrees. Molecular replacement yielded a solution with a correlation coefficient and R factor of 34.4 and 51.6%, respectively. Preliminary analysis of the molecular-replacement solution indicates a new quaternary association in the LTA structure. Crystallographic refinement is under way.

Purification, crystallization and preliminary X-ray analysis of haemoglobin I from the armoured catfish Liposarcus anisitsi

Liposarcus anisitsi is an armoured catfish that presents accessorial air oxygenation through a modified stomach, which allows this species to survive in waters with very low oxygen content. Analysis of its haemolysate has shown the presence of four haemoglobins; this work focuses on the main component, haemoglobin I. It has been crystallized in two different forms and X-ray diffraction data have been collected to 2.77 and 2.86 Angstrom resolution using synchrotron radiation. Crystals were determined to belong to the space groups C2 and P2(1) and preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit in both crystal forms. The structure was determined using a standard molecular-replacement technique.

Crystallization of the secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus 39E.

The secondary alcohol dehydrogenase from the thermophile Thermoanaerobacter ethanolicus 39E has been crystallized at 40 degrees C by vapour difussion using polyethelene glycol as a precipitant. The orthorhombic crystals belong to the space group P 2(1)2(1)2 with cell constants of a=170.0 Angstrom, b=125.7 Angstrom and c=80.5 Angstrom. A native X-ray diffraction data set has been collected to 2.7 Angstrom resolution.

Isolation, characterization and biological activity of acidic phospholipase A(2) isoforms from Bothrops jararacussu snake venom

Acidic phospholipase A(2) (PLA(2)) isoforms in snake venoms, particularly those from Bothrops jararacussu, have not been characterized. This article reports the isolation and partial biochemical, functional and structural characterization of four acidic PLA(2)s (designated SIIISPIIA, SIIISPIIB, SIIISPIIIA and SIIISPIIIB) from this venom. The single chain purified proteins contained 122 amino acid residues and seven disulfide bonds with approximate molecular masses of 15 kDa and isoelectric points of 5.3. The respective N-terminal sequences were: SIIISPIIA-SLWQFGKMIDYVMGEEGAKS; SIIISPIIB-SLWQFGKMIFYTGKNEPVLS; SIIISPIIIA-SLWQFGKMILYVMGGEGVKQ and SIIISPIIIB-SLWQFGKMIFYEMTGEGVL. Crystals of the acidic protein SIIISPIIIB diffracted beyond 1.8 Angstrom resolution. These crystals are monoclinic with unit cell dimensions of a = 40.1 Angstrom, b = 54.2 Angstrom and c = 90.7 Angstrom. The crystal structure has been refined to a crystallographic residual of 16.1% (R-free = 22.9%). Specific catalytic activity (U/mg) of the isolated acidic PLA(2)s were SIIISPIIA = 290.3 U/mg; SIIISPIIB = 279.0 U/mg; SIIISPIIIA = 270.7 U/mg and SIIISPIIIB = 96.5 U/mg. Although their myotoxic activity was low, SIIISPIIA, SIIISPIIIB and SIIISPIIIA showed significant anticoagulant activity. However, there was no indirect hemolytic activity. SIIISPIIIB revealed no anticoagulant, but presented indirect hemolytic activity. With the exception of SIIISPIIIB, which inhibited platelet aggregation, all the others were capable of inducing time-independent edema. Chemical modification with 4-bromophenacyl bromide did not inhibit the induction of edema, but did suppress other activities. (C) 2003 Editions scientifiques et medicales Elsevier SAS. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of the lectin from Canavalia gladiata seeds

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

Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A(0) fully stripped of any anions

In this work, initial crystallographic studies of human haemoglobin (Hb) crystallized in isoionic and oxygen-free PEG solution are presented. Under these conditions, functional measurements of the O-2-linked binding of water molecules and release of protons have evidenced that Hb assumes an unforeseen new allosteric conformation. The determination of the high-resolution structure of the crystal of human deoxy-Hb fully stripped of anions may provide a structural explanation for the role of anions in the allosteric properties of Hb and, particularly, for the influence of chloride on the Bohr effect, the mechanism by which Hb oxygen affinity is regulated by pH. X-ray diffraction data were collected to 1.87 Angstrom resolution using a synchrotron-radiation source. Crystals belong to the space group P2(1)2(1)2 and preliminary analysis revealed the presence of one tetramer in the asymmetric unit. The structure is currently being refined using maximum-likelihood protocols.

Crystallization, preliminary X-ray analysis and molecular-replacement solution of the carboxy form of haemoglobin I from the fish Brycon cephalus

Haemoglobin, the 'honorary enzyme' [Brunori (1999), Trends Biochem. Sci. 24, 158-161], constitutes a prime prototype for allosteric models. Here, the crystallization and preliminary X-ray analysis of haemoglobin I from the South American fish Brycon cephalus are reported. X-ray diffraction data have been collected to 2.5 Angstrom resolution using synchrotron radiation (LNLS). Crystals were determined to belong to the space group P6(1)22 and preliminary structural analysis revealed the presence of one dimer (alpha beta) in the asymmetric unit. The structure was determined using standard molecular-replacement techniques.

Disulfide Biochemistry in 2-Cys Peroxiredoxin: Requirement of Glu50 and Arg146 for the Reduction of Yeast Tsa1 by Thioredoxin

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

Structure of the polypeptide crotamine from the Brazilian rattlesnake Crotalus durissus terrificus

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

Crystallization and preliminary X-ray diffraction studies of an L-amino-acid oxidase from Lachesis muta venom

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

Preliminary X-ray crystallographic studies of a Lys49-phospholipase A(2) homologue from Bothrops pirajai venom complexed with p-bromophenacyl bromide and alpha-tocopherol inhibitors

PrTX-I, a non-catalytic and myotoxic Lys49-PLA(2) from Bothrops pirajai venom has been crystallized alone and in complex with bromophenacyl bromide (BPB), alpha-tocopherol and alpha-tocopherol acetate inhibitors. These crystals have shown to diffract X-rays between 2.34 and 1.65 angstrom resolution. All complexes crystals are isomorphous and belong to the space group P2(1) whereas native PrTX-I crystals belong to the P3(1)21.

Effect of pH and temperature on the global compactness, structure, and activity of cellobiohydrolase Cel7A from Trichoderma harzianum

Due to its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum (T. harzianum) has considerable potential in biomass hydrolysis application. Cellulases from Trichoderma reesei have been widely used in studies of cellulose breakdown. However, cellulases from T. harzianum are less-studied enzymes that have not been characterized biophysically and biochemically as yet. Here, we examined the effects of pH and temperature on the secondary and tertiary structures, compactness, and enzymatic activity of cellobiohydrolase Cel7A from T. harzianum (Th Cel7A) using a number of biophysical and biochemical techniques. Our results show that pH and temperature perturbations affect Th Cel7A stability by two different mechanisms. Variations in pH modify protonation of the enzyme residues, directly affecting its activity, while leading to structural destabilization only at extreme pH limits. Temperature, on the other hand, has direct influence on mobility, fold, and compactness of the enzyme, causing unfolding of Th Cel7A just above the optimum temperature limit. Finally, we demonstrated that incubation with cellobiose, the product of the reaction and a competitive inhibitor, significantly increased the thermal stability of Th Cel7A. Our studies might provide insights into understanding, at a molecular level, the interplay between structure and activity of Th Cel7A at different pH and temperature conditions.