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Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:23:42Z No. of bitstreams: 1 WOS000233520700034.pdf: 595992 bytes, checksum: f004788d490756675a8f76c182ea544c (MD5) Made available in DSpace on 2014-02-26T17:23:42Z (GMT). No. of bitstreams: 1 WOS000233520700034.pdf: 595992 bytes, checksum: f004788d490756675a8f76c182ea544c (MD5) Previous issue date: 2005-11-21 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:02:21Z No. of bitstreams: 1 WOS000233520700034.pdf: 595992 bytes, checksum: f004788d490756675a8f76c182ea544c (MD5) Made available in DSpace on 2014-05-20T14:02:21Z (GMT). No. of bitstreams: 1 WOS000233520700034.pdf: 595992 bytes, checksum: f004788d490756675a8f76c182ea544c (MD5) Previous issue date: 2005-11-21 The 1.7 angstrom resolution crystal structure of recombinant family G/11 beta-1,4-xylanase (rXynA) from Bacillus subtilis 1A1 shows a jellyroll fold in which two curved P-sheets form the active-site and substrate-binding cleft. The onset of thermal denaturation of rXynA occurs at 328 K, in excellent agreement with the optimum catalytic temperature. Molecular dynamics simulations at temperatures of 298-328 K demonstrate that below the optimum temperature the thumb loop and palm domain adopt a closed conformation. However, at 328 K these two domains separate facilitating substrate access to the active-site pocket, thereby accounting for the optimum catalytic temperature of the rXynA. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. UNESP, IBILCE, Dept Phys, São Paulo, Brazil Univ São Paulo, FMRP, Dept Cellular & Mol Biol, BR-14049 Ribeirao Preto, SP, Brazil Univ São Paulo, FFCLRP, Dept Chem, BR-14049 Ribeirao Preto, SP, Brazil UNESP, IBILCE, Dept Phys, São Paulo, Brazil

Suramin, a synthetic polysulfonated compound, developed initially for the treatment of African trypanosomiasis and onchocerciasis, is currently used for the treatment of several medically relevant disorders. Suramin, heparin, and other polyanions inhibit the myotoxic activity of Lys49 phospholipase A2 analogues both in vitro and in vivo, and are thus of potential importance as therapeutic agents in the treatment of viperid snake bites. Due to its conformational flexibility around the single bonds that link the central phenyl rings to the secondary amide backbone, the symmetrical suramin molecule binds by an induced-fit mechanism complementing the hydrophobic surfaces of the dimer and adopts a novel conformation that lacks C2 symmetry in the dimeric crystal structure of the suramin–Bothrops asper myotoxin II complex. The simultaneous binding of suramin at the surfaces of the two monomers partially restricts access to the nominal active sites and significantly changes the overall charge of the interfacial recognition face of the protein, resulting in the inhibition of myotoxicity. Fundação de Amparo à Pesquisa do Estado de São Paulo//FAPESP/Brasil Structural Molecular Biology Network//SMOLBNet/Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/Brasil Coordenação de Aperfeiçoamento de Pessoal de Nível Superior//CAPES/DAAD/Brasil Centros de Pesquisa, Inovação e Difusão//CEPID/Brasil Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro//FAPERJ/Brasil Programa de Apoio a Núcles de Excelência//PRONEX/Brasil Universidad de Costa Rica//UCR/Costa Rica UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

Sphingomyelinases D (SMases D) from Loxosceles spider venom are the principal toxins responsible for the manifestation of dermonecrosis, intravascular hemolysis, and acute renal failure, which can result in death. These enzymes catalyze the hydrolysis of sphingomyelin, resulting in the formation of ceramide 1-phosphate and choline or the hydrolysis of lysophosphatidyl choline, generating the lipid mediator lysophosphatidic acid. This report represents the first crystal structure of a member of the sphingomyelinase D family from Loxosceles laeta (SMase I), which has been determined at 1.75-angstrom resolution using the "quick cryo-soaking" technique and phases obtained from a single iodine derivative and data collected from a conventional rotating anode x-ray source. SMase I folds as an (alpha/beta)8 barrel, the interfacial and catalytic sites encompass hydrophobic loops and a negatively charged surface. Substrate binding and/or the transition state are stabilized by a Mg2+ ion, which is coordinated by Glu32, Asp34, Asp91, and solvent molecules. In the proposed acid base catalytic mechanism, His12 and His47 play key roles and are supported by a network of hydrogen bonds between Asp34, Asp52, Trp230, Asp233, and Asn252.

Suramin is a highly charged polysulfonated napthylurea that interferes in a number of physiologically relevant processes such as myotoxicity, blood coagulation and several kinds of cancers. This synthetic compound was complexed with a myotoxic Lys49 PLA2 from Bothrops asper venom and crystallized by the hanging-drop vapor diffusion method at 18 °C. The crystals belong to the orthorhombic space group P212121, with unit cell parameters a=49.05, b=63.84 and c=85.67 Å. Diffraction data was collected to 1.78 Å. Fundação de Amparo à Pesquisa do Estado de São Paulo//FAPESP/Brasil Conselho Nacional de Desenvolvimento Científico e Tecnológico//CNPq/Brasil Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior//CAPES/Brasil UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)

In the Americas, 7.6 million people are infected with Trypanosoma cruzi, causative agent of Chagas disease. Although the parasite life cycle was determined in 1909, the drugs developed to eliminate T. cruzi have low efficacy and high toxicity, especially when the disease is in the chronic phase. Extracellular proteins secreted by protozoan parasites are key mediators in host-parasite interactions. Proteomic approaches have been used to investigate protein secreted by parasites intra-and extra-cellular and these studies led to the identification of a large number of proteins directly involved in fundamental processes of host-parasite interactions. There is evidence that the enzyme Asparagine synthase A from Trypanosoma cruzi is secreted during parasite invasion. This enzyme produces asparagine, glutamate, AMP and pyrophosphate in the presence of aspartate, ATP and glutamine or NH3. Although asparagine is of vital importance for the correct synthesis of proteins and post-translational modifications such as N-glycosylation, there is no structural characterization of this enzyme. In this work, the protein asparagine synthase A of T. cruzi was recombinantly-produced and purified. SAXS and DLS indicate changes in quaternary structure dependent on pH. Those modifications influence protein thermal stability. Structural analysis indicates the determinants of oligomerization and indicate potential sites of inhibition of this protein.

High pressure homogenization (HPH) is a non-thermal method, which has been employed to change the activity and stability of biotechnologically relevant enzymes. This work investigated how HPH affects the structural and functional characteristics of a glucose oxidase (GO) from Aspergillus niger. The enzyme was homogenized at 75 and 150 MPa and the effects were evaluated with respect to the enzyme activity, stability, kinetic parameters and molecular structure. The enzyme showed a pH-dependent response to the HPH treatment, with reduction or maintenance of activity at pH 4.5–6.0 and a remarkable activity increase (30–300%) at pH 6.5 in all tested temperatures (15, 50 and 75°C). The enzyme thermal tolerance was reduced due to HPH treatment and the storage for 24 h at high temperatures (50 and 75°C) also caused a reduction of activity. Interestingly, at lower temperatures (15°C) the activity levels were slightly higher than that observed for native enzyme or at least maintained. These effects of HPH treatment on function and stability of GO were further investigated by spectroscopic methods. Both fluorescence and circular dichroism revealed conformational changes in the molecular structure of the enzyme that might be associated with the distinct functional and stability behavior of GO.

Xylanases have been the focus of research owing to their industrial potential in animal feed production, food processing and pulp and paper processes. In order to obtain insight into the structural stability of family 11 xylanases, the mesophilic family 11 xylanase (beta-1,4-xylan xylanohydrolase; EC 3.2.1.8) from Bacillus subtilis 1A1 has been crystallized and diffraction data have been collected to 1.7 A. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 50.93, b = 70.50, c = 40.05 A. The structure has been determined by molecular replacement, resulting in a crystallographic residual of 36.4% after rigid-body refinement.

Nucleoside diphosphate kinases play a crucial role in the purine-salvage pathway of trypanosomatid protozoa and have been found in the secretome of Leishmania sp., suggesting a function related to host-cell integrity for the benefit of the parasite. Due to their importance for housekeeping functions in the parasite and by prolonging the life of host cells in infection, they become an attractive target for drug discovery and design. In this work, we describe the first structural characterization of nucleoside diphosphate kinases b from trypanosomatid parasites (tNDKbs) providing insights into their oligomerization, stability and structural determinants for nucleotide binding. Crystallographic studies of LmNDKb when complexed with phosphate, AMP and ADP showed that the crucial hydrogen-bonding residues involved in the nucleotide interaction are fully conserved in tNDKbs. Depending on the nature of the ligand, the nucleotide-binding pocket undergoes conformational changes, which leads to different cavity volumes. SAXS experiments showed that tNDKbs, like other eukaryotic NDKs, form a hexamer in solution and their oligomeric state does not rely on the presence of nucleotides or mimetics. Fluorescence-based thermal-shift assays demonstrated slightly higher stability of tNDKbs compared to human NDKb (HsNDKb), which is in agreement with the fact that tNDKbs are secreted and subjected to variations of temperature in the host cells during infection and disease development. Moreover, tNDKbs were stabilized upon nucleotide binding, whereas HsNDKb was not influenced. Contrasts on the surface electrostatic potential around the nucleotide-binding pocket might be a determinant for nucleotide affinity and protein stability differentiation. All these together demonstrated the molecular adaptation of parasite NDKbs in order to exert their biological functions intra-parasite and when secreted by regulating ATP levels of host cells.

Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the −1 and −2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 °C, and exclusively xylobiose at 90 °C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

Iron–sulfur cluster-containing proteins are present in all living organisms and are considered to be very ancient due to their ubiquity on the three domains of life, their importance in anaerobic metabolic pathways and energy conservation mechanisms. To date, there is little information regarding these proteins in Acidithiobacillus ferrooxidans , a bacterium involved in bioleaching processes. In this study are described the cloning, expression, purification and spectroscopic characterization of a new Fe–S cluster-containing protein, a putative HdrC homologue, from A. ferrooxidans strain LR. The oligomeric state of the protein was assessed by both dynamic light scattering and size-exclusion chromatography, demonstrating that it is present as a monomer in solution. Far-UV CD measurements revealed that this protein is extremely stable at acidic pHs, in which it assumes a different structure that exhibits a predominance of α-helixes. In contrast, at basic pHs, from 9 to 12, the protein showed a spectral profile which is characteristic for proteins with high content of beta structure and random coil. Thermal unfolding studies demonstrated that this protein tolerates high temperatures at acidic conditions, with a melting temperature of 95 °C. Furthermore, bioinformatics analysis suggested a heterodisulfide reductase function for the analyzed protein and for the other identified subunits, HdrA and HdrB, which could be related to hydrogen and/or formate metabolism in A. ferrooxidans .

The eukaryotic translation initiation factor 3, subunit L (eIF3L) is one of the subunits of the eIF3 complex, an accessory protein of the Polymerase I enzyme and may have an important role in the Flavivirus replication by interaction with a viral non-structural 5 protein. Considering the importance of eIF3L in a diversity of cellular functions, we have produced the recombinant full-length eIF3L protein in Escherichia coli and performed spectroscopic and in silico analyses to gain insights into its hydrodynamic behavior and structure. Dynamic light scattering showed that eIF3L behaves as monomer when it is not interacting with other molecular partners. Circular dichroism experiments showed a typical spectrum of α-helical protein for eIF3L, which is supported by sequence-based predictions of secondary structure and the 3D in silico model. The molecular docking with the K subunit of the eIF3 complex revealed a strong interaction. It was also predicted several potential interaction sites in eIF3L, indicating that the protein is likely capable of interacting with other molecules as experimentally shown in other functional studies. Moreover, bioinformatics analyses showed approximately 8 putative phosphorylation sites and one possible N-glycosylation site, suggesting its regulation by post-translational modifications. The production of the eIF3L protein in E. coli and structural information gained in this study can be instrumental for target-based drug design and inhibitors against Flavivirus replication and to shed light on the molecular mechanisms involved in the eukaryotic translation initiation.

Background Acidithiobacillus ferrooxidans is an acidophilic, chemolithoautotrophic bacterium that has been successfully used in metal bioleaching. In this study, an analysis of the A. ferrooxidans ATCC 23270 genome revealed the presence of three sHSP genes, Afe_1009, Afe_1437 and Afe_2172, that encode proteins from the HSP20 family, a class of intracellular multimers that is especially important in extremophile microorganisms. Results The expression of the sHSP genes was investigated in A. ferrooxidans cells submitted to a heat shock at 40°C for 15, 30 and 60 minutes. After 60 minutes, the gene on locus Afe_1437 was about 20-fold more highly expressed than the gene on locus Afe_2172. Bioinformatic and phylogenetic analyses showed that the sHSPs from A. ferrooxidans are possible non-paralogous proteins, and are regulated by the σ32 factor, a common transcription factor of heat shock proteins. Structural studies using homology molecular modeling indicated that the proteins encoded by Afe_1009 and Afe_1437 have a conserved α-crystallin domain and share similar structural features with the sHSP from Methanococcus jannaschii, suggesting that their biological assembly involves 24 molecules and resembles a hollow spherical shell. Conclusion We conclude that the sHSPs encoded by the Afe_1437 and Afe_1009 genes are more likely to act as molecular chaperones in the A. ferrooxidans heat shock response. In addition, the three sHSPs from A. ferrooxidans are not recent paralogs, and the Afe_1437 and Afe_1009 genes could be inherited horizontally by A. ferrooxidans.