Effects Of High Pressure Homogenization On The Activity, Stability, Kinetics And Three-dimensional Conformation Of A Glucose Oxidase Produced By Aspergillus Niger.

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.9e10341

Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

AbstractEndo-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

Kinase Inhibitor Profile For Human Nek1, Nek6, And Nek7 And Analysis Of The Structural Basis For Inhibitor Specificity.

Human Neks are a conserved protein kinase family related to cell cycle progression and cell division and are considered potential drug targets for the treatment of cancer and other pathologies. We screened the activation loop mutant kinases hNek1 and hNek2, wild-type hNek7, and five hNek6 variants in different activation/phosphorylation statesand compared them against 85 compounds using thermal shift denaturation. We identified three compounds with significant Tm shifts: JNK Inhibitor II for hNek1(Δ262-1258)-(T162A), Isogranulatimide for hNek6(S206A), andGSK-3 Inhibitor XIII for hNek7wt. Each one of these compounds was also validated by reducing the kinases activity by at least 25%. The binding sites for these compounds were identified by in silico docking at the ATP-binding site of the respective hNeks. Potential inhibitors were first screened by thermal shift assays, had their efficiency tested by a kinase assay, and were finally analyzed by molecular docking. Our findings corroborate the idea of ATP-competitive inhibition for hNek1 and hNek6 and suggest a novel non-competitive inhibition for hNek7 in regard to GSK-3 Inhibitor XIII. Our results demonstrate that our approach is useful for finding promising general and specific hNekscandidate inhibitors, which may also function as scaffolds to design more potent and selective inhibitors.201176-9

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of the catalytic domain of a hyperthermostable endo-1,4-B-D-mannanase from Thermotoga petrophila RKU-1

Endo-1,4-[beta]-D-mannanases play key roles in seed germination and fruit ripening and have recently received much attention owing to their potential applications in the food, detergent and kraft pulp industries. In order to delineate their structural determinants for specificity and stability, X-ray crystallographic investigations combined with detailed functional studies are being performed. In this work, crystals of the catalytic domain of a hyperthermostable endo-1,4-[beta]-D-mannanase from Thermotoga petrophila RKU-1 were obtained from three different conditions, resulting in two crystalline forms. Crystals from conditions with phosphate or citrate salts as precipitant (CryP) belonged to space group P212121, with unit-cell parameters a = 58.76, b = 87.99, c = 97.34 A, while a crystal from a condition with ethanol as precipitant (CryE) belonged to space group I212121, with unit-cell parameters a = 91.03, b = 89.97, c = 97.89 A. CryP and CryE diffracted to resolutions of 1.40 and 1.45 A, respectively.Peer reviewedMicrobiology and Molecular Genetic

Crystal structure and regulation of the citrus pol III repressor MAF1 by auxin and phosphorylation

MAF1 is the main RNA polymerase (Pol) III repressor that controls cell growth in eukaryotes. The Citrus ortholog, CsMAF1, was shown to restrict cell growth in citrus canker disease but its role in plant development and disease is still unclear. We solved the crystal structure of the globular core of CsMAF1, which reveals additional structural elements compared with the previously available structure of hMAF1, and explored the dynamics of its flexible regions not present in the structure. CsMAF1 accumulated in the nucleolus upon leaf excision, and this translocation was inhibited by auxin and by mutation of the PKA phosphorylation site, S45, to aspartate. Additionally, mTOR phosphorylated recombinant CsMAF1 and the mTOR inhibitor AZD8055 blocked canker formation in normal but not CsMAF1-silenced plants. These results indicate that the role of TOR on cell growth induced by Xanthomonas citri depends on CsMAF1 and that auxin controls CsMAF1 interaction with Pol III in citrusThis work was supported by Sa˜ o Paulo Research Foundation (FAPESP grant 2011/20468-1). C.E.B. and A.F.Z.N. received a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).Peer reviewe

Estratégias de inibição, mecanismos moleculares e interações intermoleculares em complexos macromoleculares

This work presents some features of essential biological processes such as the haemostatic system, integrity of biological membranes and thermostability of proteins. Crystallographic, spectroscopic and in silico tools have been used to obtain information at the molecular level of macromolecular complexes, action mechanisms and inhibition pathways. Worms, snakes, ticks, leeches and spiders produce a variety of proteins, which interfere in the regulation of these systems. Different toxins isolated from these organisms were characterized providing necessary information for the development of a new anti-myonecrotic molecule and reveal a new factor Xa exosite that is important for macromolecular substrates recognition and inhibition. The first crystal structure of a member of the sphingomyelinases D family was determined by the quick cryo-soaking technique and the catalytic mechanism was proposed, which involves a magnesium-binding site and two catalytic histidines. An alternative activation of the protein C pathway that does not require thrombomodulin was structurally characterized and revealed the dual role of the elestrotatic surface charge around the active site and the three strategically positioned carbohydrate moieties in the approach, recognition and activation of protein C.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Expression, purification, crystallization and preliminary crystallographic analysis of an endo-1,5-α-l-arabinanase from hyperthermophilic Thermotoga petrophila

The crystallization and preliminary X-ray diffraction studies of an endo-1,5-α-arabinanase from hyperthermophilic T. petrophila are reported. The crystals diffracted to 2.86 Å resolution

Effects of HPH treatment on GO conformation: UV absorption spectra (A) and Fluorescence emission spectra (B).

<p>Effects of HPH treatment on GO conformation: UV absorption spectra (A) and Fluorescence emission spectra (B).</p

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

Snake-venom proteins form multi-component defence systems by the recruitment and rapid evolution of nonvenomous proteins and hence serve as model systems to understand the structural modifications that result in toxicity. l-Amino-acid oxidases (LAAOs) are encountered in a number of snake venoms and have been implicated in the inhibition of platelet aggregation, cytotoxicity, haemolysis, apoptosis and haemorrhage. An l-amino-acid oxidase from Lachesis muta venom has been purified and crystallized. The crystals belonged to space group P2(1), with unit-cell parameters a = 66.05, b = 79.41, c= 100.52 angstrom, beta = 96.55 degrees. The asymmetric unit contained two molecules and the structure has been determined and partially refined at 3.0 angstrom resolution.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES