Exploring the Molecular Basis for Substrate Affinity and Structural Stability in Bacterial GH39 ß-Xylosidases.

The glycoside hydrolase family 39 (GH39) is a functionally expanding family with limited understanding about the molecular basis for substrate specificity and extremophilicity. In this work, we demonstrate the key role of the positive-subsite region in modulating substrate affinity and how the lack of a C-terminal extension impacts on oligomerization and structural stability of some GH39 members. The crystallographic and SAXS structures of a new GH39 member from the phytopathogen Xanthomonas citri support the importance of an extended C-terminal to promote oligomerization as a molecular strategy to enhance thermal stability. Comparative structural analysis along with site-directed mutagenesis showed that two residues located at the positive-subsite region, Lys166 and Asp167, are critical to substrate affinity and catalytic performance, by inducing local changes in the active site for substrate binding. These findings expand the molecular understanding of the mechanisms involved in substrate recognition and structural stability of the GH39 family, which might be instrumental for biological insights, rational enzyme engineering and utilization in biorefineries.

Assessment of postural sway in people living with HIV/AIDS / Avaliação da oscilação postural em pessoas vivendo com HIV/AIDS

INTRODUCTION: Motor changes are observed in people living with HIV/AIDS. These changes may be associated with the chronicity of infection, continued use of antiretroviral medication, and/or the presence of comorbidities. OBJECTIVE: The objective of the present study was to evaluate postural sway in people living with asymptomatic HIV/AIDS under treatment with highly active antiretroviral therapy. METHODS: Twenty-seven subjects, recruited at an HIV referral center, aged between 30 and 40 years, participated in the study, divided into two groups: HIV group (n=12) and non-HIV group (n=15). The participants performed an experimental task, remaining on a force platform in a static position, in bipedal support and semi-tandem positions, in conditions with and without vision. RESULTS: The results demonstrated that visual occlusion, when the bipedal base was adopted, generated significant differences in the area of oscillation and mean velocity in both groups. Differences were also observed in the area and mean velocity of both groups when the semi-tandem position was adopted without vision. When comparing the groups, it was possible to identify significant differences in the semi-tandem base with vision. CONCLUSION: Considering these results, it was found that postural oscillation was higher in the visual occlusion condition for both groups. Concomitant to this, we conclude that in the challenging condition, where the base of support is reduced, the HIV group presents greater oscillation (medial-lateral) than the non-HIV group.

INTRODUÇÃO: Alterações motoras são observadas em pessoas vivendo com HIV/AIDS. Essas alterações podem estar associadas à cronicidade da infecção, uso contínuo da medicação antiretroviral e ou pela presença de comorbidades. OBJETIVO: O objetivo do presente estudo foi avaliar a oscilação postural de pessoas vivendo com HIV/AIDS assintomáticos em tratamento com terapia antirretroviral altamente ativa. MÉTODOS: Vinte e sete indivíduos, recrutados em um centro de referência em HIV, com idade entre 30 e 40 anos, participaram do estudo, divididos em dois grupos: grupo HIV (n=12) e grupo não HIV (n=15). Os participantes realizaram uma tarefa experimental, permanecendo em uma plataforma de força em posição estática, em posições de apoio bipodal e semi-tandem, em condições com e sem visão. RESULTADOS: Os resultados demonstraram que a oclusão visual, quando adotada a base bipodal, gerou diferenças significativas na área de oscilação e velocidade média em ambos os grupos. Diferenças também foram observadas na área e velocidade média de ambos os grupos quando a posição semi-tandem foi adotada sem visão. Ao comparar os grupos, foi possível identificar diferenças significativas na base semi-tandem com visão. CONCLUSÃO: Considerando esses resultados, constatou-se que a oscilação postural foi maior na condição de oclusão visual para ambos os grupos. Concomitante a isso, concluímos que na condição desafiadora, onde a base de suporte é reduzida, o grupo HIV apresenta maior oscilação (médiolateral) que o grupo não HIV.

The impact of systematized physical activity on parameters of health-related physical fitness in schoolchildren aged 8 to 11 years / Impacto da atividade esportiva organizada sobre parâmetros da aptidão física relacionado à saúde de escolares de 8 a 11 anos de idade

The objective of this study was to verify if there is an impact of an additional program of sports activities (GSA) in school Physical Education classes on parameters of physical fitness related to the health of children. A quasi-experimental study was carried out, including 73 children, 8 to 11 years of age, of both sexes. The intervention took place over four weeks, with two weekly sessions of training or class, lasting 50 to 60 minutes per session. The children were allocated into two groups: a group that performed the Physical Education classes exclusively (GPE, n = 39); and a sports activities group (GSA, n = 34). The sports activities included passes, reception, pitches, dribbling, and running. Physical fitness was investigated by the Fitnessgram test in the pre- and post-intervention moments. Generalized Estimating Equations were used to compare the physical fitness tests between the pre and post moments (intragroup), and the impact of the intervention was verified by the intergroup analysis. The intragroup results showed that the GSA presented significant improvements in all physical fitness tests (p < 0.01), as well as reduction in relative fat and BMI (p < 0.05). However, the GPE demonstrated worsening results in the abdominal test (p = 0.019) and an increase in relative fat (p = 0.001). In the intergroup analysis, there were significant differences in the BMI and aerobic endurance test, with superiority in the GSA (p = 0.030). It was concluded that the program with sports activities was effective in improving physical fitness levels, while PE classes in isolation were not sufficient to guarantee improvement in physical fitness related to health

O objetivo deste estudo foi verificar se existe impacto de um programa adicional de atividades esportivas (GSA) às aulas de Educação Física (EF) escolar sobre parâmetros da aptidão física relacionada à saúde de crianças. Estudo quase - experimental que envolveu 73 crianças, de 8 a 11 anos de idade, de ambos os sexos. A intervenção ocorreu durante quatro semanas, com duas sessões semanais de treinamento ou aula, com duração de 50 a 60 minutos por sessão. As crianças foram alocadas em dois grupos: grupo que realizou as aulas de Edu-cação Física (GPE, n = 39) exclusivamente; e grupo de atividades esportivas (GSA, n = 34). As atividades esportivas incluíram jogos pré-desportivos, incluindo passes, recepção, arremessos, dribles e corridas. A aptidão física foi investigada pelo Fitnessgram nos momentos pré e pós-intervenção. As Equações de Estimativa Ge-neralizadas foram utilizadas para comparar os testes de aptidão física entre os momentos pré e pós (intragru-pos), e o impacto da intervenção foi verificado pela análise intergrupos. Os resultados intragrupo revelaram que o GSA obteve uma melhora significativa em todos os testes de aptidão física (p < 0,01), além da redução da gordura relativa e no IMC (p < 0,05). No entanto, o GPE demonstrou piora no teste abdominal (p = 0,019) e aumento na gordura relativa (p = 0,001). Na análise intergrupos, houve diferenças significativas no IMC e no teste de resistência aeróbia, com superioridade no GSA (p = 0,030). Conclui-se que o programa com atividades esportivas foi efetivo na melhora dos níveis de aptidão física, enquanto que as aulas de EF não foram suficientes de forma isolada para garantir melhora na aptidão física relacionada à saúde

The mechanism by which a distinguishing arabinofuranosidase can cope with internal di-substitutions in arabinoxylans.

BACKGROUND: Arabinoxylan is an abundant polysaccharide in industrially relevant biomasses such as sugarcane, corn stover and grasses. However, the arabinofuranosyl di-substitutions that decorate the xylan backbone are recalcitrant to most known arabinofuranosidases (Abfs). RESULTS: In this work, we identified a novel GH51 Abf (XacAbf51) that forms trimers in solution and can cope efficiently with both mono- and di-substitutions at terminal or internal xylopyranosyl units of arabinoxylan. Using mass spectrometry, the kinetic parameters of the hydrolysis of 33-α-l-arabinofuranosyl-xylotetraose and 23,33-di-α-l-arabinofuranosyl-xylotetraose by XacAbf51 were determined, demonstrating the capacity of this enzyme to cleave arabinofuranosyl linkages of internal mono- and di-substituted xylopyranosyl units. Complementation studies of fungal enzyme cocktails with XacAbf51 revealed an increase of up to 20% in the release of reducing sugars from pretreated sugarcane bagasse, showing the biotechnological potential of a generalist GH51 in biomass saccharification. To elucidate the structural basis for the recognition of internal di-substitutions, the crystal structure of XacAbf51 was determined unveiling the existence of a pocket strategically arranged near to the - 1 subsite that can accommodate a second arabinofuranosyl decoration, a feature not described for any other GH51 Abf structurally characterized so far. CONCLUSIONS: In summary, this study reports the first kinetic characterization of internal di-substitution release by a GH51 Abf, provides the structural basis for this activity and reveals a promising candidate for industrial processes involving plant cell wall depolymerization.

Structural basis of exo-ß-mannanase activity in the GH2 family.

The classical microbial strategy for depolymerization of ß-mannan polysaccharides involves the synergistic action of at least two enzymes, endo-1,4-ß-mannanases and ß-mannosidases. In this work, we describe the first exo-ß-mannanase from the GH2 family, isolated from Xanthomonas axonopodis pv. citri (XacMan2A), which can efficiently hydrolyze both manno-oligosaccharides and ß-mannan into mannose. It represents a valuable process simplification in the microbial carbon uptake that could be of potential industrial interest. Biochemical assays revealed a progressive increase in the hydrolysis rates from mannobiose to mannohexaose, which distinguishes XacMan2A from the known GH2 ß-mannosidases. Crystallographic analysis indicates that the active-site topology of XacMan2A underwent profound structural changes at the positive-subsite region, by the removal of the physical barrier canonically observed in GH2 ß-mannosidases, generating a more open and accessible active site with additional productive positive subsites. Besides that, XacMan2A contains two residue substitutions in relation to typical GH2 ß-mannosidases, Gly439 and Gly556, which alter the active site volume and are essential to its mode of action. Interestingly, the only other mechanistically characterized mannose-releasing exo-ß-mannanase so far is from the GH5 family, and its mode of action was attributed to the emergence of a blocking loop at the negative-subsite region of a cleft-like active site, whereas in XacMan2A, the same activity can be explained by the removal of steric barriers at the positive-subsite region in an originally pocket-like active site. Therefore, the GH2 exo-ß-mannanase represents a distinct molecular route to this rare activity, expanding our knowledge about functional convergence mechanisms in carbohydrate-active enzymes.

Crystal structure of mature 2S albumin from Moringa oleifera seeds.

2S albumins, the seed storage proteins, are the primary sources of carbon and nitrogen and are involved in plant defense. The mature form of Moringa oleifera (M. oleifera), a chitin binding protein isoform 3-1 (mMo-CBP3-1) a thermostable antifungal, antibacterial, flocculating 2S albumin is widely used for the treatment of water and is potentially interesting for the development of both antifungal drugs and transgenic crops. The crystal structure of mMo-CBP3-1 determined at 1.7 Å resolution demonstrated that it is comprised of two proteolytically processed α-helical chains, stabilized by four disulfide bridges that is stable, resistant to pH changes and has a melting temperature (TM) of approximately 98 °C. The surface arginines and the polyglutamine motif are the key structural factors for the observed flocculating, antibacterial and antifungal activities. This represents the first crystal structure of a 2S albumin and the model of the pro-protein indicates the structural changes that occur upon formation of mMo-CBP3-1 and determines the structural motif and charge distribution patterns for the diverse observed activities.

Efeito da atividade esportiva sistematizada sobre o desenvolvimento motor de crianças de sete a 10 anos / The effect of systematic sports activities on the motor development of children from seven to 10 years

Atividades físicas programadas na infância são reconhecidas por ocasionar mudanças nos diversos aspectos do desenvolvimento das crianças. Assim, o presente estudo objetivou verificar o impacto da atividade esportiva programada de ballet clássico e de futsal sobre indicadores de motricidade global e de equilíbrio em crianças. A amostra foi composta por 160 crianças entre sete e 10 anos de idade. Oitenta crianças de ambos os sexos foram selecionadas no ambiente escolar e compuseram os grupos de escolares, caracterizado pela prática exclusiva de Educação Física escolar. Os grupos vinculados à prática esportiva foram compostos por 40 crianças do sexo feminino, praticantes de "ballet" clássico e 40 do sexo masculino, praticantes de futsal, caracterizando os grupos de prática sistematizada. Para a avaliação motora foram aplicados os testes de motricidade global e equilíbrio da Escala de Desenvolvimento Motor "EDM". Além disso, o questionário de atividade física habitual foi utilizado para calcular o gasto energético. A distribuição dos dados foi verificada através do teste Shapiro-Wilk, e em seguida foram aplicados os testes não-paramétricos Kruskall-Wallis com post hoc U de Mann-Whitney, Wilcoxon e Qui-Quadrado de Pearson. O nível de significância foi estabelecido em 5% (p ≤ 0,05). Resultados significantes foram encontrados entre os grupos de prática sistematizada com índices classificados como superiores e percentuais maiores em Idade Motora em Motricidade Global (IMMG) e Idade Motora em Equilíbrio (IME) do que idade cronológica (IC). Conclui-se que as crianças praticantes de atividades esportivas demonstraram superioridade nos testes aplicados quando comparados ao grupo controle, em que mais de 65% apresentam classificação normal.

It is recognized that systematic physical activities cause changes in many aspects of children's development. Thus, the present study aimed to verify the impact of programmed sports activities, classical ballet and futsal, on indicators of global motor function and balance in children. The sample consisted of 160 children between 7 and 10 years of age. Eighty school children of both sexes were selected, characterized by the exclusive practice of school Physical Education. The programmed sports groups were composed of 40 female children, classical ballet practitioners, and 40 males who participated in futsal, characterizing the systematized practice group. The Motor Development Scale MDS was applied to assess global motor function and balance. In addition, the habitual physical activity questionnaire was used to calculate energy expenditure. Data distribution was verified using the Shapiro-Wilk's test and then were applied non-parametric tests of the Kruskall-Wallis test with post hoc Mann-Whitney U, Wilcoxon and Chi-Square Pearson tests. The significance level was set at 5% (p ≤ 0.05). Significant results were found in the systematized practice groups, with indices classified as superior and higher percentage for Motor Age of Global Motricity (MAGM) and Motor Age of Balance (MAB), when compared to chronological age (CA). In conclusion the children who practiced programmed sports activities demonstrated superiority in the tests when compared to the control group, where more than 65% were classified as normal.

Structural basis for xyloglucan specificity and α-d-Xylp(1 → 6)-D-Glcp recognition at the -1 subsite within the GH5 family.

GH5 is one of the largest glycoside hydrolase families, comprising at least 20 distinct activities within a common structural scaffold. However, the molecular basis for the functional differentiation among GH5 members is still not fully understood, principally for xyloglucan specificity. In this work, we elucidated the crystal structures of two novel GH5 xyloglucanases (XEGs) retrieved from a rumen microflora metagenomic library, in the native state and in complex with xyloglucan-derived oligosaccharides. These results provided insights into the structural determinants that differentiate GH5 XEGs from parental cellulases and a new mode of action within the GH5 family related to structural adaptations in the -1 subsite. The oligosaccharide found in the XEG5A complex, permitted the mapping, for the first time, of the positive subsites of a GH5 XEG, revealing the importance of the pocket-like topology of the +1 subsite in conferring the ability of some GH5 enzymes to attack xyloglucan. Complementarily, the XEG5B complex covered the negative subsites, completing the subsite mapping of GH5 XEGs at high resolution. Interestingly, XEG5B is, to date, the only GH5 member able to cleave XXXG into XX and XG, and in the light of these results, we propose that a modification in the -1 subsite enables the accommodation of a xylosyl side chain at this position. The stereochemical compatibility of the -1 subsite with a xylosyl moiety was also reported for other structurally nonrelated XEGs belonging to the GH74 family, indicating it to be an essential attribute for this mode of action.

Molecular mechanisms associated with xylan degradation by Xanthomonas plant pathogens.

Xanthomonas pathogens attack a variety of economically relevant plants, and their xylan CUT system (carbohydrate utilization with TonB-dependent outer membrane transporter system) contains two major xylanase-related genes, xynA and xynB, which influence biofilm formation and virulence by molecular mechanisms that are still elusive. Herein, we demonstrated that XynA is a rare reducing end xylose-releasing exo-oligoxylanase and not an endo-ß-1,4-xylanase as predicted. Structural analysis revealed that an insertion in the ß7-α7 loop induces dimerization and promotes a physical barrier at the +2 subsite conferring this unique mode of action within the GH10 family. A single mutation that impaired dimerization became XynA active against xylan, and high endolytic activity was achieved when this loop was tailored to match a canonical sequence of endo-ß-1,4-xylanases, supporting our mechanistic model. On the other hand, the divergent XynB proved to be a classical endo-ß-1,4-xylanase, despite the low sequence similarity to characterized GH10 xylanases. Interestingly, this enzyme contains a calcium ion bound nearby to the glycone-binding region, which is required for catalytic activity and structural stability. These results shed light on the molecular basis for xylan degradation by Xanthomonas and suggest how these enzymes synergistically assist infection and pathogenesis. Our findings indicate that XynB contributes to breach the plant cell wall barrier, providing nutrients and facilitating the translocation of effector molecules, whereas the exo-oligoxylanase XynA possibly participates in the suppression of oligosaccharide-induced immune responses.

Development and biotechnological application of a novel endoxylanase family GH10 identified from sugarcane soil metagenome.

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (ß/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production.

The accessory domain changes the accessibility and molecular topography of the catalytic interface in monomeric GH39 ß-xylosidases.

ß-Xylosidases (EC 3.2.1.37) are among the principal glycosyl hydrolases involved in the breakdown of hemicelluloses, catalyzing the reduction of xylooligosaccharides to free xylose. All GH39 ß-xylosidases structurally characterized to date display a modular multi-domain organization that assembles a tetrameric quaternary structure. In this work, the crystal structure and the SAXS molecular envelope of a new GH39 ß-xylosidase from Caulobacter crescentus (CcXynB2) have been determined. Interestingly, CcXynB2 is a monomer in solution and comparative structural analyses suggest that the shortened C-terminus prevents the formation of a stable tetramer. Moreover, CcXynB2 has a longer loop from the auxiliary domain (the long α-helix-containing loop) which makes a number of polar and hydrophobic contacts with the parental (α/ß)(8)-barrel domain, modifying the accessibility and the molecular topography of the catalytic interface. These interactions also maintain the accessory domain tightly linked to the catalytic core, which may be important for enzyme function and stability.

Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-ß-D-mannanase.

The breakdown of ß-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of ß-mannanases. In this work, a two-domain ß-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/ß)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.

Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of a purine nucleoside phosphorylase from Bacillus subtilis strain 168.

Purine nucleoside phosphorylase (PNP; EC 2.4.2.1) is a key enzyme of the purine-salvage pathway. Its ability to transfer glycosyl residues to acceptor bases is of great biotechnological interest owing to its potential application in the synthesis of nucleoside analogues used in the treatment of antiviral infections and in anticancer chemotherapy. Although hexameric PNPs are prevalent in prokaryotes, some microorganisms, such as Bacillus subtilis, present both hexameric and trimeric PNPs. The hexameric PNP from B. subtilis strain 168, named BsPNP233, was cloned, expressed and crystallized. Crystals belonging to different space groups (P32(1), P2(1)2(1)2(1), P6(3)22 and H32) were grown in distinct conditions with pH values ranging from 4.2 to 10.5. The crystals diffracted to maximum resolutions ranging from 2.65 to 1.70 Å.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-ß-1,3-glucanase from Thermotoga petrophila.

1,3-ß-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal ß-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90°C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules.

Functional and biophysical characterization of a hyperthermostable GH51 α-L-arabinofuranosidase from Thermotoga petrophila.

A hyperthermostable glycoside hydrolase family 51 (GH51) α-L-arabinofuranosidase from Thermotoga petrophila RKU-1 (TpAraF) was cloned, overexpressed, purified and characterized. The recombinant enzyme had optimum activity at pH 6.0 and 70°C with linear α-1,5-linked arabinoheptaose as substrate. The substrate cleavage pattern monitored by capillary zone electrophoresis showed that TpAraF is a classical exo-acting enzyme producing arabinose as its end-product. Far-UV circular dichroism analysis displayed a typical spectrum of α/ß barrel proteins analogously observed for other GH51 α-L-arabinofuranosidases. Moreover, TpAraF was crystallized in two crystalline forms, which can be used to determine its crystallographic structure.

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.

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.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of the catalytic domain of a hyperthermostable endo-1,4-beta-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 P2(1)2(1)2(1), 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 I2(1)2(1)2(1), 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.

Crystallization and preliminary X-ray diffraction analysis of Q4DV70 from Trypanosoma cruzi, a hypothetical protein with a putative thioredoxin domain.

Q4DV70 is annotated in the Trypanosoma cruzi CL Brener genome as a hypothetical protein with a predicted thioredoxin-like fold, although the catalytic cysteine residues that are conserved in typical oxidoreductases are replaced by serine residues. Gene-expression analysis indicates that this protein is differentially expressed during the T. cruzi life cycle, suggesting that it plays an important role during T. cruzi development. The gene coding for Q4DV70 was cloned and the protein was overexpressed in Escherichia coli with an N-terminal His tag. Purification of Q4DV70 was carried out by affinity and size-exclusion chromatography and the His tag was removed by TEV protease digestion. Crystals of Q4DV70 were grown using the sitting-drop vapour-diffusion method. A diffraction data set was collected to 1.50 A resolution from a single crystal grown in 25% PEG 1500, 200 mM sodium thiocyanate pH 6.9, 10 mM phenol and 10% ethylene glycol. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 35.04, b = 50.32, c = 61.18 A. The Q4DV70 structure was solved by molecular replacement using protein disulfide isomerase from yeast (PDB code 2b5e) as a search model. Initial refinement of the model indicated that the solution was correct. These data are being used for refinement of the model of Q4DV70.

Purification, crystallization and preliminary crystallographic studies of SPCI-chymotrypsin complex at 2.8 A resolution.

A binary complex of the Schizolobium parahyba chymotrypsin inhibitor (SPCI) with chymotrypsin was purified by size-exclusion chromatography and crystallized by the sitting-drop vapour-diffusion method with 100 mM MES-NaOH pH 5.5, 20%(w/v) PEG 6000, 200 mM LiCl as precipitant and 200 mM nondetergent sulfobetaine molecular weight 201 Da (NDSB-201) as an additive. SPCI is a small protein with 180 amino-acid residues isolated from S. parahyba seeds and is able to inhibit chymotrypsin at a 1:1 molar ratio by forming a stable complex. X-ray data were collected to 2.8 A resolution from a single crystal of the SPCI-chymotrypsin binary complex under cryogenic conditions. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 45.28, b = 64.57, c = 169.23 A, and the R(merge) is 0.122 for 11 254 unique reflections. A molecular-replacement solution was found using the preliminary crystal structure of SPCI and the structure of chymotrypsin (PDB code 4cha) independently as search models.