Conformation analysis of a surface loop that controls active site access in the GH11 xylanase A from Bacillus subtilis

Xylanases (EC 3.2.1.8 endo-1,4-glycosyl hydrolase) catalyze the hydrolysis of xylan, an abundant hemicellulose of plant cell walls. Access to the catalytic site of GH11 xylanases is regulated by movement of a short beta-hairpin, the so-called thumb region, which can adopt open or closed conformations. A crystallographic study has shown that the D11F/R122D mutant of the GH11 xylanase A from Bacillus subtilis (BsXA) displays a stable "open" conformation, and here we report a molecular dynamics simulation study comparing this mutant with the native enzyme over a range of temperatures. The mutant open conformation was stable at 300 and 328 K, however it showed a transition to the closed state at 338 K. Analysis of dihedral angles identified thumb region residues Y113 and T123 as key hinge points which determine the open-closed transition at 338 K. Although the D11F/R122D mutations result in a reduction in local inter-intramolecular hydrogen bonding, the global energies of the open and closed conformations in the native enzyme are equivalent, suggesting that the two conformations are equally accessible. These results indicate that the thumb region shows a broader degree of energetically permissible conformations which regulate the access to the active site region. The R122D mutation contributes to the stability of the open conformation, but is not essential for thumb dynamics, i.e., the wild type enzyme can also adapt to the open conformation.

Evaluation of plasma homocysteine level according to the C677T and A1298C polymorphism of the enzyme MTHRF in type 2 diabetic adults

Objective: To determine plasma homocysteine levels during fasting and after methionine overload, and to correlate homocysteinemia according to methylenetetrahydrofolate reductase (MTHFR) polymorphism in type 2 diabetic adults. Subjects and methods: The study included 50 type 2 diabetic adults (DM group) and 52 healthy subjects (Control group). Anthropometric data, and information on food intake, serum levels of vitamin B 12, folic acid and plasma homocysteine were obtained. The identification of C677T and A1298C polymorphisms was carried out in the MTHFR gene. Results: There was no significant difference in homocysteinemia between the two groups, and hyperhomocysteinemia during fasting occurred in 40% of the diabetic patients and in 23% of the controls. For the same polymorphism, there was not any significant difference in homocysteine between the groups. In the Control group, homocysteinemia was greater in those subjects with C677T and A1298C polymorphisms. Among diabetic subjects, those with the A1298C polymorphism had lower levels of homocysteine compared with individuals with C677T polymorphism. Conclusion: The MTHFR polymorphism (C677T and A1298C) resulted in different outcomes regarding homocysteinemia among individuals of each group (diabetic and control). These data suggest that metabolic factors inherent to diabetes influence homocysteine metabolism. Arq Bras Endocrinol Metab. 2012;56(7):429-34

Correlation Between Molecular Conformation, Packing, and Dynamics in Oligofluorenes: A Theoretical/Experimental Study

Fluorene-based systems have shown great potential as components in organic electronics and optoelectronics (organic photovoltaics, OPVs, organic light emitting diodes, OLEDs, and organic transistors, OTFTs). These systems have drawn attention primarily because they exhibit strong blue emission associated with relatively good thermal stability. It is well-known that the electronic properties of polymers are directly related to the molecular conformations and chain packing of polymers. Here, we used three oligofluorenes (trimer, pentamer, and heptamer) as model systems to theoretically investigate the conformational properties of fluorene molecules, starting with the identification of preferred conformations. The hybrid exchange correlation functional, OPBE, and ZINDO/S-CI showed that each oligomer exhibits a tendency to adopt a specific chain arrangement, which could be distinguished by comparing their UV/vis electronic absorption and C-13 NMR spectra. This feature was used to identify the preferred conformation of the oligomer chains in chloroform-cast films by comparing experimental and theoretical UV/vis and C-13 NMR spectra. Moreover, the oligomer chain packing and dynamics in the films were studied by DSC and several solid state NMR techniques, which indicated that the phase behavior of the films may be influenced by the tendency that each oligomeric chain has to adopt a given conformation.

Fluorescence polarization assay, competitive enzyme-linked immunosorbent assay (ELISA-C) and indirect ELISA for the diagnosis of brucellosis in buffaloes (Bubalus bubalis)

The objective of the present study was to compare the performance of three serological tests for diagnosis of Brucella abortus infections in buffaloes (Bubalus bubalis). Serum samples collected from 696 adult females were submitted to the competitive enzyme-linked immunosorbent assay (ELISAC), (I-ELISA), fluorescence polarization test (FPA), 2-mercaptoethanol test (2-ME) and complement fixation test (CFT). The gold standard was the combination of CFT and 2-ME, considering as positive the reactors in both CFT and 2-ME, and as negative those non-reactors. ROC analyses were done for C-ELISA, I-ELISA and FPA and the Kappa agreement index were also calculated. The best combinations of relative sensitivity (SEr) and relative specificity (SPr) and Kappa were given by C-ELISA (96.9%, 99.1%, and 0.932, respectively) and FPA (92.2%, 97.6 and 0.836, respectively). The C-ELISA and FPA were the most promising confirmatory tests for the serological diagnosis of brucellosis in buffaloes, and for these tests, cut-off values for buffaloes may be the same as those used for bovines.

Structural role of the active-site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase

Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzymes function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches.

Immbolization of uricase enzyme in Langmuir and Langmuir-Blodgett films of fatty acids: Possible use as a uric acid sensor

Preserving the enzyme structure in solid films is key for producing various bioelectronic devices, including biosensors, which has normally been performed with nanostructured films that allow for control of molecular architectures. In this paper, we investigate the adsorption of uricase onto Langmuir monolayers of stearic acid (SA), and their transfer to solid supports as Langmuir Blodgett (LB) films. Structuring of the enzyme in beta-sheets was preserved in the form of 1-layer LB film, which was corroborated with a higher catalytic activity than for other uricase-containing LB film architectures where the beta-sheets structuring was not preserved. The optimized architecture was also used to detect uric acid within a range covering typical concentrations in the human blood. The approach presented here not only allows for an optimized catalytic activity toward uric acid but also permits one to explain why some film architectures exhibit a superior performance. (C) 2011 Elsevier Inc. All rights reserved.

Influence of the type of phospholipid head and of the conformation of the polyelectrolyte on the growth of calcium carbonate thin films on LB/LbL matrices

Calcium carbonate is one of the most important biominerals, and it is the main constituent of pearls, seashells, and teeth. The in vitro crystallization of calcium carbonate using different organic matrices as templates has been reported. In this work, the growth of calcium carbonate thin films on special organic matrices consisting of layer-by-layer (LbL) polyelectrolyte films deposited on a pre-formed phospholipid Langmuir-Blodgett (LB) film has been studied. Two types of randomly coiled polyelectrolytes have been used: lambda-carrageenan and poly(acrylic acid). A precoating comprised of LB films has been prepared by employing a negatively charged phospholipid, the sodium salt of dimyristoilphosphatidyl acid (DMPA), or a zwitterionic phospholipid, namely dimyristoilphosphatidylethanolamine (DMPE). This approach resulted in the formation of particulate calcium carbonate continuous films with different morphologies, particle sizes, and roughness, as revealed by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The crystalline structure of the calcium carbonate particles was analyzed by Raman spectroscopy. The randomly coiled conformation of the polyelectrolytes seems to be the main reason for the formation of continuous films rather than CaCO3 isolated crystals. (C) 2012 Elsevier B.V. All rights reserved.

Improving the performance of a glucose biosensor using an ionic liquid for enzyme immobilization. On the chemical interaction between the biomolecule, the ionic liquid and the cross-linking agent

The effect of the room temperature ionic liquid (1-butyl-2,3-dimethylimidazolium tetrafluoroborate ([BMMI][BF4])) on the immobilization of glucose oxidase (GOx) was studied. The electrochemical performance of biosensors prepared following different protocols indicated a beneficial effect of the ionic liquid on the analytical parameters. The chemical interaction between GOx, [BMMI][BF4] and glutaraldehyde was investigated using UV-visible spectroscopy (UV-vis) and circular dichroism (CD). Structural changes of the biomolecule were observed to depend on the method used for the immobilization. (C) 2011 Elsevier Ltd. All rights reserved.

Purification and characterization of Hb 98-114: A novel hemoglobin-derived antimicrobial peptide from the midgut of Rhipicephalus (Boophilus) microplus

The antimicrobial activity of hemoglobin fragments (hemocidins) has been reported in a variety of models. The cattle tick Rhipicephalus (Boophilus) microplus is a blood sucking arthropod from where the first in vivo-generated hemocidin was characterized (Hb 33-61). In the present work we identified a novel antimicrobial peptide from the midgut of fully engorged R. (B.) microplus females, which comprises the amino acids 98-114 of the alpha subunit of bovine hemoglobin, and was designated Hb 98-114. This peptide was active against several yeast and filamentous fungi, although no activity was detected against bacteria up to 50 mu M of the synthetic peptide. Hb 98-114 was capable of permeabilizing Candida albicans cell membrane and had a fungicidal effect against this yeast. Circulardichroism (CD) and nuclear magnetic resonance (NMR) experiments showed that Hb 98-114 has a random conformation in aqueous solution but switches to an alpha-helical conformation in the presence of sodium dodecyl sulfate (SDS). This alpha helix adopts an amphipathic structure which may be the mechanism of cell membrane permeabilization. Importantly, Hb 98-114 may play an important role in defending the tick midgut against fungal pathogens and is the first hemocidin with specific antifungal activity to be characterized. (C) 2012 Elsevier Inc. All rights reserved.

Insights into Phosphate Cooperativity and Influence of Substrate Modifications on Binding and Catalysis of Hexameric Purine Nucleoside Phosphorylases

The hexameric purine nucleoside phosphorylase from Bacillus subtilis (BsPNP233) displays great potential to produce nucleoside analogues in industry and can be exploited in the development of new anti-tumor gene therapies. In order to provide structural basis for enzyme and substrates rational optimization, aiming at those applications, the present work shows a thorough and detailed structural description of the binding mode of substrates and nucleoside analogues to the active site of the hexameric BsPNP233. Here we report the crystal structure of BsPNP233 in the apo form and in complex with 11 ligands, including clinically relevant compounds. The crystal structure of six ligands (adenine, 2'deoxyguanosine, aciclovir, ganciclovir, 8-bromoguanosine, 6-chloroguanosine) in complex with a hexameric PNP are presented for the first time. Our data showed that free bases adopt alternative conformations in the BsPNP233 active site and indicated that binding of the co-substrate (2'deoxy) ribose 1-phosphate might contribute for stabilizing the bases in a favorable orientation for catalysis. The BsPNP233-adenosine complex revealed that a hydrogen bond between the 5' hydroxyl group of adenosine and Arg(43*) side chain contributes for the ribosyl radical to adopt an unusual C3'-endo conformation. The structures with 6-chloroguanosine and 8-bromoguanosine pointed out that the Cl-6 and Br-8 substrate modifications seem to be detrimental for catalysis and can be explored in the design of inhibitors for hexameric PNPs from pathogens. Our data also corroborated the competitive inhibition mechanism of hexameric PNPs by tubercidin and suggested that the acyclic nucleoside ganciclovir is a better inhibitor for hexameric PNPs than aciclovir. Furthermore, comparative structural analyses indicated that the replacement of Ser(90) by a threonine in the B. cereus hexameric adenosine phosphorylase (Thr(91)) is responsible for the lack of negative cooperativity of phosphate binding in this enzyme.

Order-Disorder Transitions Govern Kinetic Cooperativity and Allostery of Monomeric Human Glucokinase

Glucokinase (GCK) catalyzes the rate-limiting step of glucose catabolism in the pancreas, where it functions as the body's principal glucose sensor. GCK dysfunction leads to several potentially fatal diseases including maturity-onset diabetes of the young type II (MODY-II) and persistent hypoglycemic hyperinsulinemia of infancy (PHHI). GCK maintains glucose homeostasis by displaying a sigmoidal kinetic response to increasing blood glucose levels. This positive cooperativity is unique because the enzyme functions exclusively as a monomer and possesses only a single glucose binding site. Despite nearly a half century of research, the mechanistic basis for GCK's homotropic allostery remains unresolved. Here we explain GCK cooperativity in terms of large-scale, glucose-mediated disorder-order transitions using 17 isotopically labeled isoleucine methyl groups and three tryptophan side chains as sensitive nuclear magnetic resonance (NMR) probes. We find that the small domain of unliganded GCK is intrinsically disordered and samples a broad conformational ensemble. We also demonstrate that small-molecule diabetes therapeutic agents and hyperinsulinemia-associated GCK mutations share a strikingly similar activation mechanism, characterized by a population shift toward a more narrow, well-ordered ensemble resembling the glucose-bound conformation. Our results support a model in which GCK generates its cooperative kinetic response at low glucose concentrations by using a millisecond disorder-order cycle of the small domain as a "time-delay loop," which is bypassed at high glucose concentrations, providing a unique mechanism to allosterically regulate the activity of human GCK under physiological conditions.

Application of the log-conformation tensor to three-dimensional time-dependent free surface flows

The numerical simulation of flows of highly elastic fluids has been the subject of intense research over the past decades with important industrial applications. Therefore, many efforts have been made to improve the convergence capabilities of the numerical methods employed to simulate viscoelastic fluid flows. An important contribution for the solution of the High-Weissenberg Number Problem has been presented by Fattal and Kupferman [J. Non-Newton. Fluid. Mech. 123 (2004) 281-285] who developed the matrix-logarithm of the conformation tensor technique, henceforth called log-conformation tensor. Its advantage is a better approximation of the large growth of the stress tensor that occur in some regions of the flow and it is doubly beneficial in that it ensures physically correct stress fields, allowing converged computations at high Weissenberg number flows. In this work we investigate the application of the log-conformation tensor to three-dimensional unsteady free surface flows. The log-conformation tensor formulation was applied to solve the Upper-Convected Maxwell (UCM) constitutive equation while the momentum equation was solved using a finite difference Marker-and-Cell type method. The resulting developed code is validated by comparing the log-conformation results with the analytic solution for fully developed pipe flows. To illustrate the stability of the log-conformation tensor approach in solving three-dimensional free surface flows, results from the simulation of the extrudate swell and jet buckling phenomena of UCM fluids at high Weissenberg numbers are presented. (C) 2012 Elsevier B.V. All rights reserved.

Identification of Novel Interaction between ADAM17 (a Disintegrin and Metalloprotease 17) and Thioredoxin-1

ADAM17, which is also known as TNF alpha-converting enzyme, is the major sheddase for the EGF receptor ligands and is considered to be one of the main proteases responsible for the ectodomain shedding of surface proteins. How a membrane-anchored proteinase with an extracellular catalytic domain can be activated by inside-out regulation is not completely understood. We characterized thioredoxin-1 (Trx-1) as a partner of the ADAM17 cytoplasmic domain that could be involved in the regulation of ADAM17 activity. We induced the overexpression of the ADAM17 cytoplasmic domain in HEK293 cells, and ligands able to bind this domain were identified by MS after protein immunoprecipitation. Trx-1 was also validated as a ligand of the ADAM17 cytoplasmic domain and full-length ADAM17 recombinant proteins by immunoblotting, immunolocalization, and solid phase binding assay. In addition, using nuclear magnetic resonance, it was shown in vitro that the titration of the ADAM17 cytoplasmic domain promotes changes in the conformation of Trx-1. The MS analysis of the cross-linked complexes showed cross-linking between the two proteins by lysine residues. To further evaluate the functional role of Trx-1, we used a heparin-binding EGF shedding cell model and observed that the overexpression of Trx-1 in HEK293 cells could decrease the activity of ADAM17, activated by either phorbol 12-myristate 13-acetate or EGF. This study identifies Trx-1 as a novel interaction partner of the ADAM17 cytoplasmic domain and suggests that Trx-1 is a potential candidate that could be involved in ADAM17 activity regulation.

Relationship between global structural parameters and Enzyme Commission hierarchy: Implications for function prediction

In protein databases there is a substantial number of proteins structurally determined but without function annotation. Understanding the relationship between function and structure can be useful to predict function on a large scale. We have analyzed the similarities in global physicochemical parameters for a set of enzymes which were classified according to the four Enzyme Commission (EC) hierarchical levels. Using relevance theory we introduced a distance between proteins in the space of physicochemical characteristics. This was done by minimizing a cost function of the metric tensor built to reflect the EC classification system. Using an unsupervised clustering method on a set of 1025 enzymes, we obtained no relevant clustering formation compatible with EC classification. The distance distributions between enzymes from the same EC group and from different EC groups were compared by histograms. Such analysis was also performed using sequence alignment similarity as a distance. Our results suggest that global structure parameters are not sufficient to segregate enzymes according to EC hierarchy. This indicates that features essential for function are rather local than global. Consequently, methods for predicting function based on global attributes should not obtain high accuracy in main EC classes prediction without relying on similarities between enzymes from training and validation datasets. Furthermore, these results are consistent with a substantial number of studies suggesting that function evolves fundamentally by recruitment, i.e., a same protein motif or fold can be used to perform different enzymatic functions and a few specific amino acids (AAs) are actually responsible for enzyme activity. These essential amino acids should belong to active sites and an effective method for predicting function should be able to recognize them. (C) 2012 Elsevier Ltd. All rights reserved.

Enzyme activity in the small intestine of goat kids during the period of passive immunity acquisition

Enzyme activity of protein and carbohydrate degradation in small intestinal mucosa was investigated in goat kids fed with lyophilized bovine and goat colostrum. At 0,7 and 14 h of life 15 male newborns received 5% of body weight of lyophilized bovine colostrum and 14 goat colostrum, both with 55 mg/mL of IgG. Duodenum, jejunum and ileum samples were collected at 18,36 and 96 h of life. Three animals were sampled at birth, without colostrum intake. Activity of aminopeptidase N and A, dipeptidil peptidase IV, lactase, maltase and sucrase was determined as one international unit per gram of tissue. Intracellular enzymatic activity of acid phosphatase was observed by histochemistry in tissue section. Only the activity of aminopeptidase A in the ileum was affected by treatment, with a greater value for LBC than for GC (P < 0.05). The aminopeptidase N activity was the highest at 36 h in the duodenum (P < 0.05) and lowest at 96 h in the jejunum (P < 0.05). Dipeptidil peptidase IV activity was highest at 36 h in the duodenum (P < 0.05), lowest at 96 h in the jejunum (P < 0.05) and higher at 36 h than at 96 h in the ileum (P < 0.05). Aminopeptidase A activity in the ileum was highest at 36 h (P < 0.05), followed by 18 and 96 h of life (P < 0.05). Lactase activity in the duodenum increased from 18 to 36 h and from 36 to 96 h in the jejunum (P < 0.05). Maltase activity increased only in the duodenum from 18 to 96 h (P < 0.05). Sucrase activity in the jejunum decreased from 18 to 36 h and from 36 to 96 h in the ileum (P < 0.05). At birth, activity of most enzymes was similar to that at later times (P < 0.05). Histochemistry analyses showed a higher frequency of lysosomes with acid phosphatase activity in the duodenum, especially at 36 h of life. In the jejunum, the presence of lysosomes with acid phosphatase activity was the highest at 96 h, followed by 36 and 18 h of life. In the ileum, all samples showed low presence of lysosomes with acid phosphatase activity. These results indicate that lyophilized bovine colostrum, as a heterologous source of antibodies or nutrients, is a possible alternative management tool for goats. The present work also suggests that in the first 4 days of life, enzyme activity in the intestinal epithelium of goats is still not fully stimulated, which is an important characteristic for these animals that depend on macromolecule absorption to acquire passive protection after birth. (C) 2012 Elsevier B.V. All rights reserved.