Crystallization and preliminary X-ray diffraction analysis of an l-amino-acid oxidase from Bothrops jararacussu venom

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

Isolation of a new L-amino acid oxidase from Crotalus durissus cascavella venom

A novel L-amino acid oxidase (LAO) (Casca LAO) from Crotalus durissus cascavella venom was purified to a high degree of molecular homogeneity using a combination of molecular exclusion and ion-exchange chromatography system. The purified monomer of LAO presented a molecular mass of 68 kDa and pI estimated in 5.43, which were determined by two-dimensional electrophoresis. The 71st N-terminal amino acid sequence of the LAO from Crotalus durissus cascavella presented a high amino acid sequence similarities with other LAOs from Colloselasma rhosostoma, Crotalus adamanteus, Agkistrodon h. blomhoffi, Agkistrodon h. halys and Trimeresurus stejnegeri. LAO displayed a Michaelis-Menten behavior with a kilometer of 46.7 mu M and an optimum pH for enzymatic activity of 6.5. Casca LAO induced a dose-dependent platelet aggregation, which was abolished by catalase and inhibited by indomethacin and aspirin. These results suggest that the production of H2O2 is involved in subsequent activation of inflammatory enzymes, such as thromboxane. Casca LAO also inhibited the bacterial Growth of Gram-negative (Xanthomonas axonopodis pv passiflorae) and Gram-positive (S. mutans) strains. Electron microscopy assessments of both bacterial strains suggest that the hydrogen peroxide produced by LAO induce bacterial membrane rupture and consequently loss of cytoplasmatic content. This LAO exhibited a high antileishmanic activity against the promastigote of Leishmania amazonensis in vitro, its activity was dependent on the production of hydrogen peroxide, and the 50% inhibitory concentration was estimated in 2.39 mu g/ml. (C) 2005 Elsevier Ltd. All rights reserved.

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)

l-amino acid oxidase from Bothrops marajoensis causes nephrotoxicity in isolated perfused kidney and cytotoxicity in MDCK renal cells

Renal alterations caused by Bothrops venom and its compounds are studied to understand these effects and provide the best treatment. Previously, we studied the renal effect of the whole venom of Bothrops marajoensis and its phospholipase A2 (PLA2), but these effects could not to be attributed to PLA2. To continue the study, we report in this short communication the effects of l-amino acid oxidase from B. marajoensis venom (LAAOBm) on renal function parameter alterations observed in the same model of isolated perfused kidney, as well as the cytotoxic effect on renal cells. LAAOBm caused a decrease in PP, RVR, UF, GFR, %TNa(+) and %TCl(-), very similar to the effects of whole venom using the same model. We also demonstrated its cytotoxicity in MDCK cells with IC50 of 2.5 μg/mL and late apoptotic involvement demonstrated by flow cytometry assays. In conclusion, we suggested that LAAOBm is a nephrotoxic compound of B. marajoensis venom.

Isolation and biochemical, functional and structural characterization of a novel L-amino acid oxidase from Lachesis muta snake venom

The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K-m of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 mu g of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC50: 22.7 mu g/mL) and on MCF-7 cell line (breast adenocarcinoma, IC50:1.41 mu g/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC50: 2.22 mu g/nnL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions. (C) 2012 Elsevier Ltd. All rights reserved.

L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase Cgamma2. Unlike convulxin, Fc receptor gamma chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

Crystal structure of D-amino acid oxidase: a case of active site mirror-image convergent evolution with flavocytochrome b2.

D-amino acid oxidase is the prototype of the FAD-dependent oxidases. It catalyses the oxidation of D-amino acids to the corresponding alpha-ketoacids. The reducing equivalents are transferred to molecular oxygen with production of hydrogen peroxide. We have solved the crystal structure of the complex of D-amino acid oxidase with benzoate, a competitive inhibitor of the substrate, by single isomorphous replacement and eightfold averaging. Each monomer is formed by two domains with an overall topology similar to that of p-hydroxybenzoate hydroxylase. The benzoate molecule lays parallel to the flavin ring and is held in position by a salt bridge with Arg-283. Analysis of the active site shows that no side chains are properly positioned to act as the postulated base required for the catalytic carboanion mechanism. On the contrary, the benzoate binding mode suggests a direct transfer of the substrate alpha-hydrogen to the flavin during the enzyme reductive half-reaction.The active site Of D-amino acid oxidase exhibits a striking similarity with that of flavocytochrome b2, a structurally unrelated FMN-dependent flavoenzyme. The active site groups (if these two enzymes are in fact superimposable once the mirror-image of the flavocytochrome b2 active site is generated with respect to the flavin plane. Therefore, the catalytic sites of D-amino acid oxidase and flavocytochrome b2 appear to have converged to a highly similar but enantiomeric architecture in order to catalvze similar reactions (oxidation of alpha-amino acids or alpha-hydroxy acids), although with opposite stereochemistry.

Glycerol-induced development of catalytically active conformation of Crotalus adamanteus L-amino acid oxidase in vitro.

The reconstitutable apoprotein of Crotalus adamanteus L-amino acid oxidase was prepared using hydrophobic interaction chromatography. After reconstitution with flavin adenine dinucleotide, the resulting protein was inactive, with a perturbed conformation of the flavin binding site. Subsequently, a series of cosolvent-dependent compact intermediates was identified. The nearly complete activation of the reconstituted apoprotein and the restoration of its native flavin binding site was achieved in the presence of 50% glycerol. We provide evidence that in addition to a merely stabilizing effect of glycerol on native proteins, glycerol can also have a restorative effect on their compact equilibrium intermediates, and we suggest the hydrophobic effect as a dominating force in this in vitro-assisted restorative process.

Structural insights into selectivity and cofactor binding in snake venom l-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH 3) and hydrogen peroxide (H 2O 2). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62-71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode. © 2012 Elsevier Inc.

Phtx-ii A Basic Myotoxic Phospholipase A2 From Porthidium Hyoprora Snake Venom, Pharmacological Characterization And Amino Acid Sequence By Mass Spectrometry.

A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca2+-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca2+. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.

Energy and amino acid content in phase 1 nursery diet: piglet performance and body chemical composition

It was evaluated the effects of metabolizable energy (ME) and digestible lysine (dLYS) densities on performance and body composition of weaned piglets. The study used 114 piglets weaned at 7.4 ± 0.80 kg, out of which 108 were allotted in the nursery and 6 were slaughtered on the weaning day to determine comparative data of body chemical composition. Six nutrients densities were stipulated from a previous study based on the highest nitrogen retention, maintaining the following ME:LYS relationship in the experimental diets: 3,390:1.291; 3,450:1.409; 3,650:1.411; 3,780:1.461; 3,940:1.507; and 4,109 kcal/kg ME:1.564% dLYS. The experimental diets were offered for 13 days when the piglets reached 12.986 ± 1.449 kg of body weight. The probable residual effects of nutritional density on the subsequent performance of the piglets were evaluated. At the end of initial phase 1, six piglets from each density were slaughtered to determine their chemical composition in body fractions and empty body. There was no significant influence of nutritional levels on the performance of the piglets at the end of the evaluation. The results of food conversion and body composition confirm the level indicated in the previous study, 4 g dLYS/Mcal of ME. The increase of energy and lysine densities confirms the need for a correct relationship among both of them to assure better performance of the piglets at the beginning of the growing phase.

The Monocarboxylate Transporter 8 and L-Type Amino Acid Transporters 1 and 2 Are Expressed in Mouse Skeletons and in Osteoblastic MC3T3-E1 Cells

Background: Several plasma membrane transporters have been shown to mediate the cellular influx and/or efflux of iodothyronines, including the sodium-independent organic anion co-transporting polypeptide 1 (OATP1), the sodium taurocholate co-transporting polypeptide (NTCP), the L-type amino acid transporter 1 (LAT1) and 2 (LAT2), and the monocarboxylate transporter 8 (MCT8). The aim of this study was to investigate if the mRNAs of these transporters were expressed and regulated by thyroid hormone (TH) in mouse calvaria-derived osteoblastic MC3T3-E1 cells and in the fetal and postnatal bones of mice. Methods: The mRNA expression of the iodothyronine transporters was investigated with real-time polymerase chain reaction analysis in euthyroid and hypothyroid fetuses and litters of mice and in MC3T3-E1 cells treated with increasing doses of triiodothyronine (T(3); 10(-10) to 10(-6) M) or with 10(-8) M T(3) for 1-9 days. Results: MCT8, LAT1, and LAT2 mRNAs were detected in fetal and postnatal femurs and in MC3T3-E1 cells, while OATP1 and NTCP mRNAs were not. LAT1 and LAT2 mRNAs were not affected by TH status in vivo or in vitro or by the stage of bone development or osteoblast maturation (analyzed by the expression of osteocalcin and alkaline phosphatase, which are key markers of osteoblastic differentiation). In contrast, the femoral mRNA expression of MCT8 decreased significantly during post-natal development, whereas MCT8 mRNA expression increased as MC3T3-E1 cells differentiated. We also showed that MCT8 mRNA was up-regulated in the femur of hypothyroid animals, and that it was down-regulated by treatment with T(3) in MC3T3-E1 cells. Conclusions: This is the first study to demonstrate the mRNA expression of LAT1, LAT2, and MCT8 in the bone tissue of mice and in osteoblast-like cells. In addition, the pattern of MCT8 expression observed in vivo and in vitro suggests that MCT8 may be important to modulate TH effects on osteoblast differentiation and on bone development and metabolism.

Positive Selection Results in Frequent Reversible Amino Acid Replacements in the G Protein Gene of Human Respiratory Syncytial Virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a ""flipflop'' phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Effects of branched-chain amino acid (BCAA) supplementation on endurance exercise performance of pregnant rats

Aims. - The present study evaluated the effects of BCAA supplementation on exercise performance of pregnant rats. Methods. - In order to assess these effects, Wistar rats were divided into four groups: sedentary not-supplemented (SNS, n = 8); sedentary supplemented (SS, n = 8); trained not-supplemented (TNS, n = 8) and trained supplemented (TS, n = 8). All groups were submitted to the endurance test until exhaustion (ET) and post-effort lactate (PEL) determination before pregnancy (ET-B and PEL-B) and at the 19th day of pregnancy (ET-19 and PEL-19). Results. - The endurance training significantly increased the ET time to exhaustion (p<0.05). Regardless of BCAA supplementation, both endurance trained groups (TS and TNS) showed a longer time to exhaustion, assessed by ET, compared with the sedentary groups (SS and SNS) (p < 0.05). In the TNS, ET-19 time to exhaustion decreased when compared with the period before pregnancy. On the other hand, ET-19 time to exhaustion was not affected in the TS at the end of the pregnancy period. In addition, TS showed a marked PEL-19 reduction when compared with PEL-B. The data presented herein suggest that BCAA supplementation plays an ergogenic role in the maintenance of exercise performance during pregnancy in rats. (C) 2008 Elsevier Masson SAS. All rights reserved.

Estimating Amino Acid Requirement of Brazilian Freshwater Fish from Muscle Amino Acid Profile

Information on nutritional requirement of some Brazilian farmed fish species, especially essential amino acids (EAA) requirements, is scarce. The estimation of amino acids requirements based on amino acid composition of fish is a fast and reliable alternative. Matrinxa, Brycon amazonicus, and curimbata, Prochilodus lineatus, are two important Brazilian fish with potential for aquaculture. The objective of the present study was to estimate amino acid requirements of these species and analyze similarities among amino acid composition of different fish species by cluster analysis. To estimate amino acid requirement, the following formula was used: amino acid requirement = [(amount of an individual amino acid in fish muscle tissue) x (average totalEAA requirement among channel catfish, Ictalurus punctatus, Nile tilapia, Oreochromis niloticus, and common carp, Cyprinus carpio)]/(average fish muscle totalEAA). Most values found lie within the range of requirements determined for other omnivorous fish species, in exception of leucine requirement estimated for both species, and arginine requirement estimated for matrinxa alone. Rather than writing off the need for regular dose-response assays under the ideal protein concept to determine EAA requirements of curimbata and matrinxa, results set solid base for the study of tropical species dietary amino acids requirements.