Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia)

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85 angstrom. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP. (C) 2009 Elsevier Inc. All rights reserved.

Isozyme markers and genetic variability in three species of Centrosema (Leguminosae)

Isozyme patterns and their genetic control in three Centrosema species are described. Seven isozymatic systems (aspartate aminotransferase, glucose-6-phosphate isomerase, phosphoglucomutase, anodal peroxidase, malate dehydrogenase, 6-phosphogluconate dehydrogenase, and isocitrate dehydrogenase) were studied in 18 populations and several breeding lines of C. acutifolium, C. brasilianum and C. pubescens, using starch gel electrophoresis techniques. All systems, except glucose-6-phosphate isomerase, are described for the first time in these species. A total of 17 isozyme loci were scored; this represents the largest set of Mendelian loci known up to now in Centrosema species. Isozyme polymorphism and variability within and between populations and species were relatively high and allowed discrimination among species

Characterization of peroxidase changes in tolerant and susceptible soybeans challenged by soybean aphid (Hemiptera: Aphididae)

Changes in protein content, peroxidase activity, and isozyme profiles in response to soybean aphid feeding were documented at V1 (fully developed leaves at unifoliate node, first trifoliate leaf unrolled) and V3 (fully developed leaf at second trifoliate node, third trifoliate leaf unrolled) stages of soybean aphid-tolerant (KS4202) and -susceptible (SD76R) soybeans. Protein content was similar between infested and control V1 and V3 stage plants for both KS4202 and SD76R at 6, 16, and 22 d after aphid introduction. Enzyme kinetics studies documented that control and aphid-infested KS4202 V1 stage and SD76R V1 and V3 stages had similar levels of peroxidase activity at the three time points evaluated. In contrast, KS4202 aphid-infested plants at the V3 stage had significantly higher peroxidase activity levels than control plants at 6 and 22 d after aphid introduction. The differences in peroxidase activity observed between infested and control V3 stage KS4202 plants at these two time points suggest that peroxidases may be playing multiple roles in the tolerant plant. Native gels stained for peroxidase were able to detect differences in the isozyme profiles of aphid-infested and control plants for both KS4202 and SD76R.

Removal of the N-linked glycan structure from the peanut peroxidase prxPNC2: Influence on protein stability and activity

Lines of transgenic tobacco have been generated that are transformed with either the wild-type peanut peroxidase prxPNC2 cDNA, driven by the CaMV3 5S promoter (designated 35S::prxPNC2-WT) or a mutated PNC2 cDNA in which the asparagine residue (Asn(189)) associated with the point of glycan attachment (Asn(189)) has been replaced with alanine (designated 35S::prxPNC2-M). PCR, using genomic DNA as template, has confirmed the integration of the 35S::prxPNC2-WT and 35::prxPNC2-M constructs into the tobacco genome, and western analysis using anti-PNC2 antibodies has revealed that the prxPNC2-WT protein product (PNC2-WT) accumulates with a molecular mass of 34,670 Da, while the prxPNC2-M protein product (PNC2-M) accumulates with a molecular mass of 32,600 Da. Activity assays have shown that both PNC2-WT and PNC2-M proteins accumulate preferentially in the ionically-bound cell wall fraction, with a significantly higher relative accumulation of the PNC2-WT isoenzyme in the ionically-bound fraction when compared with the PNC2-M isoform. Kinetic analysis of the partially purified PNC2-WT isozyme revealed an affinity constant (apparent K-m) of 11.2 mM for the reductor substrate guaiacol and 1.29 mM for H2O2, while values of 11.9 mM and 1.12 mM were determined for the PNC2-M isozyme. A higher Arrenhius activation energy (E,,) was determined for the PNC2-M isozyme (22.9 kJ mol(-1)), when compared with the PNC2-WT isozyme (17.6 kJ mol(-1)), and enzyme assays have determined that the absence of the glycan influences the thermostability of the PNC2-M isozyme. These results are discussed with respect to the proposed roles of N-linked glycans attached to plant peroxidases. (c) 2005 Elsevier Ltd. All rights reserved.

Extraction, purification and biochemical characterization of a peroxidase from Copaifera langsdorffii leaves

The aim of this work is to obtain, purify and characterize biochemically a peroxidase from Copaifera langsdorffii leaves (COP). COP was obtained by acetone precipitation followed by ion-exchange chromatography. Purification yielded 3.5% of peroxidase with the purification factor of 46.86. The COP optimum pH is 6.0 and the temperature is 35 ºC. COP was stable in the pH range of 4.5 to 9.3 and at temperatures below 50.0 ºC. The apparent Michaelis-Menten constants (Km) for guaiacol and H2O2 were 0.04 mM and 0.39 mM respectively. Enzyme turnover was 0.075 s-1 for guaiacol and 0.28 s-1 for hydrogen peroxide. Copaifera langsdorffii leaves showed to be a rich source of active peroxidase (COP) during the whole year. COP could replace HRP, the most used peroxidase, in analytical determinations and treatment of industrial effluents at low cost.

Ecdysteroid-Dependent Expression of the Tweedle and Peroxidase Genes during Adult Cuticle Formation in the Honey Bee, Apis mellifera

Cuticle renewal is a complex biological process that depends on the cross talk between hormone levels and gene expression. This study characterized the expression of two genes encoding cuticle proteins sharing the four conserved amino acid blocks of the Tweedle family, AmelTwdl1 and AmelTwdl2, and a gene encoding a cuticle peroxidase containing the Animal haem peroxidase domain, Ampxd, in the honey bee. Gene sequencing and annotation validated the formerly predicted tweedle genes, and revealed a novel gene, Ampxd, in the honey bee genome. Expression of these genes was studied in the context of the ecdysteroid-coordinated pupal-to-adult molt, and in different tissues. Higher transcript levels were detected in the integument after the ecdysteroid peak that induces apolysis, coinciding with the synthesis and deposition of the adult exoskeleton and its early differentiation. The effect of this hormone was confirmed in vivo by tying a ligature between the thorax and abdomen of early pupae to prevent the abdominal integument from coming in contact with ecdysteroids released from the prothoracic gland. This procedure impaired the natural increase in transcript levels in the abdominal integument. Both tweedle genes were expressed at higher levels in the empty gut than in the thoracic integument and trachea of pharate adults. In contrast, Ampxd transcripts were found in higher levels in the thoracic integument and trachea than in the gut. Together, the data strongly suggest that these three genes play roles in ecdysteroid-dependent exoskeleton construction and differentiation and also point to a possible role for the two tweedle genes in the formation of the cuticle (peritrophic membrane) that internally lines the gut.

Extraction of manganese peroxidase produced by Lentinula edodes

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 degrees C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to UP of Phanerochaete chrysosporium and a total inhibition of UP was observed. (C) 2007 Elsevier Ltd. All rights reserved.

Optimal recovery process conditions for manganese-peroxidase obtained by solid-state fermentation of eucalyptus residue using Lentinula edodes

Enzyme production is a growing field in biotechnology and increasing attention has been devoted to the solid-state fermentation (SSF) of lignocellulosic biomass for production of industrially relevant lignocellulose deconstruction enzymes, especially manganese-peroxidase (MnP), which plays a crucial role in lignin degradation. However, there is a scarcity of studies regarding extraction of the secreted metabolities that are commonly bound to the fermented solids, preventing their accurate detection and limiting recovery efficiency. In the present work, we assessed the effectiveness of extraction process variables (pH, stirring rate, temperature, and extraction time) on recovery efficiency of manganese-peroxidase (MnP) obtained by SSF of eucalyptus residues using Lentinula edodes using statistical design of experiments. The results from this study indicated that of the variables studied, pH was the most significant (p < 0.05%) parameter affecting MnP recovery yield, while temperature, extraction time, and stirring rate presented no statistically significant effects in the studied range. The optimum pH for extraction of MnP was at 4.0-5.0, which yielded 1500-1700 IU kg (1) of enzyme activity at extraction time 4-5 h, under static condition at room temperature. (C) 2011 Elsevier Ltd. All rights reserved.

Thermal inactivation of polyphenoloxidase and peroxidase in green coconut (Cocos nucifera) water

P>Coconut water is an isotonic beverage naturally obtained from the green coconut. After extracted and exposed to air, it is rapidly degraded by enzymes peroxidase (POD) and polyphenoloxidase (PPO). To study the effect of thermal processing on coconut water enzymatic activity, batch process was conducted at three different temperatures, and at eight holding times. The residual activity values suggest the presence of two isoenzymes with different thermal resistances, at least, and a two-component first-order model was considered to model the enzymatic inactivation parameters. The decimal reduction time at 86.9 degrees C (D(86.9 degrees C)) determined were 6.0 s and 11.3 min for PPO heat labile and heat resistant fractions, respectively, with average z-value = 5.6 degrees C (temperature difference required for tenfold change in D). For POD, D(86.9 degrees C) = 8.6 s (z = 3.4 degrees C) for the heat labile fraction was obtained and D(86.9 degrees C) = 26.3 min (z = 6.7 degrees C) for the heat resistant one.

Photochemical damage and comparative performance of superoxide dismutase and ascorbate peroxidase in sugarcane leaves exposed to paraquat-induced oxidative stress

The physiological responses of sugarcane (Succharion officinarum L.) to oxidative stress induced by methyl viologen (paraquat) were examined with respect to photochemical activity, chlorophyll content, lipid peroxidation and superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities. Thirty-day-old sugarcane plants were sprayed with 0, 2, 4, 6 and 8 mM methyl viologen (MV). Chlorophyll fluorescence was measured after 18 It and biochemical analyses were performed after 24 and 48 h. Concentrations of MV above 2 mM caused significant damage to photosystem II (PSII) activity. Potential and effective quantum efficiency of PSII and apparent electron transport rate were greatly reduced or practically abolished. Both chlorophyll and soluble protein contents steadily decreased with MV concentrations above 2 mM after 24 It of exposure, which became more pronounced after 48 It, achieving a 3-fold decrease. Insoluble protein contents were little affected by MV. Oxidative stress induced by MV was evidenced by increases in lipid peroxidation. Specific activity of SOD increased, even after 48 h of exposure to the highest concentrations of MV, but total activity on a fresh weight basis did not change significantly. Nondenaturing YAGE assayed with H2O2 and KCN showed that treatment with MV did not change Cu/Zn-SOD and MnSOD isoform activities. In contrast, APX specific activity increased at 2 mM MV but then dropped at higher doses. Oxidative damage induced by MV was inversely related to APX activity. It is suggested that the major MV-induced oxidative damages in sugarcane leaves were related to excess H2O2, probably in chloroplasts, caused by an imbalance between SOD and APX activities, in which APX was a limiting step. Reduced photochemical activity allowed the early detection of the ensuing oxidative stress. (c) 2007 Elsevier Inc. All rights reserved.

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil

Chitinase and peroxidase activity in different stages of eucalypt leaves after inoculation with Puccinia psidii and acibenzolar-S-metil To elucidate some biochemical processes during infection in the pathosystem Puccinia psidii x eucalyptus, the defense metabolism in different-stage leaves was compared between rust-resistant and susceptible clones, respectively. In addition, chitinase and peroxidase activities were assayed. Each treatment consisted of 4 replicates, in a completely randomized design: 2 clones, inoculated and not inoculated with P. psidii; sprayed with acibenzolar-S-methyl (ASM) and distilled water; and represented by the 1(st) leaf pair (size equivalent to 1/5 total leaf development), 2(nd) pair (2/5 total development), and 4(th) pair (4/5 total leaf length). Leaves were harvested in 4 periods: 0, 24, 72 and 96 hours after inoculation. Results indicated that ASM treatment or P. psidii action led to higher chitinase and peroxidase activity level but did not alter the expression of these activities in developed leaves (4(th) pair) during the experiment. Alterations in enzyme levels after inoculation were only observed in developing leaves (1(st) and 2(nd) pairs), which suggests that the response to infection was concomitant to chitinase and peroxidase synthesis. The highest increases in enzymatic activities were observed in resistant clones at 72 hours after inoculation and in susceptible ones previously treated with ASM and later inoculated with the pathogen.

Effect of Brazil nut supplementation on the blood levels of selenium and glutathione peroxidase in hemodialysis patients

Objective: In patients who have undergone hemodialysis, large amounts of reactive oxygen species (ROS) are produced and, at higher concentrations, ROS are thought to be involved in the pathogenesis of cardiovascular disease. It has been proposed that selenium (Se) may exert an anti-atherogenic influence by reducing oxidative stress. The richest known food source of selenium is the Brazil nut (Bertholletia excelsa, family Lecythidaceae), found in the Amazon region. We evaluated the effect of Brazil nut supplementation on blood levels of Se and glutathione peroxidase (GSH-Px) activity in patients on hemodialysis. Methods: A total of 81 patients on hemodialysis (52.0 +/- 15.2 y old, average time on dialysis 82.3 +/- 91.4 mo, body mass index 24.9 +/- 4.4 kg/m(2)) from the RenalCor and RenalVida Clinics in Rio de Janeiro, Brazil, were studied. All patients received one nut (around 5 g, averaging 58.1 mu g Se/g) a day for 3 mo. The Se concentrations in the nuts and in plasma and erythrocytes were determined by atomic absorption spectrophotometry with hydride generation (Hitachi, Z-500). GSH-Px levels were measured using Randox commercial kits. Results: Plasma Se (18.8 +/- 17.4 mu g/L) and erythrocyte (72.4 +/- 37.9 mg/L) levels were below the normal, range before nut supplementation. After supplementation, the plasma level increased to 104.0 +/- 65.0 mu g/L and erythrocytes to 244.1 +/- 119.5 mg/L (P<0.0001). The activity of GSH-Px also increased after supplementation, from 46.6 +/- 14.9 to 55.9 +/- 23.6 U/g of hemoglobin (P<0.0001). Before supplementation, 11% of patients had GSH-Px activity below the normal range (27.5-73.6 U/g of hemoglobin). After supplementation, all patients showed GSH-Px activity within the normal range. Conclusion: The data revealed that the investigated patients presented Se deficiency and that the consumption of only one Brazil nut a day (5 g) during 3 mo was effective to increase the Se concentration and GSH-Px activity in these patients, thus improving their antioxidant status. (C) 2010 Elsevier Inc. All rights reserved.

Associations between glutathione peroxidase-1 Pro198Leu polymorphism, selenium status, and DNA damage levels in obese women after consumption of Brazil nuts

Objective: Alterations in selenium (Se) status may result in suboptimal amounts of selenoproteins, which have been associated with increased oxidative stress levels. The Pro198Leu polymorphism at the glutathione peroxidase-1 (GPx1) gene is supposed to be functional. The response of Se status, GPx activity, and levels of DNA damage to a Se supplementation trial between the genotypes related to that polymorphism was investigated. Methods: A randomized trial was conducted with 37 morbidly obese women. Participants consumed one Brazil nut, which provided approximately 290 mu g of Se a day, for 8 wk. Blood Se concentrations, erythrocyte GPx activity, and DNA damage levels were measured at baseline and at 8 wk. The results were compared by genotypes. Results: The genotype frequencies were 0.487, 0.378, and 0.135 for Pro/Pro (the wild-type genotype), Pro/Leu, and Leu/Leu, respectively. At baseline, 100% of the subjects were Se deficient, and after the supplementation, there was an improvement in plasma Se (P < 0.001 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), erythrocyte Se (P = 0.00 for Pro/Pro and Pro/Leu, P < 0.05 for Leu/Leu), and GPx activity (P = 0.00 for Pro/Pro, P < 0.00001 for Pro/Leu, P < 0.001 for Leu/Leu). In addition, the Pro/Pro group showed a decrease in DNA damage after Brazil nut consumption compared with baseline (P < 0.005), and those levels were higher in Leu/Leu subjects compared with those with the wild-type genotype (P < 0.05). Conclusion: Consumption of one unit of Brazil nuts daily effectively increases Se status and increases GPx activity in obese women, regardless of GPx1 Pro198Leu polymorphism. However, the evaluated biomarkers showed distinct results in response to the supplementation when the polymorphism was considered. (c) 2011 Elsevier Inc. All rights reserved.

Real-time monitoring and kinetic parameter estimation of the affinity interaction of jArtinM and rArtinM with peroxidase glycoprotein by the electrogravimetric technique

ArtinM is a D-mannose binding lectin that has been arousing increasing interest because of its biomedical properties, especially those involving the stimulation of Th1 immune response, which confers protection against intracellular pathogens The potential pharmaceutical applications of ArtinM have motivated the production of its recombinant form (rArtinM) so that it is important to compare the sugar-binding properties of jArtinM and rArtinM in order to take better advantage of the potential applications of the recombinant lectin. In this work, a biosensor framework based on a Quartz Crystal Microbalance was established with the purpose of making a comparative study of the activity of native and recombinant ArtinM protein The QCM transducer was strategically functionalized to use a simple model of protein binding kinetics. This approach allowed for the determination of the binding/dissociation kinetics rate and affinity equilibrium constant of both forms of ArtinM with horseradish peroxidase glycoprotein (HRP), a N-glycosylated protein that contains the trimannoside Man alpha 1-3[Man alpha 1-6]Man, which is a known ligand for jArtinM (Jeyaprakash et al, 2004). Monitoring of the real-time binding of rArtinM shows that it was able to bind HRP, leading to an analytical curve similar to that of jArtinM, with statistically equivalent kinetic rates and affinity equilibrium constants for both forms of ArtinM The lower reactivity of rArtinM with HRP than jArtinM was considered to be due to a difference in the number of Carbohydrate Recognition Domains (CRDs) per molecule of each lectin form rather than to a difference in the energy of binding per CRD of each lectin form. (C) 2010 Elsevier B V. All rights reserved

Role of promoter polymorphisms in the plasma glutathione peroxidase (GPx-3) gene as a risk factor for cerebral venous thrombosis

Background and Purpose-Plasma glutathione peroxidase (GPx-3) is a major antioxidant enzyme in plasma and the extracellular space that scavenges reactive oxygen species produced during normal metabolism or after oxidative insult. A deficiency of this enzyme increases extracellular oxidant stress, promotes platelet activation, and may promote oxidative posttranslational modification of fibrinogen. We recently identified a haplotype (H-2) in the GPx-3 gene promoter that increases the risk of arterial ischemic stroke among children and young adults. Methods-The aim of this study is to identify possible relationships between promoter haplotypes in the GPx-3 gene and cerebral venous thrombosis (CVT). We studied the GPx-3 gene promoter from 23 patients with CVT and 123 young controls (18 to 45 years) by single-stranded conformational polymorphism and sequencing analysis. Results-Over half of CVT patients (52.1%) were heterozygous (H1H2) or homozygous (H2H2) carriers of the H-2 haplotype compared with 12.2% of controls, yielding a more than 10-fold independent increase in the risk of CVT (OR=10.7; 95% CI, 2.70 to 42.36; P<0.0001). Among women, the interaction of the H2 haplotype with hormonal risk factors increased the OR of CVT to almost 70 (P<0.0001). Conclusions-These findings show that a novel GPx-3 promoter haplotype is a strong, independent risk factor for CVT. As we have previously shown that this haplotype is associated with a reduction in transcriptional activity, which compromises antioxidant activity and antithrombotic benefits of the enzyme, these results suggest that a deficiency of GPx-3 leads to a cerebral venous thrombophilic state.