Thermodynamic characterization of the palm tree Roystonea regia peroxidase stability

The structural stability of a peroxidase, a dimeric protein from royal palm tree (Roystonea regia) leaves, has been characterized by high-sensitivity differential scanning calorimetry, circular dichroism, steady-state tryptophan fluorescence and analytical ultracentifugation under different solvent conditions. It is shown that the thermal and chemical (using guanidine hydrochloride (Gdn-HCl)) folding/unfolding of royal palm tree peroxidase (RPTP) at pH 7 is a reversible process involving a highly cooperative transition between the folded dimer and unfolded monomers, with a free stabilization energy of about 23 kcal per mol of monomer at 25 degrees C. The structural stability of RPTP is pH-dependent. At pH 3, where ion pairs have disappeared due to protonation, the thermally induced denaturation of RPTP is irreversible and strongly dependent upon the scan rate, suggesting that this process is under kinetic control. Moreover, thermally induced transitions at this pH value are dependent on the protein concentration, allowing it to be concluded that in solution RPTP behaves as dimer, which undergoes thermal denaturation coupled with dissociation. Analysis of the kinetic parameters of RPTP denaturation at pH 3 was accomplished on the basis of the simple kinetic scheme N ->(k) D, where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state, and thermodynamic information was obtained by extrapolation of the kinetic transition parameters to an infinite heating rate. Obtained in this way, the value of RPTP stability at 25 degrees C is ca. 8 kcal per mole of monomer lower than at pH 7. In all probability, this quantity reflects the contribution of ion pair interactions to the structural stability of RPTP. From a comparison of the stability of RPTP with other plant peroxidases it is proposed that one of the main factors responsible for the unusually high stability of RPTP which enhances its potential use for biotechnological purposes, is its dimerization. (c) 2008 Elsevier Masson SAS. All rights reserved.

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.

Peroxymonocarbonate and Carbonate Radical Displace the Hydroxyl-like Oxidant in the Sod1 Peroxidase Activity under Physiological Conditions

Despite being one of the most important antioxidant defenses, Cu,Zn-superoxide dismutase (Sod1) has been frequently associated with harmful effects, including neurotoxicity. This toxicity has been attributed to immature forms of Sod1 and extraneous catalytic activities. Among these, the ability of Sod1 to function as a peroxidase may be particularly relevant because it is increased in bicarbonate buffer and produces the reactive carbonate radical. Despite many studies, how this radical forms remains unknown. To address this question, we systematically studied hSod1 peroxidase activity in the presence of nitrite, formate, and bicarbonate-carbon dioxide. Kinetic analyses of hydrogen peroxide consumption and of nitrite, formate, and bicarbonate-carbon dioxide oxidation showed that the Sod1-bound hydroxyl-like oxidant functions in the presence of nitrite and formate. In the presence of bicarbonate-carbon dioxide, this oxidant is replaced by peroxymonocarbonate, which is then reduced to the carbonate radical. Peroxymonocarbonate intermediacy was evidenced by (13)C NMR experiments showing line broadening of its peak in the presence of Zn,ZnSod1. In agreement, peroxymonocarbonate was docked into the hSod1 active site, where it interacted with the conserved Arg(143). Also, a reaction between peroxymonocarbonate and Cu(I)Sod1 was demonstrated by stopped-flow experiments. Kinetic simulations indicated that peroxymonocarbonate is produced during Sod1 turnover and not in bulk solution. In the presence of bicarbonate-carbon dioxide, sustained hSod1-mediated oxidations occurred with low steady-state concentrations of hydrogen peroxide (4-10 mu M). Thus, carbonate radical formation through peroxymonocarbonate may be a key event in Sod1-induced toxicity.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.

A ditryptophan cross-link is responsible for the covalent dimerization of human superoxide dismutase 1 during its bicarbonate-dependent peroxidase activity

Unlike intermolecular disulfide bonds, other protein cross-links arising from oxidative modifications cannot be reversed and are presumably more toxic to cells because they may accumulate and induce protein aggregation. However, most of these irreversible protein cross-links remain poorly characterized. For instance, the antioxidant enzyme human superoxide dismutase 1 (hSod1) has been reported to undergo non-disulfide covalent dimerization and further oligomerization during its bicarbonate-dependent peroxidase activity. The dimerization was shown to be dependent on the oxidation of the single, solvent-exposed TrP(32) residue of hSod1, but the covalent dimer was not isolated nor was its structure determined. In this work, the hSod1 covalent dimer was isolated, digested with trypsin in H(2)O and H(2)(18)O, and analyzed by UV-Vis spectroscopy and mass spectrometry (MS). The results demonstrate that the covalent dimer consists of two hSod1 subunits cross-linked by a ditryptophan, which contains a bond between C3 and N1 of the respective Trp(32) residues. We further demonstrate that the cross-link cleaves under usual MS/MS conditions leading to apparently unmodified Trp(32), partially hinders proteolysis, and provides a mechanism to explain the formation of hSod1 covalent trimers and tetramers. This characterization of the covalent hSod1 dimer identifies a novel oxidative modification of protein Trp residues and provides clues for studying its occurrence in vivo. (C) 2010 Elsevier Inc. All rights reserved.

Inactivation kinetics of polyphenol oxidase and peroxidase in green coconut water by microwave processing

The inactivation kinetics of enzymes polyphenol oxidase (PPO) and peroxidase (POD) was studied for the batch (discontinuous) microwave treatment of green coconut water. Inactivation of commercial PPO and POD added to sterile coconut water was also investigated. The complete time-temperature profiles of the experimental runs were used for determination of the kinetic parameters D-value and z-value: PPO (D(92.20 degrees C) = 52 s and z = 17.6 degrees C); POD (D(92.92 degrees C) = 16 s and z = 11.5 degrees C); PPO/sterile coconut water: (D(84.45 degrees C) = 43 s and z = 39.5 degrees C) and POD/sterile coconut water: (D(86.54 degrees C) = 20 s and z = 19.3 degrees C). All data were well fitted by a first order kinetic model. The enzymes naturally present in coconut water showed a higher resistance when compared to those added to the sterilized medium or other simulated solutions reported in the literature. The thermal inactivation of PPO and POD during microwave processing of green coconut water was significantly faster in comparison with conventional processes reported in the literature. (C) 2008 Elsevier Ltd. All rights reserved.

The co-catalytic effect of chlorpromazine on peroxidase-mediated oxidation of melatonin: enhanced production of N-1-acetyl-N (2)-formyl-5-methoxykynuramine

Accumulating evidence points to relationships between increased production of reactive oxygen or decreased antioxidant protection in schizophrenic patients. Chlorpromazine (CPZ), which remains a benchmark treatment for people with schizophrenia, has been described as a pro-oxidant compound. Because the antioxidant compound melatonin exerts protective effects against CPZ-induced liver disease in rats, in this investigation, our main objective was to study the effect of CPZ as a co-catalyst of peroxidase-mediated oxidation of melatonin. We found that melatonin was an excellent reductor agent of preformed CPZ cation radical (CPZ(center dot+)). The addition of CPZ during the horseradish peroxidase (HRP)-catalyzed oxidation of melatonin provoked a significant increase in the rate of oxidation and production of N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK). Similar results were obtained using myeloperoxidase. The effect of CPZ on melatonin oxidation was rather higher at alkaline pH. At pH 9.0, the efficiency of oxidation of melatonin was 15 times higher and the production of AFMK was 30 times higher as compared with the assays in the absence of CPZ. We suggest that CPZ is able to exacerbate the rate of oxidation of melatonin by an electron transfer mechanism where CPZ(center dot+), generated during the peroxidase-catalyzed oxidation, is able to efficiently oxidize melatonin.

Efeito de indutores bióticos e abióticos na atividade de quitinase e peroxidase e no controle da ferrugem causada por Puccinia psidii em eucalipto

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

Inhibition of peroxidase activity and scavenging of reactive oxygen species by astilbin isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Antibody-targeted horseradish peroxidase associated with indole-3-acetic acid induces apoptosis in vitro in hematological malignancies

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

The effect of pH on horseradish peroxidase-catalyzed oxidation of melatonin: production of N-1-acetyl-N-2-formyl-5-methoxykynuramine versus radical-mediated degradation

There is a g-rowing body of evidence that melatonin and its oxidation product, N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5. 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin a dimer of, 2-hydroxymelatonin and O-demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. on the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle.

Oxidation of acetylacetone catalyzed by horseradish peroxidase in the absence of hydrogen peroxide

Horseradish peroxidase (HRP) is a plant enzyme widely used in biotechnology, including antibody-directed enzyme prodrug therapy (ADEPT). Here, we showed that HRP is able to catalyze the autoxidation of acetylacetone in the absence of hydrogen peroxide. This autoxidation led to generation of methylglyoxal and reactive oxygen species. The production of superoxide anion was evidenced by the effect of superoxide dismutase and by the generation of oxyperoxidase during the enzyme turnover. The HRP has a high specificity for acetylacetone, since the similar beta-dicarbonyls dimedon and acetoacetate were not oxidized. As this enzyme prodrug combination was highly cytotoxic for neutrophils and only requires the presence of a non-human peroxidase and acetylacetone, it might immediately be applied to research on the ADEPT techniques. The acetylacetone could be a starting point for the design of new drugs applied in HRP-related ADEPT techniques. (c) 2006 Elsevier B.V. All rights reserved.

Ionically bound peroxidase from peach fruit

Soluble, ionically bound peroxidase (POD) and polyphenoloxidase (PPO) were extracted from the pulp of peach fruit during ripening at 20degreesC Ionically bound form was purified 6.1 -fold by DEAE-cellulose and Sephadex G-100 chromatography. The purified enzyme showed only one peak of activity on Sephadex G-100 and PAGE revealed that the enzyme was purified by the procedures adopted. The purified enzyme showed a molecular weight of 29000 Da, maximum activity at pH 5.0 and at 40degreesC the calculated apparent activation energy (Ea) for the reaction was 10.04 kcal/mol. The enzyme was heat-labile in the temperature range of 60 to 75degreesC with a fast inactivation at 75degreesC Measurement of residual activity showed a stabilizing effect of sucrose at various temperature/sugar concentrations (0, 10, 20 %, w/w), with an activation energy (Ea) for inactivation increasing with sucrose concentration from 0 to 20% (w/w). The Km and V-max values were 9.35 and 15.38 mM for O-dianisidine and H2O2, respectively. The bound enzyme was inhibited competitively by (.)ferulic, caffeic and protocatechuic acids with different values of Ki,. L-cysteine, p-coumaric and indolacetic acid and Fe++ also inhibited the enzyme but at a lower grade. N-ethylmaleimide and p-CMB were not effective to inhibit the enzyme demonstrating the non-essentiality of SH groups.

Peroxidase and phenylalanine ammonia-lyase activities, phenolic acid contents, and allelochemicals-inhibited root growth of soybean

The influence of the allelochemicals ferulic (FA) and vanillic (VA) acids on peroxidase (POD, EC 1.11.1.7) and phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activities and their relationships with phenolic acid (PhAs) contents and root growth of soybean (Glycine max (L.) Merr.) were examined. Three-day-old seedlings were cultivated in nutrient solution containing FA or VA (0.1 to 1 mM) for 48 h. Both compounds (at 0.5 and 1 mM) decreased root length (RL), fresh weight (FW) and dry weight (DW) and increased PhAs contents. At 0.5 and 1 mM, FA increased soluble POD activity (18% and 47%, respectively) and cell wall (CW)-bound POD activity (61% and 34%), while VA increased soluble POD activity (33% and 17%) but did not affect CW-bound POD activity. At I mM, FA increased (82%) while VA reduced (32%) PAL activities. The results are discussed on the basis of the role of these compounds on phenylpropanoid metabolism and root growth and suggest that the effects caused on POD and PAL activities are some of the many mechanisms by which allelochemicals influence plant growth.