Peroxidase from leaves of royal palm tree Roystonea regia: purification and some properties

Screening of tropical plants demonstrated high peroxidase activity in leaves of some species of palms. Using the leaves of royal palm Roystonea regia as a source, the peroxidase has been isolated to homogeneity. The enzyme purification steps included homogenization, (NH₄)₂SO₄ precipitation, extraction of palm leaf colored compounds and consecutive chromatography on Phenyl-Sepharose, Sephacryl S100 and DEAE-Toyopearl. The novel peroxidase was characterized as having a specific activity of 6170 U/mg, RZ 3.0, molecular weight of 51 kDa and isoelectric point pI 3.5. The electronic spectrum of RPP is characteristic for plant peroxidases with a Soret maximum at 403 nm and maxima in a visible region at 492 and 633 nm, respectively. The substrate specificity of royal palm tree peroxidase (RPTP) is distinct from the specificity of other plant peroxidases. The best substrates for RPTP are ferulic acid and 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid). The palm peroxidase exhibits an unusually high thermostability inactivating at 90 °C with k_inac of 1.5×10⁻² min⁻¹.     

Papaya fruit ripening: ROS metabolism,gene cloning,characterization and molecular docking of peroxidase

Climacteric fruit ripening has been characterized by oxidative burst and involve active oxidative metabolism with generation of reactive oxygen species (ROS). In the present paper, the papaya fruit ripening was found to be associated with increase in polygalacturonase (PG), pectate lyase (PEL), catalase (CAT), ascorbate peroxidase (APX), H₂O₂ and lipid peroxidation concomitant with decrease in the activities of superoxide dismutase (SOD) and guaiacol peroxidase (GPX). Furthermore, a cDNA (903 bp) of GPX from unripe papaya fruit pulp was isolated and cloned. On BLAST analysis, the deduced protein exhibited homology with various peroxidases and specific hits for plant heme peroxidase family namely heme and calcium binding domains. GPX of papaya was modeled and docked with various substrates and inhibitors among which guaiacol and cysteine were found to be the best substrate and inhibitor, respectively.     

植物过氧化物酶超家族的分子结构

过氧化物酶广泛存在于生物中。基于序列相似性比较,可将真菌、细菌和植物来源的过氧化物酶归为一个超家族－植物过氧化物酶超家族。作者对近几年来植物过氧化物酶超家族的分子结构与功能研究进展,从过氧化物酶的辅基（血红素）微循环结构、过氧化物酶超家族的序列结构域,以及酶分子中底物结合位点和Ca^2＋结合位点的结构等方面作了简要评述。     

Zo-peroxidase: Crystal structure and sequence of a highly-glycosylated peroxidase resistant to high concentrations of H2O2 from Japanese radish

Understanding Peroxidase (PRXs) enzymatic diversity and functional significance from a three-dimensional point of view is a key point for structural and mechanistic studies. In this context, Zo-peroxidase (ZoPrx) a member of the class III peroxidases and secreted by plants, differs from all previously described PRXs because of its remarkable catalytic stability in the presence of hydrogen peroxide. In this work, we present the crystallographic structure of ZoPrx isolated from Japanese radish, at 2.05 Å resolution. The mature enzyme consists of a single monomer of 308 residues exhibiting the same fold as all previously described members of the plant PRXs superfamily. Furthermore, the enzyme contains a heme b group as the prosthetic group and two Ca²⁺ binding sites. Moreover, seven N-glycosylation sites were found in the structure, and 49 glycans bound to the two ZoPrx molecules found in the asymmetric unit are clearly visible in the electron density map. The comparison of ZoPrx coordinates with homologous enzymes revealed minor structural changes, in which the residue 177 appears to be responsible for enlarging the access to the heme cavity, the only structural finding which may be related to the H₂O₂ tolerance of ZoPrx and detected by X-ray crystallography. Because of its characteristics, ZoPrx has a broad range of potential applications from chemical synthesis to environmental biocatalysis, thus its aminoacidic sequence, partially completed using the electron density, and the three-dimensional structure itself, become a possible starting point to engineering heme-peroxidases to enhance oxidative stability.     

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-->kD, 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.     

Naturally-occurring tetrahydro-β-carboline alkaloids derived from tryptophan are oxidized to bioactive β-carboline alkaloids by heme peroxidases

β-Carbolines are indole alkaloids that occur in plants, foods, and endogenously in mammals and humans, and which exhibit potent biological, psychopharmacological and toxicological activities. They form from naturally-occurring tetrahydro-β-carboline alkaloids arising from tryptophan by still unknown way and mechanism. Results in this research show that heme peroxidases catalyzed the oxidation of tetrahydro-β-carbolines (i.e. 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid and 1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid) into aromatic β-carbolines (i.e. norharman and harman, respectively). This oxidation followed a typical catalytic cycle of peroxidases through redox intermediates I, II, and ferric enzyme. Both, plant peroxidases (horseradish peroxidase, HRP) and mammalian peroxidases (myeloperoxidase, MPO and lactoperoxidase, LPO) catalyzed the oxidation in an efficient manner as determined by kinetic parameters (V_MAX and K_M). Oxidation of tetrahydro-β-carbolines was inhibited by peroxidase inhibitors such as sodium azide, ascorbic acid, hydroxylamine and excess of H₂O₂. The formation of aromatic β-carbolines by heme peroxidases can help to explain the presence and activity of these compounds in biological systems.     

Phylogenetic relationships in class I of the superfamily of bacterial, fungal, and plant peroxidases.

Molecular phylogeny among catalase-peroxidases, cytochrome c peroxidases, and ascorbate peroxidases was analysed. Sixty representative sequences covering all known subgroups of class I of the superfamily of bacterial, fungal, and plant heme peroxidases were selected. Each sequence analysed contained the typical peroxidase motifs evolved to bind effectively the prosthetic heme group, enabling peroxidatic activity. The N-terminal and C-terminal domains of catalase-peroxidases matching the ancestral tandem gene duplication event were treated separately in the phylogenetic analysis to reveal their specific evolutionary history. The inferred unrooted phylogenetic tree obtained by three different methods revealed the existence of four clearly separated clades (C-terminal and N-terminal domains of catalase-peroxidases, ascorbate peroxidases, and cytochrome c peroxidases) which were segregated early in the evolution of this superfamily. From the results, it is obvious that the duplication event in the gene for catalase-peroxidase occurred in the later phase of evolution, in which the individual specificities of the peroxidase families distinguished were already formed. Evidence is presented that class I of the heme peroxidase superfamily is spread among prokaryotes and eukaryotes, obeying the birth-and-death process of multigene family evolution.     

The rate-limiting step in O2 reduction by cytochrome ba3 from Thermus thermophilus

Cytochrome ba₃ (ba₃) of Thermus thermophilus (T. thermophilus) is a member of the heme–copper oxidase family, which has a binuclear catalytic center comprised of a heme (heme a₃) and a copper (Cu_B). The heme–copper oxidases generally catalyze the four electron reduction of molecular oxygen in a sequence involving several intermediates. We have investigated the reaction of the fully reduced ba₃ with O₂ using stopped-flow techniques. Transient visible absorption spectra indicated that a fraction of the enzyme decayed to the oxidized state within the dead time (~ 1 ms) of the stopped-flow instrument, while the remaining amount was in a reduced state that decayed slowly (k = 400 s^? 1) to the oxidized state without accumulation of detectable intermediates. Furthermore, no accumulation of intermediate species at 1 ms was detected in time resolved resonance Raman measurements of the reaction. These findings suggest that O₂ binds rapidly to heme a₃ in one fraction of the enzyme and progresses to the oxidized state. In the other fraction of the enzyme, O₂ binds transiently to a trap, likely Cu_B, prior to its migration to heme a₃ for the oxidative reaction, highlighting the critical role of Cu_B in regulating the oxygen reaction kinetics in the oxidase superfamily. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Evolution of structure and function of Class I peroxidases

The phylogenetics of Class I of the heme peroxidase-catalase superfamily currently representing over 940 known sequences in all available genomes of prokaryotes and eukaryotes has been analysed. The robust reconstructed tree for 193 Class I peroxidases with 6 selected Class II representatives reveals all main trends of molecular evolution. It suggests how the ancestral peroxidase gene might have been transferred from prokaryotic into eukaryotic genomes. Besides well known families of catalase-peroxidases, cytochrome c peroxidases and ascorbate peroxidases, the phylogenetic analysis shows for the first time the presence of two new well separated clades of hybrid-type peroxidases that might represent evolutionary bridges between catalase-peroxidases and cytochrome c peroxidases (type A) as well as between ascorbate peroxidases and Class II peroxidases (type B). Established structure-function relationships are summarized. Presented data give useful hints on the origin and evolution of catalytic promiscuity and specificity and will be a valuable basis for future functional analysis of Class I enzymes as well as for de novo design.     

Identification of a new member of the dye-decolorizing peroxidase family from <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

Dye-decolorizing peroxidases (DyP) are atypical peroxidases showing no homology to other fungal peroxidases and lacking the typical heme binding region conserved among plant peroxidase superfamily. The gene and the corresponding cDNA encoding DyP from Pleurotus ostreatus have been identified on the basis of sequence homology analyses. The deduced amino acid sequence shares 43% identity with DyP from the ascomycete Thanatephorus cucumeris Dec 1. Analyses of the protein sequence by homology searches pointed out some properties of the DyP-type peroxidase family, which includes members from bacteria, ascomycete, and basidiomycete fungi. Some amino acids (C374, H379, and Y501 in the P. ostreatus DyP sequence) are proposed as candidates for the heme ligand, providing a basis for further investigations on the structure of the DyP type peroxidase family members.     

Kinetic analysis for suicide-substrate inactivation of microperoxidase-11: A modified model for bisubstrate enzymes in the presence of reversible inhibitors

Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H₂O₂. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism.The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor.Kinetic parameters of inactivation including the initial activity of MP-11, α₀, the apparent inactivation rate constant, k_i and the apparent inhibition rate constant for cysteine, k_I were obtained 0.282 ± 0.006 min^?1, 0.497 ± 0.013 min^?1 and 1.374 ± 0.007 min^?1 at [H₂O₂] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation.     

Role of Tyr residues on the protein surface of cationic cell-wall-peroxidase (CWPO-C) from poplar: potential oxidation sites for oxidative polymerization of lignin

It was previously reported that an unique peroxidase isoenzyme, cationic cell-wall-bound peroxidase (CWPO-C), from poplar callus oxidizes sinapyl alcohol, ferrocytochrome c and synthetic lignin polymers, unlike other plant peroxidases. Here, the catalytic mechanism of CWPO-C was investigated using chemical modification and homology modeling. The simulated CWPO-C structure predicts that the entrance to the heme pocket of CWPO-C is the same size as those of other plant peroxidases, suggesting that ferrocytochrome c and synthetic lignin polymers cannot interact with the heme of CWPO-C. Since Trp and Tyr residues are redox-active, such residues located on the protein surface were predicted to be active sites for CWPO-C. Modification of CWPO-C Trp residues did not suppress its oxidation activities toward guaiacol and syringaldazine. On the other hand, modification of CWPO-C Tyr residues using tetranitromethane strongly suppressed its oxidation activities toward syringaldazine and 2,6-dimethoxyphenol by 90%, respectively, and also suppressed its guaiacol oxidation activity to a lesser extent. Ferrocytochrome c was not oxidized by Tyr-modified CWPO-C. These results indicate that the Tyr residues in CWPO-C mediate its oxidation of syringyl compounds and high-molecular-weight substrates. Homology modeling indicates that Tyr-177 and Tyr-74 are located near the heme and exposed on the protein surface of CWPO-C. These results suggest that Tyr residues on the protein surface are considered to be important for the oxidation activities of CWPO-C with a wide range of substrates, and potentially unique oxidation sites for the plant peroxidase family.     

Identification and characterization of VPO1, a new animal heme-containing peroxidase

Animal heme-containing peroxidases play roles in innate immunity, hormone biosynthesis, and the pathogenesis of inflammatory diseases. Using the peroxidase-like domain of Duox1 as a query, we carried out homology searching of the National Center for Biotechnology Information database. Two novel heme-containing peroxidases were identified in humans and mice. One, termed VPO1 for vascular peroxidase 1, exhibits its highest tissue expression in heart and vascular wall. A second, VPO2, present in humans but not in mice, is 63% identical to VPO1 and is highly expressed in heart. The peroxidase homology region of VPO1 shows 42% identity to myeloperoxidase and 57% identity to the insect peroxidase peroxidasin. A molecular model of the VPO1 peroxidase region reveals a structure very similar to that of known peroxidases, including a conserved heme binding cavity, critical catalytic residues, and a calcium binding site. The absorbance spectra of VPO1 are similar to those of lactoperoxidase, and covalent attachment of the heme to VPO1 protein was demonstrated by chemiluminescent heme staining. VPO1 purified from heart or expressed in HEK cells is catalytically active, with a K_m for H₂O₂ of 1.5 mM. When co-expressed in cells, VPO1 can use H₂O₂ produced by NADPH oxidase enzymes. VPO1 is likely to carry out peroxidative reactions previously attributed exclusively to myeloperoxidase in the vascular system.     

Concerted involvement of cooperative proton–electron linkage and water production in the proton pump of cytochrome c oxidase

In cytochrome c oxidase, oxido-reductions of heme a/Cu_A and heme a₃/Cu_B are cooperatively linked to proton transfer at acid/base groups in the enzyme. H⁺/e⁻ cooperative linkage at Fe_a3/Cu_B is envisaged to be involved in proton pump mechanisms confined to the binuclear center. Models have also been proposed which involve a role in proton pumping of cooperative H⁺/e⁻ linkage at heme a (and Cu_A). Observations will be presented on: (i) proton consumption in the reduction of molecular oxygen to H₂O in soluble bovine heart cytochrome c oxidase; (ii) proton release/uptake associated with anaerobic oxidation/reduction of heme a/Cu_A and heme a₃/Cu_B in the soluble oxidase; (iii) H⁺ release in the external phase (i.e. H⁺ pumping) associated with the oxidative (R → O transition), reductive (O → R transition) and a full catalytic cycle (R → O → R transition) of membrane-reconstituted cytochrome c oxidase. A model is presented in which cooperative H⁺/e⁻ linkage at heme a/Cu_A and heme a₃/Cu_B with acid/base clusters, C₁ and C₂ respectively, and protonmotive steps of the reduction of O₂ to water are involved in proton pumping.     

Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA

TyrA is a member of the dye-decolorizing peroxidase (DyP) family, a new family of heme-dependent peroxidase recently identified in fungi and bacteria. Here, we report the crystal structure of TyrA in complex with iron protoporphyrin (IX) at 2.3 A. TyrA is a dimer, with each monomer exhibiting a two-domain, alpha/beta ferredoxin-like fold. Both domains contribute to the heme-binding site. Co-crystallization in the presence of an excess of iron protoporphyrin (IX) chloride allowed for the unambiguous location of the active site and the specific residues involved in heme binding. The structure reveals a Fe-His-Asp triad essential for heme positioning, as well as a novel conformation of one of the heme propionate moieties compared to plant peroxidases. Structural comparison to the canonical DyP family member, DyP from Thanatephorus cucumeris (Dec 1), demonstrates conservation of this novel heme conformation, as well as residues important for heme binding. Structural comparisons with representative members from all classes of the plant, bacterial, and fungal peroxidase superfamily demonstrate that TyrA, and by extension the DyP family, adopts a fold different from all other structurally characterized heme peroxidases. We propose that a new superfamily be added to the peroxidase classification scheme to encompass the DyP family of heme peroxidases.     

Bacterial catalase-peroxidases are gene duplicated members of the plant peroxidase superfamily

Bacterial catalase-peroxidases are enzymes containing 0.5-1.0 heme per subunit. The identical subunits are generally 80 kDa in size, and the sequenced subunits of E. coli, S. typhimurium and B. stearothermophilus contain 726-731 amino acid residues per subunit. The heme-containing peroxidases of plants, fungi and yeast are monomeric, homologous and 290-350 residues in size. Analyses of the amino acid sequences indicate that the double length of the bacterial peroxidases can be ascribed to gene duplication. Each half is homologous to eukaryotic, monomeric peroxidase and can be modelled into the high-resolution crystal structure of yeast cytochrome c peroxidase. The comparisons and modelling have predicted: (1) the C-terminal half does not bind heme, and bacterial peroxidases have one heme per subunit; (2) the ten dominating helices observed in the yeast enzyme are highly conserved and connected by surface loops which are often longer in the bacterial peroxidases; and (3) yeast cytochrome c peroxidase has evolved more slowly than other known peroxidases. The study has revealed ten invariant residues and a number of highly conserved residues present in peroxidases of the plant peroxidase superfamily and provides a basis for rationally engineered peroxidases.     

Extracellular Heme Peroxidases in Actinomycetes: a Case of Mistaken Identity

Actinomycetes secrete into their surroundings a suite of enzymes involved in the biodegradation of plant lignocellulose; these have been reported to include both hydrolytic and oxidative enzymes, including peroxidases. Reports of secreted peroxidases have been based upon observations of peroxidase-like activity associated with fractions that exhibit optical spectra reminiscent of heme peroxidases, such as the lignin peroxidases of wood-rotting fungi. Here we show that the appearance of the secreted pseudoperoxidase of the thermophilic actinomycete Thermomonospora fusca BD25 is also associated with the appearance of a heme-like spectrum. The species responsible for this spectrum is a metalloporphyrin; however, we show that this metalloporphyrin is not heme but zinc coproporphyrin. The same porphyrin was found in the growth medium of the actinomycete Streptomyces viridosporus T7A. We therefore propose that earlier reports of heme peroxidases secreted by actinomycetes were due to the incorrect assignment of optical spectra to heme groups rather than to non-iron-containing porphyrins and that lignin-degrading heme peroxidases are not secreted by actinomycetes. The porphyrin, an excretory product, is degraded during peroxidase assays. The low levels of secreted peroxidase activity are associated with a nonheme protein fraction previously shown to contain copper. We suggest that the role of the secreted copper-containing protein may be to bind and detoxify metals that can cause inhibition of heme biosynthesis and thus stimulate porphyrin excretion.     

Pleurotus ostreatus heme peroxidases: an in silico analysis from the genome sequence to the enzyme molecular structure

An exhaustive screening of the Pleurotus ostreatus genome was performed to search for nucleotide sequences of heme peroxidases in this white-rot fungus, which could be useful for different biotechnological applications. After sequence identification and manual curation of the corresponding genes and cDNAs, the deduced amino acid sequences were converted into structural homology models. A comparative study of these sequences and their structural models with those of known fungal peroxidases revealed the complete inventory of heme peroxidases of this fungus. This consists of cytochrome c peroxidase and ligninolytic peroxidases, including manganese peroxidase and versatile peroxidase but not lignin peroxidase, as representative of the "classical" superfamily of plant, fungal, and bacterial peroxidases; and members of two relatively "new" peroxidase superfamilies, namely heme-thiolate peroxidases, here described for the first time in a fungus from the genus Pleurotus, and dye-decolorizing peroxidases, already known in P.?ostreatus but still to be thoroughly explored and characterized.     

Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity

BackgroundHorseradish peroxidase (HRP) catalyzes H₂O₂ dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H₂O₂-induced inactivation, have been investigated.MethodsHRP reaction with H₂O₂ was studied by following H₂O₂ depletion, O₂ evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow.ResultsNitroxide protects HRP against H₂O₂-induced inactivation. The rate of H₂O₂ dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H₂O₂. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO) > 4-OH-TPO > 3-carbamoyl proxyl > 4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III.ConclusionsNitroxide catalytically protects HRP against inactivation induced by H₂O₂ while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex.General SignificanceNitroxides catalytically protect heme proteins against inactivation induced by H₂O₂ revealing an additional role played by nitroxide antioxidants in vivo.