Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction

The wild-type anionic tobacco peroxidase and its Glu141Phe mutant have been expressed in Escherichia coli, and reactivated to yield active enzymes. A Glu141Phe substitution was made with the tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases, such as horseradish peroxidase (HRP). Both recombinant forms of tobacco peroxidase show extremely high activity in luminol oxidation with hydrogen peroxide, and thus, preserve the unique property of the native tobacco peroxidase, a superior chemiluminescent reagent. The chemiluminescent signal intensity for both recombinant forms of TOP is orders of magnitude higher than that for wild-type recombinant HRP. The substitution slightly increases TOP activity and stability in the reaction course, but has almost no effect on the optimal parameters of the reaction (pH, luminol and hydrogen peroxide concentrations) and calibration plot. Comparison of substrate specificity profiles for recombinant TOP and HRP demonstrates that Glu141 has no principal effect on the enzyme activity. It is not the presence of the negative charge at the haem edge, but the high redox potential of TOP Compounds I and II that provides high activity towards aromatic amines and aminophenols, and luminol in particular.     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

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.     

The impact of thiol peroxidases on redox regulation

The biology of glutathione peroxidases and peroxiredoxins is reviewed with emphasis on their role in metabolic regulation. Apart from their obvious function in balancing oxidative challenge, these thiol peroxidases are not only implicated in orchestrating the adaptive response to oxidative stress, but also in regulating signaling triggered by hormones, growth factors and cytokines. The mechanisms presently discussed comprise dampening of redox-sensitive regulatory processes by elimination of hydroperoxides, suppression of lipoxygenase activity, committing suicide to save H₂O₂ for signaling, direct binding to receptors or regulatory proteins in a peroxidase activity-independent manner, or acting as sensors for hydroperoxides and as transducers of oxidant signals. The various mechanistic proposals are discussed in the light of kinetic data, which unfortunately are scarce. Taking into account pivotal criteria of a meaningful regulatory circuit, kinetic plausibility and specificity, the mechanistic concepts implying a direct sensor/transducer function of the thiol peroxidases appear most appealing. With rate constants for the reaction with hydroperoxide of 10⁵–10⁸ M^{? 1} s^{? 1}, thiol peroxidases are qualified as kinetically preferred hydroperoxide sensors, and the ability of the oxidized enzymes to react with defined protein thiols lends specificity to the transduction process. The versatility of thiol peroxidases, however, allows multiple ways of interaction with regulatory pathways.     

Glutamic acid-141: a heme 'bodyguard' in anionic tobacco peroxidase

The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it 'guards' heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.     

Biocatalytic properties of recombinant tobacco peroxidase in chemiluminescent reaction

The wild-type anionic tobacco peroxidase and its Glu141Phe mutant have been expressed in Escherichia coli, and reactivated to yield active enzymes. A Glu141Phe substitution was made with the tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases, such as horseradish peroxidase (HRP). Both recombinant forms of tobacco peroxidase show extremely high activity in luminol oxidation with hydrogen peroxide, and thus, preserve the unique property of the native tobacco peroxidase, a superior chemiluminescent reagent. The chemiluminescent signal intensity for both recombinant forms of TOP is orders of magnitude higher than that for wild-type recombinant HRP. The substitution slightly increases TOP activity and stability in the reaction course, but has almost no effect on the optimal parameters of the reaction (pH, luminol and hydrogen peroxide concentrations) and calibration plot. Comparison of substrate specificity profiles for recombinant TOP and HRP demonstrates that Glu141 has no principal effect on the enzyme activity. It is not the presence of the negative charge at the haem edge, but the high redox potential of TOP Compounds I and II that provides high activity towards aromatic amines and aminophenols, and luminol in particular.     

Oxidative reactions in axenically seedling roots of olive (Olea europaea, L.)

The production of reactive oxygen species (ROS) plays important roles in the life cycle and in the stress response and defence mechanisms of plants. Various enzyme systems are involved in the formation of ROS in the apoplast, including plasmalemma NADPH oxidase and apoplastic peroxidases. The production of O ₂ ^·? and apoplastic peroxidase and exogenous NADH oxidation activities are all strongly dependent on the age of roots??the younger the root, the greater the activity. Apoplastic production of ROS is shown in the root by using specific histochemical probes, this ROS production is growing zone dependent. In the present study, using olive seedlings, differences were also observed between cultivars, especially in O ₂ ^·? production by the Verdial cultivar which was well above that of other cultivars studied. In all the cultivars, treatment of roots with methyl jasmonate (MeJA) or methyl salicylate (MeSA) increased O ₂ ^·? production. Similar results were observed for peroxidase activity, but not for the oxidation of exogenous NADH which was either unaffected (MeJA) or even partially inhibited (MeSA). A conclusion was that MeJA or MeSA induced apoplastic production of ROS does not use exogenous NADH. Treatment with diphenylene iodonium (DPI) reduced the formation of O ₂ ^·? , but affected neither peroxidase nor NADH oxidation activities. Cyanide inhibited O ₂ ^·? production and peroxidase and NADH oxidation activities. Treatment with MnCl₂ had a strong stimulatory effect on peroxidase and NADH oxidation activities, but much less on O ₂ ^·? production. Finally, azide greatly reduced all activities, but especially O ₂ ^·? production. Together, these results indicate a relationship between oxidative activities and the processes of root growth, and that those activities are also dependent on the cultivar, as well as an involvement of peroxidases and plasmalemma NADPH oxidase in apoplast ROS production which is sensitive to DPI, azide, and cyanide but relatively insensitive to MnCl₂, while exogenous NADH oxidation is linked to peroxidase activity.     

In situ Inactivation of the Oxidase Activity of Xylem Peroxidases by H2O2 in the H2O2-Producing Xylem of Zinnia elegans

Zinnia elegans stems with 3,3′, 5, 5′-tetramethylbenzidine (TMB) in the presence and in the absence of catalase reveals the presence of xylem oxidase activities in the H₂O₂-producing lignifying xylem cells. This staining of lignifying xylem cells with TMB is the result of two independent mechanisms: one is the catalase-sensitive (H₂O₂-dependent) peroxidase-mediated oxidation of TMB, and the other the catalase-insensitive (H₂O₂-independent) oxidation of TMB, probably due to the oxidase activity of xylem peroxidases. The response of this TMB-oxidase activity of xylem peroxidases to different exogenous H₂O₂ concentrations was studied, and the results showed that H₂O₂ at high concentrations (100–1,000 mM) clearly acted as an inactivator of this xylem TMB-oxidase activity, although some inhibitory effect could still be appreciated at 10 mM H₂O₂. This xylem TMB-oxidase activity resided in a strongly basic cell wall-bound peroxidase (pl about 10.5). Given such a scenario, it may be concluded that this TMB-oxidase activity of peroxidase is located in tissues capable of sustaining H₂O₂ production, and that the in situ oxidase activity shown by this enzyme is inactivated by high H₂O₂ concentrations. Received 20 April 1999/ Accepted in revised form 16 August 1999     

Characterization of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> adhesin thiol peroxidase (HP0390) purified from <Emphasis Type="Italic">Escherichia coli</Emphasis>

The antioxidant protein, adhesin thiol peroxidase (HpTpx or HP0390), plays an important role in enabling Helicobacter pylori to survive gastric oxidative stress. The bacterium colonizes the host stomach and produces gastric cancer. However, little information is available about the biochemical characteristics of HpTpx. We expressed recombinant HpTpx in Escherichia coli, purified to homogeneity, and characterized it. The results showed that HpTpx existed in a monomeric hydrodynamic form and the enzyme fully retained its peroxidase and antioxidant activities. The catalytic reaction of the enzyme was similar to an atypical 2-cysteine peroxiredoxin (Prx). The conformation of the enzyme was observed in the presence and absence of dithiothreitol (DTT); similar to other known thiol peroxidases, conformational change was observed in HpTpx by the addition of DTT.     

Characterization of a Selenium-Independent Glutathione Peroxidase From Euglena gracilis

Light or dark grown Euglena gracilis strains contain similar levels of glutathione (GSH) peroxidase. Cells in midstationary phase of growth contained the highest level of the enzyme. The enzyme was purified 280-fold to homogeneity from the permanently bleached strain, E. gracilis var bacillaris W₃BUL. The native enzyme has a molecular weight of 130,000 as measured by gel permeation chromatography, and contains four subunits (mol wt 31,500) as measured by sodium dodecyl sulfate gel electrophoresis. A variable amount of a higher molecular weight form of the enzyme (approximate mol wt 250,000) was detected but not further characterized. The enzyme has an isoelectric point of 4.7. No selenium could be detected in the purified enzyme. The enzyme is active with H₂O₂ and a variety of organic hydroperoxides, including 13-hydroperoxylinoleic acid, and is specific for GSH as the thiol substrate. Apparent K_m values for H₂O₂, t-butyl hydroperoxide, and GSH were 0.03, 1.5, and 0.7 millimolar, respectively. A comparison of selenium-dependent and selenium-independent GSH peroxidases from various eukaryotic sources is presented.     

Lignin synthesis: The generation of hydrogen peroxide and superoxide by horseradish peroxidase and its stimulation by manganese (II) and phenols

The enzyme horseradish peroxidase (EC 1.11.1.7) catalyses oxidation of NADH. NADH oxidation is prevented by addition of the enzyme superoxide dismutase (EC 1.15.1.1) to the reaction mixture before adding peroxidase but addition of dismutase after peroxidase has little inhibitory effect. Catalase (EC 1.11.1.6) inhibits peroxidase-catalysed NADH oxidation when added at any time during the reaction. Apparently the peroxidase uses hydrogen peroxide (H₂O₂) generated by non-enzymic breakdown of NADH to catalyse oxidation of NADH to a free-radical, NAD., which reduces oxygen to the superoxide free-radical ion, O₂ ^.-. Some of the O₂ ^.- reacts with peroxidase to give peroxidase compound III, which is catalytically inactive in NADH oxidation. The remaining O₂ ^.- undergoes dismutation to O₂ and H₂O₂. O₂ ^.- does not react with NADH at significant rates. Mn²⁺ or lactate dehydrogenase stimulate NADH oxidation by peroxidase because they mediate a reaction between O₂ ^.- and NADH. 2,4-Dichlorophenol, p-cresol and 4-hydroxycinnamic acid stimulate NADH oxidation by peroxidase, probably by breaking down compound III and so increasing the amount of active peroxidase in the reaction mixture. Oxidation in the presence of these phenols is greatly increased by adding H₂O₂. The rate of NADH oxidation by peroxidase is greatest in the presence of both Mn²⁺ and those phenols which interact with compound III. Both O₂ ^.- and H₂O₂ are involved in this oxidation, which plays an important role in lignin synthesis.     

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO₂ from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.     

Purification,characterization and catalytic activity of anionic zucchini peroxidase

The isolation and purification, by preparative electrofocusing, of the major anionic (ZPOA) and cationic (ZPOC) isoenzymes, collected from young zucchini squash, are reported. The M _r and sugar content are similar to those found previously for the major isoenzymes from the ripe fruits and in the range commonly observed for plant peroxidases. The amount of the two cationic enzymes was very low compared with that of anionic ZPOA. The anionic enzyme has been characterized by electronic, circular dichroism, proton NMR and electron paramagnetic resonance spectroscopy. The spectra are qualitatively similar to those of the corresponding anionic horseradish peroxidase (HRPA) derivatives, with minor differences attributable to the particular protein environment around the heme. The kinetics of the enzymatic oxidation of a series of phenols by H₂O₂ have been studied. ZPOA shows a parallel behavior to HRPA, but it is systematically more active than HRPA, indicating that the zucchini enzymes have a marked tendency to carry out oxidation of this type of compounds.     

Effect of thiocyanate on the peroxidase and pseudocatalase activities of Leishmania major ascorbate peroxidase

We report here that the Leishmania major ascorbate peroxidase (LmAPX), having similarity with plant ascorbate peroxidase, catalyzes the oxidation of suboptimal concentration of ascorbate to monodehydroascorbate (MDA) at physiological pH in the presence of added H₂O₂ with concurrent evolution of O₂. This pseudocatalatic degradation of H₂O₂ to O₂ is solely dependent on ascorbate and is blocked by a spin trap, α-phenyl-n-tert-butyl nitrone (PBN), indicating the involvement of free radical species in the reaction process. LmAPX thus appears to catalyze ascorbate oxidation by its peroxidase activity, first generating MDA and H₂O with subsequent regeneration of ascorbate by the reduction of MDA with H₂O₂ evolving O₂ through the intermediate formation of O₂⁻. Interestingly, both peroxidase and ascorbate-dependent pseudocatalatic activity of LmAPX are reversibly inhibited by SCN⁻ in a concentration dependent manner. Spectral studies indicate that ascorbate cannot reduce LmAPX compound II to the native enzyme in presence of SCN⁻. Further kinetic studies indicate that SCN⁻ itself is not oxidized by LmAPX but inhibits both ascorbate and guaiacol oxidation, which suggests that SCN⁻ blocks initial peroxidase activity with ascorbate rather than subsequent nonenzymatic pseudocatalatic degradation of H₂O₂ to O₂. Binding studies by optical difference spectroscopy indicate that SCN⁻ binds LmAPX (Kd = 100 ± 10 mM) near the heme edge. Thus, unlike mammalian peroxidases, SCN⁻ acts as an inhibitor for Leishmania peroxidase to block ascorbate oxidation and subsequent pseudocatalase activity.     

Specificity of an HPETE peroxidase from rat PMN

The 15,000xg supernatant of sonicated rat PMN contains 5-lipoxygenase that converts arachidonic acid to 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene A₄ and an HPETE peroxidase that catalyzes reduction of the 5-HPETE. The specificity of this HPETE peroxidase for peroxides, reducing agents, and inhibitors has been characterized to distinguish this enzyme from other peroxidase activities. In addition to 5-HPETE, the HPETE peroxidase will catalyze reduction of 15-hydroperoxyeicosatetraenoic acid, 13-hydroperoxyoctadecadienoic acid, and 15-hydroperoxy-8,11,13-eicosatrienoic acid, but not cumene or t-butylhydroperoxides. The HPETE peroxidase accepted 5 of 11 thiols tested as reducing agents. However, glutathione is >15 times more effective than any other thiol tested. Other reducing agents, ascorbate, NADH, NADPH, phenol, p-cresol, and homovanillic acid, were not accepted by HPETE peroxidase. This enzyme is not inhibited by 10 mM KCN, 2 mM aspirin, 2 mM salicylic acid, or 0.5 mM indomethacin. When 5-[¹⁴C]HPETE is generated from [¹⁴C]arachidonic acid in the presence of unlabeled 5-HPETE and the HPETE peroxidase, the 5-[¹⁴C]HETE produced is of much lower specific activity than the [¹⁴C]arachidonic acid. This indicates that the 5-[¹⁴C]HPETE leaves the active site of 5-lipoxygenase and mixes with the unlabeled 5-HPETE in solution prior to reduction and is a kinetic demonstration that 5-lipoxygenase has no peroxidase activity. Specificity for peroxides, reducing agents, and inhibitors differentiates HPETE peroxidase from glutathione peroxidase, phospholipid-hydroperoxide glutathione peroxidase, a 12-HPETE peroxidase, and heme peroxidases. The HPETE peroxidase could be a glutathione S-transferase selective for fatty acid hydroperoxides.     

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, H₂O₂, and organic solvents. Thus, RPTP is a promising candidate for developing H₂O₂-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 Å. 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.     

Hydroquinone peroxidase activity of maize root mitochondria

Summary. The oxidation of hydroquinone with H₂O₂ in the presence of mitochondria isolated from maize (Zea mays L.) roots was studied. The results indicate that a reduced form of quinone may be a substrate of mitochondrial peroxidases. Specific activities in different mitochondrial isolates, the apparent K _m for hydrogen peroxide and hydroquinone, and the influence of some known peroxidase inhibitors or effectors are presented. Zymographic assays revealed that all mitochondrial peroxidases, which were stained with 4-chloro-1-naphthol, were capable of oxidizing hydroquinone. A possible antioxidative role of hydroquinone peroxidase in H₂O₂ scavenging within the mitochondria, in cooperation with ascorbate or coupled with mitochondrial NAD(P)H dehydrogenases, is proposed. Correspondence: M. Vuletić, Laboratory of Plant Physiology, Maize Research Zemun Polje, P.O. Box 89, 11185 Belgrade, Serbia.     

Nickel-induced oxidative stress and the role of antioxidant defence in rice seedlings

Seedlings of rice (Oryza sativa L.) cv. Pant-12 grown in sand cultures containing 200 and 400 μM NiSO₄, showed a decrease in length and fresh weight of roots and shoots. Nickel was readily taken up by rice seedlings and the concentration was higher in roots than shoots. Nickel-treated seedlings showed increased rates of superoxide anion (O₂ ^•− ) production, elevated levels of H₂O₂ and thiobarbituric acid reactive substances (TBARS) demonstrating enhanced lipid peroxidation, and a decline in protein thiol levels indicative of increased protein oxidation compared to controls. With progressively higher Ni concentrations, non-protein thiol and ascorbate (AsA) increased, whereas the level of low-molecular-weight thiols (such as glutathione and hydroxyl-methyl glutathione), the ratio of these thiols to their corresponding disulphides, and the ratio of AsA to dehydroascorbic acid declined in the seedlings. Among the antioxidant enzymes studied, the activities of all isoforms of superoxide dismutase (Cu-Zn SOD, Mn SOD and Fe SOD), guaiacol peroxidases (GPX) and ascorbate peroxidase (APX) increased in Ni-treated seedlings, while no clear alteration in catalase activity was evident. Activity of the ascorbate-glutathione cycle enzymes monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)—significantly increased in Ni-treated seedlings. However such increase was apparently insufficient to maintain the intracellular redox balance. Results suggest that Ni induces oxidative stress in rice plants, resulting in enhanced lipid peroxidation and decline in protein thiol levels, and that (hydroxyl-methyl) glutathione and AsA in conjunction with Cu-Zn SOD, GPX and APX are involved in stress response.