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At least 19 recordsLinked to original sources
PubMed1996

Heterologous expression and characterization of soybean cytosolic ascorbate peroxidase.

Ascorbate peroxidase is a widespread plant enzyme that catalyzes the removal of potentially harmful H2O2. This enzyme is particularly important in legume root nodules due to their high potential for generating activated forms of oxygen. A cDNA clone of soybean nodule ascorbate peroxidase was used to construct an expression system in Escherichia coli. The recombinant protein had an N-terminal tag of six consecutive histidine residues to allow for purification by Ni(2+)-agarose affinity chromatography. Large amounts of recombinant peroxidase (about 27% of total soluble protein) were produced but most of the peroxidase was present in the apo-form (without heme). Addition of delta-aminolevulinic acid to the growth media resulted in an increase in production of holoprotein. Apoprotein was easily converted to the holo-form by in vitro reconstitution with hemin. The reconstituted protein was catalytically, spectrally, and immunologically indistinguishable from native ascorbate peroxidase.

Amino Acid Sequence↗
PubMed2000

Common phylogeny of catalase-peroxidases and ascorbate peroxidases.

Catalase-peroxidases belong to Class I of the plant, fungal, bacterial peroxidase superfamily, together with yeast cytochrome c peroxidase and ascorbate peroxidases. Obviously these bifunctional enzymes arose via gene duplication of an ancestral hydroperoxidase. A 230-residues long homologous region exists in all eukaryotic members of Class I, which is present twice in both prokaryotic and archaeal catalase-peroxidases. The overall structure of eukaryotic Class I peroxidases may be retained in both halves of catalase-peroxidases, with major insertions in several loops, some of which may participate in inter-domain or inter-subunit interactions. Interspecies distances in unrooted phylogenetic trees, analysis of sequence similarities in distinct structural regions, as well as hydrophobic cluster analysis (HCA) suggest that one single tandem duplication had already occurred in the common ancestor prior to the segregation of the archaeal and eubacterial lines. The C-terminal halves of extant catalase-peroxidases clearly did not accumulate random changes, so prolonged periods of independent evolution of the duplicates can be ruled out. Fusion of both copies must have occurred still very early or even in the course of the duplication. We suggest that the sparse representatives of eukaryotic catalase-peroxidases go back to lateral gene transfer, and that, except for several fungi, only single copy hydroperoxidases occur in the eukaryotic lineage. The N-terminal halves of catalase-peroxidases, which reveal higher homology with the single-copy members of the superfamily, obviously are catalytically active, whereas the C-terminal halves of the bifunctional enzymes presumably control the access to the haem pocket and facilitate stable folding. The bifunctional nature of catalase-peroxidases can be ascribed to several unique sequence peculiarities conserved among all N-terminal halves, which most likely will affect the properties of both haem ligands.

Amino Acid Motifs↗
PubMed2006

QM/MM modeling of compound I active species in cytochrome P450, cytochrome C peroxidase, and ascorbate peroxidase.

QM/MM calculations provide a means for predicting the electronic structure of the metal center in metalloproteins. Two heme peroxidases, Cytochrome c Peroxidase (CcP) and Ascorbate Peroxidase (APX), have a structurally very similar active site, yet have active intermediates with very different electronic structures. We review our recent QM/MM calculations on these systems, and present new computational data. Our results are in good agreement with experiment, and suggest that the difference in electronic structure is due to a large number of small differences in structure from one protein to another. We also discuss recent QM/MM calculations on the active species of cytochrome P450, in which a similar sensitivity of the electronic structure to the environment is found. However, this does not appear to explain different catalytic profiles of the different drug-metabolizing isoforms of this class of enzyme.

Ascorbate Peroxidases↗
PubMed2005

QM/MM studies of the electronic structure of the compound I intermediate in cytochrome c peroxidase and ascorbate peroxidase.

Cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX) both involve reactive haem oxoferryl intermediates known as 'compound I' species. These two enzymes also have a very similar structure, especially in the vicinity of the haem group. Despite this similarity, the electronic structure of compound I in the two enzymes is known to be very different. Compound I intermediates have three unpaired electrons, two of which are always situated on the Fe-O core, whilst the third is located in a porphyrin orbital in APX and many other compound I species. In CcP, however, this third unpaired electron is positioned on a tryptophan residue lying close to the haem ring. The same residue is present in the same position in APX, yet it is not oxidized in that case. We report QM/MM calculations, using accurate B3LYP density functional theory for the QM region, on the active intermediate for both enzymes. We reproduce the observed difference in electronic structure, and show that it arises as a result of subtle electrostatic effects which affect the ionization potential of both the tryptophan and porphyrin groups. The computed structures of both enzymes do not involve deprotonation of the tryptophan group, or protonation of the oxoferryl oxygen.

Ascorbate Peroxidases↗
PubMed2003

Crystal structure of the ascorbate peroxidase-ascorbate complex.

Heme peroxidases catalyze the H2O2-dependent oxidation of a variety of substrates, most of which are organic. Mechanistically, these enzymes are well characterized: they share a common catalytic cycle that involves formation of a two-electron, oxidized Compound I intermediate followed by two single-electron reduction steps by substrate. The substrate specificity is more diverse--most peroxidases oxidize small organic substrates, but there are prominent exceptions--and there is a notable absence of structural information for a representative peroxidase-substrate complex. Thus, the features that control substrate specificity remain undefined. We present the structure of the complex of ascorbate peroxidase-ascorbate. The structure defines the ascorbate-binding interaction for the first time and provides new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase catalysis for more than 20 years. A new mechanism for electron transfer is proposed that challenges existing views of substrate oxidation in other peroxidases.

Amino Acid Sequence↗
PubMed1997

From sequence analysis of three novel ascorbate peroxidases from Arabidopsis thaliana to structure, function and evolution of seven types of ascorbate peroxidase.

Ascorbate peroxidases are haem proteins that efficiently scavenge H2O2 in the cytosol and chloroplasts of plants. Database analyses retrieved 52 expressed sequence tags coding for Arabidopsis thaliana ascorbate peroxidases. Complete sequencing of non-redundant clones revealed three novel types in addition to the two cytosol types described previously in Arabidopsis. Analysis of sequence data available for all plant ascorbate peroxidases resulted in the following classification: two types of cytosol soluble ascorbate peroxidase designated cs1 and cs2; three types of cytosol membrane-bound ascorbate peroxidase, namely cm1, bound to microbodies via a C-terminal membrane-spanning segment, and cm2 and cm3, both of unknown location; two types of chloroplast ascorbate peroxidase with N-terminal transit sequences, the stromal ascorbate peroxidase (chs), and the thylakoid-bound ascorbate peroxidase showing a C-terminal transmembrane segment and designated cht. Further comparison of the patterns of conserved residues and the crystal structure of pea ascorbate peroxidase showed that active site residues are conserved, and three peptide segments implicated in interaction with reducing substrate are similar, excepting cm2 and cm3 types. A change of Phe-175 in cytosol types to Trp-175 in chloroplast types might explain the greater ascorbate specificity of chloroplast compared with cytosol ascorbate peroxidases. Residues involved in homodimeric subunit interaction are conserved only in cs1, cs2 and cm1 types. The proximal cation (K+)-binding site observed in pea ascorbate peroxidase seems to be conserved. In addition, cm1, cm2, cm3, chs and cht ascorbate peroxidases contain Asp-43, Asn-57 and Ser-59, indicative of a distal monovalent cation site. The data support the hypothesis that present-day peroxidases evolved by an early gene duplication event.

Amino Acid Sequence↗
PubMed2005

Response of ascorbate peroxidase isoenzymes and ascorbate regeneration system to abiotic stresses in Cucumis sativus L.

Ascorbate peroxidase (APX) isoenzymes, distributing in at least four distinct cell compartments, the chloroplastic stroma (sAPX) and thylakoid membrane (tAPX), microbody (mAPX) and cytosol (cAPX), catalyze the reduction of H(2)O(2) to water by using ascorbic acid (AsA) as specific electron donor. In order to better clarify the response of APX isoenzymes and AsA regeneration enzymes to abiotic stresses, the activities of APX isoenzymes as well as monodehydroascorbate reductase (MDAR), glutathione reductase (GR) and dehydroascorbate reductase (DHAR) were investigated in cucumber plants after heat, methyl viologen (MV) and H(2)O(2) treatments. The activities of cAPX, sAPX, mAPX increased after a slight decline throughout the experiment. Consistent closely with sAPX activity, the expression of sAPX followed a similar change pattern, indicating that sAPX was regulated at the transcriptional level. In contrast, constitutive expression was observed in tAPX activity and no significant changes in tAPX activity were found throughout the experiment. The increases in MDAR and GR were accompanied with enhanced level of AsA/DHA, implying that the AsA regeneration system plays an essential role in compensating AsA degradation.

Ascorbate Peroxidases↗
PubMed2000

Peroxidase-catalyzed oxidation of ascorbate. Structural, spectroscopic and mechanistic correlations in ascorbate peroxidase.

Ascorbate-dependent peroxidase activity was first reported in 1979 (Groden and Beck, 1979; Kelly and Latzko, 1979) and ascorbate peroxidase (APX) is, therefore, a relative newcomer to the peroxidase field--horseradish (HRP) and cytochrome c (CcP) peroxidases were, for example, first identified in 1903 (Bach and Chodat, 1903) and 1940 (Altschul et al., 1940) respectively. The APX area was reviewed by Dalton in 1991 (Dalton, 1991): at that time, there was very little detailed kinetic, spectroscopic or functional information available and no structural information had been published. Since 1991, there have been some major advances in the field, most notably with the publication, in 1995, of the first crystal structure for an APX enzyme (Patterson and Poulos, 1995). This information, together with the availability of new recombinant expression systems (Yoshimura et al., 1998; Caldwell et al., 1998; Dalton et al., 1996; Patterson and Poulos, 1994), served as a catalyst for the publication of new functional and spectroscopic data and has meant these data could be sensibly rationalized at the molecular level. The aim of this review is to summarize the more recent advances in the APX area and, as far as possible, to draw comparisons with other, more well-characterized peroxidases. The review will concentrate on the ways in which structural, spectroscopic and mechanistic information have been used in a complementary way to provide a more detailed picture of APX catalysis. The more biological and physiological aspects of APX enzymes have been previously covered in a comprehensive manner (Dalton, 1991) and will not, therefore, be dealt with in detail here.

Ascorbate Peroxidases↗
PubMed1998

Class I heme peroxidases: characterization of soybean ascorbate peroxidase.

An efficient expression system [D. A. Dalton et al. Arch. Biochem. Biophys. 328, 1-8, 1996) for soybean nodule ascorbate peroxidase (APX) has, for the first time, been used to generate enzyme in large enough quantities for detailed biophysical analysis. The recombinant APX has been characterized by electronic absorption, EPR, NMR and circular dichroism spectroscopies, and by electrochemistry. Electronic, EPR, and NMR spectra are consistent with a high-spin ferric resting state for the enzyme at 298 K. Low-temperature EPR (7 K) and electronic absorption (77 K) experiments indicate formation of a low-spin heme derivative at these temperatures. The midpoint reduction potential for the Fe(III)/Fe(II) redox couple, determined by spectroelectrochemistry, is -159 +/- 2 mV vs SHE (pH 7.0, 25.0 degrees C, mu = 0.10 M). Circular dichroism spectra of pea and soybean APXs are very similar, indicating common structural features for the two enzymes. The melting temperature of soybean APX, as monitored by circular dichroism spectroscopy, is 49 degrees C. These results represent the first detailed spectroscopic and electrochemical analysis of soybean ascorbate peroxidase and are discussed in the broader context of other class I peroxidases.

Amino Acid Sequence↗
PubMed2002

Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites.

The catalytic mechanism of recombinant soybean cytosolic ascorbate peroxidase (rsAPX) and a derivative of rsAPX in which a cysteine residue (Cys32) located close to the substrate (L-ascorbic acid) binding site has been modified to preclude binding of ascorbate [Mandelman, D., Jamal, J., and Poulos, T. L. (1998) Biochemistry 37, 17610-17617] has been examined using pre-steady-state and steady-state kinetic techniques. Formation (k1 = 3.3 +/- 0.1 x 10(7) M(-1) s(-1)) of Compound I and reduction (k(2) = 5.2 +/- 0.3 x 10(6) M(-1) s(-1)) of Compound I by substrate are fast. Wavelength maxima for Compound I of rsAPX (lambda(max) (nm) = 409, 530, 569, 655) are consistent with a porphyrin pi-cation radical. Reduction of Compound II by L-ascorbate is rate-limiting: at low substrate concentration (0-500 microM), kinetic traces were monophasic but above approximately 500 microM were biphasic. Observed rate constants for the fast phase overlaid with observed rate constants extracted from the (monophasic) dependence observed below 500 microM and showed saturation kinetics; rate constants for the slow phase were linearly dependent on substrate concentration (k(3-slow)) = 3.1 +/- 0.1 x 10(3) M(-1) s(-1)). Kinetic transients for reduction of Compound II by L-ascorbic acid for Cys32-modified rsAPX are monophasic at all substrate concentrations, and the second-order rate constant (k(3) = 0.9 +/- 0.1 x 10(3) M(-1) s(-1)) is similar to that obtained from the slow phase of Compound II reduction for unmodified rsAPX. Steady-state oxidation of L-ascorbate by rsAPX showed a sigmoidal dependence on substrate concentration and data were satisfactorily rationalized using the Hill equation; oxidation of L-ascorbic acid by Cys32-modified rsAPX showed no evidence of sigmoidal behavior. The data are consistent with the presence of two kinetically competent binding sites for ascorbate in APX.

Ascorbate Peroxidases↗
PubMed2002

Double antisense plants lacking ascorbate peroxidase and catalase are less sensitive to oxidative stress than single antisense plants lacking ascorbate peroxidase or catalase.

The plant genome is a highly redundant and dynamic genome. Here, we show that double antisense plants lacking the two major hydrogen peroxide-detoxifying enzymes, ascorbate peroxidase (APX) and catalase (CAT), activate an alternative/redundant defense mechanism that compensates for the lack of APX and CAT. A similar mechanism was not activated in single antisense plants that lacked APX or CAT, paradoxically rendering these plants more sensitive to oxidative stress compared to double antisense plants. The reduced susceptibility of double antisense plants to oxidative stress correlated with suppressed photosynthetic activity, the induction of metabolic genes belonging to the pentose phosphate pathway, the induction of monodehydroascorbate reductase, and the induction of IMMUTANS, a chloroplastic homologue of mitochondrial alternative oxidase. Our results suggest that a co-ordinated induction of metabolic and defense genes, coupled with the suppression of photosynthetic activity, can compensate for the lack of APX and CAT. In addition, our findings demonstrate that the plant genome has a high degree of plasticity and will respond differently to different stressful conditions, namely, lack of APX, lack of CAT, or lack of both APX and CAT.

Antisense Elements (Genetics)↗
PubMed1996

Molecular characterization of Euglena ascorbate peroxidase using monoclonal antibody.

Ascorbate peroxidase (EC 1.11.1.11) has been purified to electrophoretic homogeneity from Euglena gracilis Z. The enzyme showed a molecular mass of 58 kDa on SDS-PAGE and gel filtration, indicating that Euglena ascorbate peroxidase exists as a monomeric form. The substrate specificity for an electron donor and the stability of the purified enzyme were similar to those of cytosolic isozymes from higher plants. One of the characteristic properties was that Euglena ascorbate peroxidase reduces organic hydroperoxides as well as hydrogen peroxide. The N-terminal amino-acid sequence showed no significant similarity to any other ascorbate peroxidase from higher plants. However, the sequence of the peptides from the purified enzyme exhibited a high degree of homology to sequences of cytosolic and chloroplastic ascorbate peroxidases. Monoclonal antibodies against the purified Euglena ascorbate peroxidase were prepared. Two monoclonal antibodies (EAP1 and EAP2) showed high homology to cytosolic ascorbate peroxidases of higher plants, as judged by Western blot analysis. The EAP1 was also specific for chloroplastic ascorbate peroxidase from spinach. These findings indicate that Euglena ascorbate peroxidase exists in highly homologous regions with the ascorbate peroxidases of higher plants.

Amino Acid Sequence↗
PubMed2005

Silicon-mediated alleviation of Mn toxicity in Cucumis sativus in relation to activities of superoxide dismutase and ascorbate peroxidase.

The effects of exogenous silicon (Si) on plant growth, activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and catalase, and concentrations of ascorbate and glutathione were investigated in cucumber (Cucumis sativus L.) plants treated with excess manganese (Mn) (600 microM). Compared with the treatment of normal Mn (10 microM), excess Mn significantly increased H2O2 concentration and lipid peroxidation indicated by accumulation of thiobarbituric acid reactive substances. The leaves showed apparent symptoms of Mn toxicity and the plant growth was significantly inhibited by excess Mn. The addition of Si significantly decreased lipid peroxidation caused by excess Mn, inhibited the appearance of Mn toxicity symptoms, and improved plant growth. This alleviation of Mn toxicity by Si was related to a significant increase in the activities of SOD, APX, DHAR and GR and the concentrations of ascorbate and glutathione.

Ascorbate Peroxidases↗
PubMed1991

Purification and characterization of pea cytosolic ascorbate peroxidase.

The cytosolic isoform of ascorbate peroxidase was purified to homogeneity from 14-day-old pea (Pisum sativum L.) shoots. The enzyme is a homodimer with molecular weight of 57,500, composed of two subunits with molecular weight of 29,500. Spectral analysis and inhibitor studies were consistent with the presence of a heme moiety. When compared with ascorbate peroxidase activity derived from ruptured intact chloroplasts, the purified enzyme was found to have a higher stability, a broader pH optimum for activity, and the capacity to utilize alternate electron donors. Unlike classical plant peroxidases, the cytosolic ascorbate peroxidase had a very high preference for ascorbate as an electron donor and was specifically inhibited by p-chloromercurisulfonic acid and hydroxyurea. Antibodies raised against the cytosolic ascorbate peroxidase from pea did not cross-react with either protein extracts obtained from intact pea chloroplasts or horseradish peroxidase. The amino acid sequence of the N-terminal region of the purified enzyme was determined. Little homology was observed among pea cytosolic ascorbate peroxidase, the tea chloroplastic ascorbate peroxidase, and horseradish peroxidase; homology was, however, found with chloroplastic ascorbate peroxidase isolated from spinach leaves.

Journal Article↗
PubMed1993

Detection of ascorbate peroxidase activity in native gels by inhibition of the ascorbate-dependent reduction of nitroblue tetrazolium.

A method for the detection of ascorbate peroxidase activity in native electrophoretic gels is described. The assay is based on the ability of ascorbate peroxidase to prevent the ascorbate-dependent reduction of nitroblue tetrazolium in the presence of H2O2. The method was found to be both sensitive (detection of less than 0.01 units of ascorbate peroxidase activity) and specific for ascorbate peroxidase activity. The application of the method for the detection of ascorbate peroxidase activity in protein extracts from several plant sources was investigated by comparing staining for activities of ascorbate peroxidase, horseradish peroxidase, and ascorbate oxidase and by immunodetection of ascorbate peroxidase in these extracts.

Acrylic Resins↗
PubMed2006

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 H(2)O(2) with concurrent evolution of O(2). This pseudocatalatic degradation of H(2)O(2) to O(2) is solely dependent on ascorbate and is blocked by a spin trap, alpha-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 H(2)O with subsequent regeneration of ascorbate by the reduction of MDA with H(2)O(2) evolving O(2) through the intermediate formation of O(2)(-). 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 H(2)O(2) to O(2). 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.

Animals↗
PubMed1998

Purification and molecular properties of ascorbate peroxidase from bovine eye.

Ascorbate peroxidase (APX) is a hydrogen peroxide-scavenging peroxidase which uses ascorbate (AsA) as the specific electron donor. APX has not been isolated in mammals. Ocular tissue contains AsA at high concentrations, and we detected APX activity in bovine retinal pigment epithelium (RPE) and choroid. We purified APX from bovine RPE and choroid by four chromatographic steps. The purified APX was a monomeric hemoprotein with a molecular mass of 43 kDa. The amino acid sequence of the amino-terminal region of the purified APX showed a high degree of homology to that of plants. The primary product of the APX reaction was identified as the monodehydroascorbate radical. The APX showed high specificity for AsA as an electron donor. This is the first isolation and characterization of APX from mammals, and its role in the protection against active species of oxygen in ocular tissue is discussed.

Amino Acid Sequence↗
PubMed2006

An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation.

Ascorbate peroxidase isoforms localized in the stroma and thylakoid of higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if the second substrate, ascorbate, is depleted. However, cytosolic and microbody-localized isoforms from higher plants as well as ascorbate peroxidase B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively tolerant. We constructed various chimeric ascorbate peroxidases in which regions of ascorbate peroxidase B, from sites internal to the C-terminal end, were exchanged with corresponding regions of the stromal ascorbate peroxidase of spinach. Analysis of these showed that a region between residues 245 and 287 was involved in the inactivation by hydrogen peroxide. A 16-residue amino acid sequence (249-264) found in this region of the stromal ascorbate peroxidase was not found in other ascorbate peroxidase isoforms. A chimeric ascorbate peroxidase B with this sequence inserted was inactivated by hydrogen peroxide within a few minutes. The sequence forms a loop that binds noncovalently to heme in cytosolic ascorbate peroxidase of pea but does not bind to it in stromal ascorbate peroxidase of tobacco, and binds to cations in both ascorbate peroxidases. The higher susceptibility of the stromal ascorbate peroxidase may be due to a distorted interaction of the loop with the cation and/or the heme.

Amino Acid Sequence↗