Peanut Peroxidase Research Articles

Enzymatic method for the determination of phenols with the use of peanut peroxidase

Cationic peanut peroxidase was used for the first time for the determination of phenols at a level of 0.5–10μM. The examined phenols were found to be inhibitors or second substrates of peanut peroxidase in the indicator reaction of the oxidation ofo-dianisidine by hydrogen peroxide. The effect of phenols on the rate of the indicator reaction depends on their redox properties. The data on the effects of phenols on the catalytic activities of peroxidases isolated from different sources (peanut, horseradish roots,Medicago sativa alfalfa cells, and the xylotrophic fungusPhellinius igniarius) were compared

Sequence specific analysis of the heterogeneous glycan chain from peanut peroxidase by 1H-NMR spectroscopy

The cationic peanut peroxidase is a complex enzyme consisting of a heme group, two calcium ions and three complex carbohydrate chains at positions Asn60, 144 and 185. Details of the heme and calcium ligation, necessary for oxidation, have recently been revealed from the three-dimensional structure of the peroxidase. However, the three glycans that may be important for the stability of the enzyme as well as its activity were not resolved. In order to determine the configuration of one of these glycans, PNGase A was used to cleave the glycan from the enzyme at Asn-144. This glycan was studied by two dimensional 1H-NMR spectroscopy to identify the sugar linkages. The results indicated a glycan structure comprising a Manα1-6(Xylβ1-2)Manβ1-4GlcNAcβ1-4(Fucα1-3)GlcNAcβ core but with an additional Manα1-3 appendage linked to Man3. The glycan also appeared to be heterogeneous as was noted from a single terminating galactose being linked to approximately 20–25% glycan.

Aerobic oxidation of indole-3-acetic acid catalysed by anionic and cationic peanut peroxidase

The catalytic properties of anionic and cationic peanut peroxidases with regards to the oxidation of indole-3-acetic acid (IAA) by molecular oxygen at low pH have been studied. Transient kinetic studies demonstrate that only cationic peroxidases (peanut and horseradish) but not anionic peroxidases (such as anionic tobacco and anionic peanut peroxidases) form a stable compound III in the course of IAA oxidation. The failure to observe inhibition in the presence of superoxide dismutase is consistent with the formation of compound III from a ternary complex comprising ferric enzyme, IAA and dioxygen at the initiation step. Product analysis by HPLC showed an enhanced rate of IAA oxidation in the presence of superoxide dismutase. Co-addition of superoxide dismutase and catalase demonstrates that this stimulation is not due to the formation of hydrogen peroxide. The correlation between initial rates of IAA degradation and product accumulation indicates that skatole hydroperoxide is a primary reaction product and indole-3-methanol is the product of its subsequent enzymatic reduction. The relative catalytic activities for IAA oxidation by tobacco:horseradish isoenzyme c:anionic peanut:cationic peanut peroxidase are 28:20:2:1.

Cationic peanut peroxidase: expression and characterization in transgenic tobacco and purification of the histidine-tagged protein

Cationic peanut ( Arachis hypogaea) peroxidase (CPRX) is the major isozyme of peanut peroxidases secreted into the culture medium from the cells growing in liquid suspension culture. In this report, CPRX was expressed in transgenic tobacco ( Nicotiana tabacum) plants and represented about 0.3% of the total soluble proteins. In order to purify the expressed protein, a sequence encoding a C-terminal tag of six consecutive histidine residues was introduced into the corresponding cDNA bearing a native N-terminal signal sequence facilitating secretion of CPRX. The expressed CPRX-his6 was purified under nondenaturing conditions by a single-step affinity chromatography using NTA resin. The purified CPRX-his6 was fully functional as native CPRX. CPRX is a glycoprotein. Monoclonal antibodies specific for epitope occurring at the region where oligosaccharide is linked to the polypeptide backbone recognized CPRX-his6, indicating the protein was also glycosylated properly in tobacco cells. Western blotting analysis on proteins from spent medium confirmed CPRX-his6 was secreted into the liquid medium, indicating the native CPRX signal peptide was sufficient to direct the secretion of CPRX-his6 in tobacco cells.

Expression of the peanut peroxidase cDNA in Escherichia coli and purification of the protein by nickel affinity

A cDNA sequence containing the entire cationic peanut ( Arachis hypogaea) peroxidase (EC 1.11.1.7, CPRX) coding sequence, including a 22-amino acid signal peptide, was extended using a synthetic oligonucleotide with a six histidine-tag at the C-terminal and expressed in bacteria. The expressed protein was purified on a Ni 2+-nitrilotriacetic acid (NTA) column and the resulting protein had a molecular mass corresponding to an unglycosylated polypeptide. Western blot analysis showed the protein to be recognized by polyclonal antibodies raised against native CPRX and monoclonal antibodies specifically recognizing six histidine-tag, but not by monoclonal antibody which recognizes carbohydrate-containing epitopes of CPRX.

Effect of calcium and magnesium ions on radiation-induced inactivation of plant peroxidases

Radiation-induced inactivation of soybean, horseradish, peanut, and tobacco peroxidases in the presence and in the absence of calcium and magnesium salts involves two steps which differ in the character of the effect of the metal cation. The effect of calcium cations is determined by the total charge of the enzyme molecule under experimental conditions. In general, calcium stabilizes the peroxidases, although in the case of anionic soybean and tobacco peroxidases it destabilizes the enzymes at the first inactivation step, probably due to absorption on their surface and changes in the native conformation. The effect of magnesium ions is determined by the specific features of the enzyme structure, and the data obtained allow us to suggest the existence of additional metal-binding sites in tobacco and peanut peroxidases.

Enzymatic determination of α- and β-naphthols using peanut peroxidase

Novel cationic peanut peroxidase has been used for developing a technique for the determination of α- and β-naphthols based on their differing effect towards enzyme activity in the o -dianisidine oxidation reaction.

Glycans of higher plant peroxidases: recent observations and future speculations.

Plant peroxidases are composed of a peptide and associated heme, calcium and glycans. The 3D structure of the major cationic peanut peroxidase has revealed the sites of the heme and calcium. But the diffraction of the glycans was not sufficient to show their structure. This review presents research that has been executed to obtain putative glycans and their binding sites, and to gain an indirect insight into these glycans. It also offers approaches that will be used to determine the function of the glycans on the peanut peroxidase. Some comparisons are made with other plant glycoproteins including peroxidases from plants other than peanut.

Structure of Barley Grain Peroxidase Refined at 1.9-Å Resolution: A PLANT PEROXIDASE REVERSIBLY INACTIVATED AT NEUTRAL pH

The crystal structure of the major peroxidase of barley grain (BP 1) has been solved by molecular replacement and phase combination and refined to an R-factor of 19.2% for all data between 38 and 1.9 A. The refined model includes amino acid residues 1-309, one calcium ion, one sodium ion, iron-protoporphyrin IX, and 146 solvent molecules. BP 1 has the apparently unique property of being unable to catalyze the reaction with the primary substrate hydrogen peroxide to form compound I at pH values > 5, a feature investigated by obtaining crystal structure data at pH 5.5, 7.5, and 8.5. Structural comparison shows that the overall fold of inactive barley grain peroxidase at these pH values resembles that of both horseradish peroxidase C and peanut peroxidase. The key differences between the structures of active horseradish peroxidase C and inactive BP 1 include the orientation of the catalytic distal histidine, disruption of a hydrogen bond between this histidine and a conserved asparagine, and apparent substitution of calcium at the distal cation binding site with sodium at pH 7.5. These profound changes are a result of a dramatic structural rearrangement to the loop region between helices B and C. This is the first time that structural rearrangements linked to active site chemistry have been observed by crystallography in the peroxidase domain distal to heme.

Analysis of the murine intercellular adhesion molecule-1 promoter in H441 cells exposed to TNF-alpha: [formula omitted] and Bei Wu. Baylor College of Medicine, Houston, TX

The feasibility and expediency of enzymatic methods application in food analysis is demonstrated by the example of ascorbic acid (AsA) determination in foods. Enzymatic determination of ascorbic acid is based on its action as a second substrate of horseradish (HRP) and peanut (PNP) peroxidases in the reactions of o-dianisidine (OD) and 3,3’,5,5’-tetramethylbenzidine (TMB) oxidation with hydrogen peroxide. The rates of the reactions are monitored spectrophotometrically by measuring the duration of the induction period on kinetic curves plotted in coordinates absorption-time. The proposed procedures are sensitive (cL = 0.1 μM), simple, and rapid. The procedure using horseradish peroxidase and the reaction of TMB oxidation was used to determine ascorbic acid in fruit juices, milk and sour-milk products for babies’ nutrition.

HPLC Determination of the Sugar Compositions of the Glycans on the Cationic Peanut Peroxidase

Cationic peanut peroxidase has three N-linked glycans. Two glycoforms, CP− and CP+, are known to occur. In this study, the glycans of CP− and CP+ were sequentially separated and identified by trypsin digestion, Bio-Gel P-6 filtration, and reverse phase HPLC. Sugar composition analyses of the glycans were carried out by hydrolysis with TFA, labeling the released sugars with ABEE, and reverse phase HPLC of the ABEE-sugar derivatives. Five different sugars (GlcNAc, Gal, Man, Xyl, Fuc) were found in each of the six glycans investigated. Mannose residues accounted for 31 ± 1.8% of CP− and 40 ± 2.2% of CP+ glycans in term of molar content. The galactose content was 57% lower in CP+ glycans as compared to CP−. The Xyl and Fuc contents were also lower in CP+ than in CP− glycans. A hypothesis made has been confirmed one step further, that is the Gal is one of the terminal sugars of the glycans and the removal of the Gal as a terminal sugar residue in CP− leads to the exposure of Man residue, which is then able to bind to Con-A. Keywords: Cationic peanut peroxidases; glycans; HPLC; sugar compositions

Characterization of anionic soybean (Glycine max) seed coat peroxidase

Soybean (Glycine max) seed coats may contain large amounts of peroxidase enzyme. The release of peroxidase from whole seeds upon imbibition and the catalytic and antigenic properties of this enzyme were studied. Comparisons between high (Ep) and low (epep) peroxidase activity seeds demonstrated a lengthy (336 h) release of anionic peroxidase by Ep (Harovinton) cultivars following incubation in an aqueous environment. In its purified state, soybean seed coat peroxidase exhibited good catalytic activity towards phenolic substrates including eugenol, caffeic acid, and ferulic acid. Both leaf and stem cationic peroxidases behaved similarly to seed coat peroxidase in their substrate specificity. Furthermore, using guaiacol as a substrate at various pH levels and temperatures, soybean seed coat peroxidase had a greater enzyme stability and a wider range of action than other peroxidase enzymes. Polyclonal antibodies raised against cationic peanut peroxidase cross-reacted with soybean peroxidase in the presence and the absence of its glycosidic chains. This suggests homology in epitopes between the soybean and peanut polypeptide and (or) glycan chains. Key words: cross-reactivity, enzyme activity, peroxidase, polymerization, substrate.

Solution NMR Study of the Electronic and Molecular Structure of the Heme Cavity in High-Spin, Resting State Horseradish Peroxidase

Three resting state horseradish peroxidase isozymes (HRPC, HRPA1, and HRPA2) have been investigated by solution 1D and 2D NMR to determine the scope and limitation of these methods for large (∼44 kDa), high-spin ferric heme enzymes and to develop an interpretive basis of the hyperfine shifts in terms of the molecular and electronic structure of the active site. Definitive assignments are attained for the resolved heme and axial His resonances, as well as several residues more than 7 A from the iron. Four Phe side chains located in HRPC by scalar correlation and characteristic NOEs to the heme are identified as Phe 152, Phe 172, and two unassigned Phes X and W, in contact with pyrrole D. The temperature dependence of the hyperfine shifted aromatic rings shows that dipolar shift arises from zero-field splitting; a value of D ∼ 7 cm-1 models the observed dipolar shift with use of a homology model constructed from peanut peroxidase. The combined use of steady-state NOEs, paramagnetic relaxation, and the predi...

Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase.

Manganese-binding sites are found in several heme peroxidases, namely manganese peroxidase (MnP), chloroperoxidase, and the cationic isozyme of peanut peroxidase. The Mn-binding site in MnP is of particular interest. Oxidation of Mn(II) to Mn(III) is a key step in the biodegradation of lignin, a complex phenylpropanoid polymer, as well as many aromatic pollutants. Cytochrome c peroxidase (CcP), which is structurally homologous to MnP despite a poor sequence homology, does not bind manganese. Thus, engineering a Mn-binding site into CcP will allow us to elucidate principles behind designing metal-binding sites in proteins, to understand the structure and function of this class of Mn-binding centers, and to prepare novel enzymes that can degrade both lignin and other xenobiotic compounds. Based on a comparison of the crystal structures of CcP and MnP, a site-directed triple mutant (Gly41-->Glu, Val45-->Glu, His181-->Asp) of residues near the putative Mn-binding site in CcP was prepared and purified to homogeneity. Titrating MnSO4 into freshly prepared mutant CcP resulted in electronic absorption spectral changes similar to those observed in MnP. The calculated apparent dissociation constant and the stoichiometry of Mn-binding of CCP were also similar to MnP. Titration with MnSO4 resulted in the disappearance of specific paramagnetically shifted nuclear magnetic resonance spectroscopy signals assigned to residues close to the putative Mn-binding site in the mutant CcP. None of the spectral features were observed in wild-type CcP. In addition, the triple mutant was capable of oxidizing Mn(II) at least five times more efficiently than the native CcP. A Mn-binding site has been created in CcP and based on our spectroscopic studies the designed Mn-binding site is similar to the Mn-binding site in MnP. The results provide a basis for understanding the structure and function of the Mn-binding site and its role in different heme peroxidases.

The crystal structure of peanut peroxidase

Background: Peroxidases catalyze a wide variety of peroxide-dependent oxidations. Based on sequence alignments, heme peroxidases have been divided into three classes. Crystal structures are available for peroxidases of classes I and II, but until now no structure has been determined for class III, the classical extracellular plant peroxidases. Results The crystal structure of peanut peroxidase has been solved to 2.7 å resolution. The helical fold is similar to that of known peroxidase structures. The 294-residue polypeptide chain is accompanied by a heme and two calcium ions, and there is some evidence of glycosylation. Conclusion This is the first complete structure of a class III peroxidase and as such should serve as a model for other class III enzymes including the much-studied horseradish peroxidase. It may also aid in the interpretation of functional differences between the peroxidase classes. Ten helices conserved in class I and II peroxidases are also found in peanut peroxidase. Key residues of the heme environment and the location of two calcium ions are shared with class II peroxidases. Peanut peroxidase contains three unique helices, two of which contribute to the substrate access channel leading to the heme edge.

Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by 1H-NMR Spectroscopy

Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by 1H-NMR Spectroscopy

Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by 1H-NMR Spectroscopy

Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by 1H-NMR Spectroscopy

Nucleotide sequence of a cationic peroxidase gene from the tropical forage legume Stylosanthes humilis.

Peroxidases (EC 1.1 1.1.7; donor: hydrogen-peroxide oxidoreductase) have been implicated in numerous physiological processes of potential importance in plant-pathogen interactions, including lignification (Walter, 1992), crosslinking of cell-wall components (Bradley et al., 1992), wound healing (Sherf et al., 1993), auxin oxidation (Grambow and Langenbeck-Schwich, 1979), and systemic acquired resistance responses (Irving and Kuc, 1990). A complete description of peroxidase activity in vivo is not available because multiple isoenzymes exist with large numbers of potential substrates. A cDNA (Shpx6, EMBL accession No. L36110) encoding a putative cationic peroxidase was isolated from Stylosanthes humilis. Transcripts hybridizing to this cDNA were shown to be expressed in young leaves and roots of mature plants and were strongly induced in young leaves as a response to wounding and infection with the funga1 phytopathogen Colletotrichton gloeosporioides, which causes anthracnose disease in Stylosanthes spp (Harrison et al., 1994). Southern hybridization analysis with genomic DNA of S. humilis indicated that there are probably two closely related copies of this cationic peroxidase gene (Curtis et al., 1995). We report the isolation of one of the cationic peroxidase genes from S. humilis. A single cationic peroxidase gene with 83% nucleotide sequence homology in the coding region to the cDNA ShpxG was isolated from a genomic DNA library of S. humilis constructed in the A phage EMBL3. The gene encodes a peroxidase proprotein with a putative 25 amino acid signal sequence. The predicted mature protein is 26.2 kD with a pI of 8.71 and contains the three peroxidase signature sequences at amino acid positions 56 to 73, 114 to 151, and 184 to 194 (Buffard et al., 1990). The predicted mature protein also contains the eight Cys residues involved in disulfide bridges and the two His residues involved with binding heme, both of which are characteristic of plant peroxidases (Morgens et al., 1990). The deduced amino acid sequence of this clone, AShpxG, described herein (Table I) has 80% homology to the deduced amino acid sequence of the cDNA Shpx6 and 90 and 48% homology to protein sequences of the peanut peroxidase Pncl (Buffard et al., 1990) and wheat peroxidase POXl (Hertig et al., 1991), respectively. The hShpx6 gene sequence reported is 1848 nucleotides long with two introns

Immunogenicity of the N-glycans of peanut peroxidase

The three tryptic glycopeptides of cationic peanut peroxidase (C. PRX) and the sole one of anionic peanut peroxidase (A. PRX) were individually coupled to bovine serum albumin to raise antisera. The three categories of antibodies directed towards three N-glycans of C. PRX (anti-GLa, anti-GLb and anti-GLc) were isolated from antisera with glycan-conjugated ECH Sepharose 4B affinity columns and the distribution of epitopes on the N-glycans was investigated. The reactivity of anti-GLa, anti-GLb and anti-GLc is inhibited 25–40% by 1 M fucose, compared with a slight inhibition by N-acetylglycosamine and xylose. Mannose and galactose showed no inhibition to anti-GLa and only a slight inhibition to anti-GLb and anti-GLc. All of anti-GLa, anti-GLb and anti-GLc recognize A. PRX and horseradish peroxidase but do not recognize fetuin. Also, their reactivity is inhibited by bromelain by more than 70%. The three categories of antibodies present high homogeneity and appear to be directed mainly towards the core structure [Xyl] (Man) 3 [Fuc] (GlcNAc) 2. An effective and simple method to screen antibodies with carbohydrate specificities is described herein.

Isolation of peroxidase from bean and millet compared with that from peanut

Purification of peanut (Arachis hypogaea L.) peroxidase in milligram amounts is feasible through its isolation from spent medium of cultured cells. Using the same and modified approaches for another cultured dicot and a monocot yielded only low amounts of peroxidase for bean (Phaseolus vulgaris) and millet (Setaria italica Beav.). We showed that the procedures used for millet rendered a purer fraction than the commercial crude fraction available for horseradish. The data suggest that cultured peanut cells in suspension culture are an ideal system for isolation of peroxidase in terms of the ease of purification and yield. Key words: peanut, bean, millet, peroxidase, purification, tissue.