Cationic Peanut Peroxidase Research Articles

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

The cationic isozyme of peanut peroxidase (CPRx) is one of many peroxidases which requires calcium for enzyme activity. It has been previously shown that it requires 2 mol calcium to coordinate to 1 mol CPRx, and its related peroxidases from the basidiomycete Phanerochaete chrysosporium (LiP) and isozyme C of horseradish (HRPc). X-ray crystallographic studies of LiP have shown that calcium is ligated near the C-terminus of helices proximal and distal to the heme, where it has been suggested to maintain the active site. To determine if such a mechanism was possible in CPRx, high resolution 1H-NMR spectroscopy was used to study the effect of calcium on the environment of its heme group and the coordinating histidine residues. The low-spin cyano complex of the enzyme (CPRxCN) was studied in order to assign the majority of the resonances arising from the protons in the heme pocket in both the presence and absence of bound calcium ions using two dimensional nuclear Overhauser effect spectroscopy (NOESY). The two calcium ions present in CPRxCN were removed by a non-denaturing method and a calcium titration was performed and monitored by 1H-NMR spectroscopy. These studies showed that the binding of both calcium ions in CPRx influenced the heme environment in a similar manner (Kd = 0.1 microM). In particular, calcium-dependent changes in several heme resonances and the proximal and distal histidine residues suggest that calcium binding to CPRx causes some reorientation of these residues with respect to the active site.

Investigation of cationic peanut peroxidase glycans by electrospray ionization mass spectrometry

Cationic peanut peroxidase (CP) was isolated from peanut ( Arachis hypogaea) cell suspension culture medium. CP is a glycoprotein with three N-linked glycan sites at Asn60, Asn144, and Asn185. ESI-MS of the intact purified protein reveals the microheterogeneity of the glycans. Tryptic digestion of CP gave a near complete sequence coverage by ESI-MS. The glycopeptides from the tryptic digestion were separated by RP HPLC identified by ESI-MS and the structure of the glycan chains determined by ESI-MS/MS. The glycans are large structures of up to 16 sugars, but most of their non-reducing ends have been modified giving a mixture of shorter chains at each site. Good agreement was found with the one glycan previously analyzed by 1H NMR. This work is the basis for the future studies on the role of the glycans on stability and folding of CP and is another example of a detailed structural characterization of complex glycoproteins by mass spectrometry.

A Retrospective Look at the Cationic Peanut Peroxidase Structure

ABSTRACT: The cationic peanut peroxidase has been studied in detail, not only with regard to its peptide structure, but also to the sites and role of the three moieties linked to it. Peanut peroxidase lends itself well to a close examination as a potential example for other plant peroxidase studies. It was the first plant peroxidase for which a 3-D structure was derived from crystals, with the glycans intact. Subsequent analysis of peroxidases structures from other plants have not shown great differences to that of the peanut peroxidase. As the period of proteomics follows on the era of genomics, the study of glycans has been brought back into focus. With the potential use of peroxidase as a polymerization agent for industry, there are some aspects of the overall structure that should be kept in mind for successful use of this enzyme. A variety of techniques are now available to assay for these structures/ moieties and their roles. Peanut peroxidase data are reviewed in that light, as well as defining some true terms for isozymes. Because a high return of the enzyme in a pure form has been obtained from cultured cells in suspension culture, a brief review of this is also offered.

Enzymatic determination of phenols using peanut peroxidase

The influence of phenol and its derivatives on the kinetics of oxidation of aryldiamines (indicator-substrates) catalyzed by novel plant peroxidase—cationic peanut peroxidase—was studied. The character of influence of phenols on the kinetics of enzymatic oxidation of benzidine, o-dianisidine, and 3,3′,5,5′-tetramethylbenzidine (TMB) with hydrogen peroxide was found to depend on a correlation between redox properties of phenols and the indicator-substrate of peroxidase. Thus, the catalytic activity of peanut peroxidase is inhibited by phenols with redox potentials higher than that of aryldiamines mentioned above, whereas phenols with potentials below those of aryldiamines, play the role of second substrates of the enzyme. The enzymatic procedures for the determination of numerous phenols on the level of their concentrations 0.05–80 μM were developed using the reactions of benzidine, o-dianisidine, and TMB oxidation. Different analytical signals—the indicator reaction rate and the induction period duration—were used for the determination of phenols, belonging to various groups—the inhibitors and second substrates of the enzyme, respectively.

The Effects of the Site-Directed Removal of N-Glycosylation from Cationic Peanut Peroxidase on Its Function

Peanut peroxidase has been diffracted. The location of its heme and calcium moieties have been shown and their role demonstrated. However, the structure and role of its glycans is only now being elucidated. The role of three N-linked complex glycans on cationic peroxidase (cPrx) of peanut (Arachis hypogaea L cv. Valencia), as expressed by prxPNC1 in transgenic tobacco, was analyzed by site-directed replacement of each of the three glycosylation sites, N-60, N-144, and N-185 with Q, individually. The mutant prxPNC1 cDNAs with a 3′ histidine-tag were expressed in transgenic tobacco. The effect on the catalytic ability, thermal stability, and unfolding properties of the mutant peroxidases, isolated from the medium of transgenic tobacco cell suspension cultures were compared with those of the wild cPrx from peanut. It was found that the ablation of the glycans at N-60 and N-144 influences the full expression of the cPrx catalytic ability. The glycan at N-185 is important for the thermostability, as is the removal of the carbohydrate chain at N-185, resulting in rapid enzymatic decrease at temperatures of 50°C. All three glycans appeared to influence the folding of the protein.

Glycosylation of the cationic peanut peroxidase gene expressed in transgenic tobacco

The major cationic peanut ( Arachis hypogaea) peroxidase, secreted into the extracellular space, is a glycoprotein with three N-linked glycans (polysaccharides) which are connected to the peptide backbone at Asn-60, Asn-144 and Asn-185. In this report, a C-terminal histidine-tagged cationic peanut peroxidase gene was expressed in transgenic tobacco ( Nicotiana tabacum). Tissue of the transgenic tobacco was cultured in suspension culture and the his-tagged peroxidase was purified in large quantities from 14-day-old suspension culture. The number of glycans, glycosylation sites and the chemical nature of glycan moieties attached to cationic peanut peroxidase expressed in transgenic tobacco were examined. Cationic peanut peroxidase isolated from the above transgenic tobacco had the identical number of complex glycans, attached at the same glycosylation sites as on cationic peanut peroxidase isolated from peanut suspension culture. Monosaccharide components of these glycans are N-acetylglucosamine (GlcNAc), mannose (Man), fucose (Fuc), xylose (Xyl) and galactose (Gal), the same sugars as found in native cationic peanut peroxidase.

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.

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.

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.

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

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.

Heterogeneous glycosylation of cationic peanut peroxidase.

Cationic peanut peroxidase (CPrx) from a cell suspension culture is N-glycosylated at Asn60, Asn144, and Asn185. All three N-glycans are complex type and galactose rich, and show heterogeneity in length and ConA (concanavalin A) binding property. The glycan heterogeneity causes a polymorphism of the enzyme. Based on its behavior on ConA columns, CPrx can be grouped into two fractions: nonbinding (CPrx-) and binding (CPrx+) types. A synchronously cosecreted beta-galactosidase has been discovered in the culture medium; there are two isozymes of 60 kDa (pI 7.3) and 66 kDa (pI 7.6). This beta-galactosidase has been partially purified by a combination of ion-exchange and size-exclusion chromatographies and preparative isoelectrofocusing. In vitro experiments indicate that the cosecreted beta-galactosidase is able to convert peroxidase from CPrx- to CPrx+ and may, to some extent, contribute to the glycan heterogeneity of peroxidase in the cell culture.

Cationic peanut peroxidase and the oxidation of ferulic acid

Free ferulic acid as well as ferulic acid bound to pentosans isolated from wheat flour was used in oxidation assays with highly purified peanut peroxid