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 A 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. CONCLUSIONS: 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.

Hormonal control of transcription in higher plants.

1. Nucleolar RNA polymerase Ib obtained from auxin-treated lentil roots exhibits a higher transcriptional activity than the enzyme obtained from control roots. This difference is due to a change in the enzyme properties after auxin treatment. It is suggested that the hormonal effect is mediated by a factor that changes the molecular properties of nucleolar RNA polymerase. 2. Four fractions, alpha, beta, gamma and delta, that stimulate the activity of RNA polymerase Ib, have been extracted from lentil roots. Two of them, gamma and delta have been studied. Factor delta can stimulate nucleolar polymerase Ib and the nucleoplasmic enzyme II equally well, while factor gamma is specific for polymerase Ib. 3. The curve of UMP incorporation in vitro, with and without factors gamma or delta suggests that they are initiation factors. This conclusion is reinforced by the analysis of simultaneous incorporation of [gamma-32P]ATP and [3H]UMP in the RNAs synthesized in vitro. 4. Although the level of factor delta is independent of auxin treatment, that of factor gamma is doubled in auxin-treated roots. These results suggest that factor gamma is an auxin-induced protein that modulates the specific activity of the nucleolar RNA polymerase. 5. A general model of the mode of action of auxins at the molecular level is proposed. It integrates into a unified scheme the above results as well as those obtained by other workers.

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.

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 Man alpha1-6(Xyl beta1-2)Man beta1-4GlcNAc beta1-4(Fuc alpha1-3)GlcNAc beta core but with an additional Man alpha1-3 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.

Immunoaffinity studies on cationic peanut peroxidase fraction.

Cationic peroxidase immobilized on sepharose has been used to separate specific antibodies from the antiperoxidase whole rabbit serum immunoglobulins (IgG) raised against it. The IgGs so separated completely pellet the peroxidase activity of the cationic peroxidase at a ratio of 6 : 1 (w/w). This shows the specificity of purified IgGs which is also shown by the single reaction arc obtained by immunoelectrophoresis of the isolated IgGs followed by challenging with crude peroxidase. These specific IgGs when linked to cyanogen bromide activated sepharose have been used to purify the cationic peroxidase from the spent medium proteins and from the cell extract of the peanut cells grown in suspension culture.

A study on glycosylation of cationic peanut peroxidase.

Cationic peroxidase from a cell suspension culture has two heterogeneously glycosylated forms, based on their Concanavalin A column binding property, namely CP-and CP+. In these two forms three out of five potential glycosylation sites are used. Asn60 and Asn185 are located in hydrophilic beta-turns, while Asn144 in a hydrophobic beta-sheet. Asn209 and Asn275 lacking glycans all have a proline residue at the C-terminal of the consensus sequence. The difference between CP- and CP+ is due to the heterogeneous glycosylation at individual glycosylation sites.

Peroxidase biosynthesis as part of protein synthesis by cultured peanut cells.

Membrane-bound and free ribosomes from cultured peanut cells were separated by differential centrifugation. The former ribosomes were liberated from the 27000 X g pellet with 1% Triton X100. Purification of both polysome populations was obtained by passage through a 1.5 M sucrose cushion. Both populations have a high rate of protein synthesis in vitro in the presence of a wheat germ S30 fraction. Comparisons of protein synthesis in vitro with that in vivo, and also with labelled proteins released by these cells into the medium did not provide significant information regarding peroxidase biosynthesis. However, immunoprecipitation of the products formed in vitro with antibodies raised against a purified cationic peroxidase fraction, resulted in isolating one major radioactive product. The higher molecular weight of this isolated product compared with the molecular weight of the purified peroxidase fraction suggests the occurrence of a single peptide for peroxidase commensurate with its being a secreted protein.

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.

Characterization of epitopes on the cationic peanut peroxidase by four monoclonal antibodies.

The epitope sites on the cationic peanut peroxidase were characterized by four monoclonal antibodies raised against this isozyme. Evidence is presented showing that the epitope for monoclonal antibody 1B is located on the polypeptide. Sensitivity of the epitopes recognized by 1M and 2F to 0.1M HCl, boiling, 10 mM periodate and trifluoromethane sulfonic acid treatment indicate that they occur at regions where oligosaccharides are linked to the polypeptide backbone. The antigenic specificity of 2A is, in addition, dependent on the conformation of the epitope site which is destroyed after partial proteolysis of the peroxidase.

Role of carbohydrate moieties in peanut (Arachis hypogaea) peroxidases.

The activities of a cationic (C.PRX) and an anionic peroxidase isolated from peanut (Arachis hypogaea)-cell suspension culture were drastically reduced when they were deglycosylated with glycopeptidase F or oxidized by 10 mM-periodate. In contrast with the controls, the deglycosylated or the oxidized peroxidases were much more susceptible to proteolytic degradation. In radiolabelling experiments with [35S]methionine, the non-glycosylated C.PRX was synthesized in the tunicamycin-treated cultures and secreted into the medium. Examination of the C.PRX polypeptides by SDS/polyacrylamide-gel electrophoresis followed by fluorography showed that the non-glycosylated form had an Mr of approx. 31,000, which is about 78% of that of the glycosylated form. Our results suggest that carbohydrates may not be essential for peroxidase secretion, but that stabilization of the peroxidase molecules and acquisition by these isoenzymes of a catalytically active conformation is linked directly or indirectly to glycosylation.

Immunological similarity of the cationic and the anionic peanut peroxidase isozymes.

Antibodies against both the native and the deglycosylated cationic peanut peroxidase (C.PRX) were used to probe the structural relationship of this isozyme with its anionic counterpart. Not only the native but also the deglycosylated forms of the cationic and the anionic peroxidases reacted with both antibodies. The activity of the cationic isozymes was inhibited by anti-native C.PRX. Similar but nevertheless distinct immunodetection patterns resulted from reaction of the partially digested cationic and anionic peroxidase peptides with antibodies directed to the deglycosylated as well as to the native C.PRX, suggesting a similarity in their polypeptide structures.

Polyadenylated RNA from Vicia faba meristematic root cells. Localization and size estimation of the poly (A) segment.

After incubating root apices from two-day-old bean seedlings with [3H] adenine the RNA was extracted from whole cells or polysomes, and the poly (A) sequences were isolated by nuclease digestion followed by poly(U)-Sepharose chromatography. The alterations of the RNA molecules due to the various treatments were monitored by sucrose density gradients. It was found that sequential extraction first at pH 7.6 then at pH 9.0 did not result in a separation between RNA poor in poly(A) sequences and poly(A)-rich RNA. Furthermore chromatography analysis of hydrolysates from nuclease-resistant RNA extracted either at pH 7.6 or pH 9.0 revealed that AMP constituted nearly 95% of the bases and that the poly(A) sequences, about 200 bases, were located at the 3' terminus of the polyadenylated RNA. No size difference was found for the poly(A) segment between the pH-7.6-extracted RNA and that extracted at pH 9.0.

Characterization of peroxidase in plant cells.

Two peroxidases, one anionic and one cationic, have been purified from the proteins secreted by peanut (Arachis hypogaea L. var Virginia 56R) cells in suspension culture. These two peroxidases apparently have identical catalytic properties.

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.

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 degrees C. All three glycans appeared to influence the folding of the protein.

Evidence for the presence of Mn(II) in a peanut peroxidase. An EPR study.

The characteristics of the paramagnetic centers of the main cationic isozyme of peanut peroxidase are analyzed using electron paramagnetic resonance. Two main paramagnetic species have been detected. The EPR spectrum of the native cationic peanut peroxidase shows features which correspond to those of high spin ferric heme iron. A second paramagnetic center has been identified as manganese (Mn2+). The EPR spectra of the reduced enzyme and its fluoride, cyano, carbon monoxide, thiolate and hydroxy derivatives have been studied. The signal attributed to Mn(II) disappeared after dialysis against 50 mM EDTA, which removed the Mn ions, as it was confirmed by atomic absorption spectroscopy. The disappearance of the Mn signal after the formation of the cyano, thiolate and hydroxy complexes suggest a transfer of electrons from the Mn(II) ions. The significance of manganese in the catalytic cycle of cationic peanut peroxidase discussed.

Analysis of the optical absorption and magnetic-circular-dichroism spectra of peanut peroxidase: electronic structure of a peroxidase with biochemical properties similar to those of horseradish peroxidase.

The electronic structures of the cationic isoenzyme of peanut peroxidase, horseradish peroxidase (isoenzyme C) and bovine liver catalase are compared through analysis of their optical absorption and magnetic c.d. (m.c.d.) spectral properties. The spectral data for the native resting states and compounds I and II of peanut peroxidase (PeP) are reported. The absorption and m.c.d. data for the native PeP exhibit bands characteristic of the high-spin ferric haem. The absorption spectrum of PeP compound I closely resembles that observed for the HRP compound I species. The m.c.d. data for PeP I clearly identifies that ring oxidation has occurred. One-electron reduction forms the PeP compound II species. The absorption and m.c.d. spectra recorded for PeP II exhibit the well-resolved spectral characteristics previously observed for both HRP compound II and catalase compound II. The spectral data of PeP with HRP and catalase are compared. The data clearly indicate that the m.c.d. spectral patterns of both plant peroxidases (PeP and HRP) are very similar and, therefore, the electronic structures of their resting states, and as well their primary and secondary compounds, must be similar. The m.c.d. data suggest that, while the compound I species of PeP and HRP belong to one electronic class, catalase compound I belongs to a different class. These data emphasize how the ground states of these two classes of oxidized haem, may be characterized as predominantly 2A2u (PeP I and HRP I) or 2A1u (catalase I). Peanut peroxidase is the second plant peroxidase for which the electronic structure of the compound I intermediate has been studied using the m.c.d. technique. The similarities with horseradish peroxidase allow us to suggest that plant peroxidases may operate by the same general mechanism, in spite of the low degree of sequence similarity between their polypeptide chains.