Purification, cloning and characterization of a novel peroxidase isozyme from sweetpotatoes (Ipomoea batatas)

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Biochimica et Biophysica Acta 1774 (2007) 1422 – 1430 www.elsevier.com/locate/bbapap

Purification, cloning and characterization of a novel peroxidase isozyme from sweetpotatoes (Ipomoea batatas) ☆ Annette Rompel a,⁎, Michael Albers a,b , Joseph I. Naseri a,b , Carsten Gerdemann a,b , Klaudia Büldt-Karentzopoulos a , Beate Jasper a,b , Bernt Krebs b,⁎ b

a Institut für Biochemie, Westfälische Wilhelms-Universität, Wilhelm-Klemm-Strasse 2, D-48149 Münster, Germany Institut für Anorganische und Analytische Chemie, Westfälische Wilhelms-Universität, Wilhelm-Klemm-Strasse 8, D-48149 Münster, Germany

Received 24 November 2006; received in revised form 15 August 2007; accepted 15 August 2007 Available online 28 August 2007

Abstract An anionic peroxidase from sweetpotato tubers is purified and characterized. The isozyme ibPrx15 is purified to homogeneity by affinity chromatography using a concanavalin A column. The isoelectric point was determined to pI 4.9. MALDI-MS detected a singly charged molecule with a mass of 42 029 Da. Absorption spectra of ibPrx15 compounds I, II and III were obtained after treatment with H2O2 at room temperature. Comparative data of ibPrx15 on substrate specificity to tobacco anionic peroxidase (TOP) and horseradish peroxidase (HRP) reveal similar specific activity towards a series of conventional substrates except for iodide, which is a two-electron donor interacting directly with the compound I derivative in the catalytic cycle. ibPrx15 exhibits a high specific activity towards iodide about 103-fold to that of tobacco peroxidase. The amino acid sequence of the main isozyme ibPrx15 was determined by Edman degradation and by sequencing the amplified cDNA fragments. ibPrx15 has 86% identity to another Ipomoea sequence ibPrx05 and 72% identity with a sequence from Populus trichocarpa (PtPrx72). © 2007 Elsevier B.V. All rights reserved. Keywords: Peroxidase; Sweetpotato; Ipomoea batatas; cDNA; Sequence

1. Introduction Peroxidase (Prx, EC 1.11.1.7) is a monomeric heme-containing enzyme with a molecular mass between 32 and 45 kDa [1–4]. They use H2O2 as an electron acceptor and oxidize a multitude of donor compounds. They are found widespread in nature and are expressed in eukaryotic and prokaryotic cells. The plant peroxidase superfamily can be divided into three families (classes) Abbreviations: ibPOD, peroxidase isolated from sweetpotatoes (Ipomoea batatas); TOP, tobacco peroxidase; HRP, horseradish peroxidase; rpm, rotations per minute; HPLC, high-pressure liquid chromatography; MALDI-MS, matrixassisted laser desorption/ionization mass spectrometry; Prx, peroxidase; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; fw, forward; bw, backward; TFA, trifluoroacetic acid; RZ, Reinheitszahl; ABTS, 2,2′-azino-bis-(3-ethyl-6-benzothiazolinsulphonate) ☆ The nucleotide sequence reported in this paper has been submitted to the Genebank with accession number AJ242742. ⁎ Corresponding authors. Rompel is to be contacted at Fax: +49 251 8333206. Krebs, Fax: +49 251 8338366. E-mail addresses: [email protected] (A. Rompel), [email protected] (B. Krebs). 1570-9639/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2007.08.013

[5,6]: class I peroxidases are intracellular peroxidases of prokaryotic origin, class II peroxidases consist of secreted fungal peroxidases and class III peroxidases are the higher plant peroxidases targeted for the secretory pathway. The most thoroughly studied example of a higher plant peroxidase is horseradish peroxidase (HRP) [7]. HRP catalyzes the oxidative coupling of phenolic compounds using H2O2 as the oxidizing agent [8]. The reaction is a three-step cyclic reaction in which the enzyme is first oxidized by H2O2 and then reduced in two one-electron transfer steps by reducing substrates, typically a small molecule phenol derivative. The oxidized phenolic radicals can polymerize, with the final product depending on the chemical character of the radical, the environment and the peroxidase isozyme used. The optical spectra of native HRP, compound I and compound II characterize the three steps in the enzymic reaction cycle [9]. The main features of resting state HRP are the Soret band with a maximum at 403 nm and the charge transfer bands at 498 and 643 nm. The Soret band has a shoulder at 380 nm, which is characteristic of five coordinated Fe(III) in peroxidase. The ratio of

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the absorbances at 403 nm to 280 nm is called the Reinheitszahl (RZ) number [10]. Pure enzyme preparations exhibit a RZ value between 3.0 and 3.4. On reaction with a stoichiometric amount of H2O2 the intensity of the Soret band is halved and shifted to 400 nm, characteristic for the formation of compound I. Upon formation of compound II [11] the Soret band regains the intensity seen from the resting state enzyme, but it is shifted to 420 nm. The first class III peroxidase structure to be determined was that of peanut peroxidase (PP) [12]. The crystal structure of HRP was reported in 1997 [13]. A barley grain peroxidase [14] and a soybean peroxidase [15] were reported in 1998 and 2001, respectively. Most higher plants have several Prx isozymes. They are usually classified into three subgroups: anionic, neutral and cationic peroxidases based on their isoelectric points [16]. However, their actual physiological role is still unclear. Several plant Prx cDNAs and genes have been isolated by molecular cloning. Prx sequences from sweetpotatoes are of special interest, because of its importance as a food crop. So far 10 Prx genes (6 anionic Prxs, 3 basic Prxs and 1 neutral Prx) were isolated and characterized from suspension cultures of sweetpotato in order to investigate the physiological function of Prx on the level of isoenzyme [17–20]. 2. Materials and methods 2.1. Materials All chemicals used were of analytical grade and purchased from Sigma (St. Louis, MO, USA) or Merck. The chromatographic media used were from Pharmacia Biotech (Uppsala, Sweden) and Sigma. All buffers used in this study were degassed and adjusted to their respective pH values at 20 °C. Mature tubers of sweetpotatoes, Ipomoea batatas (Bushbuck, South Africa), were purchased at a local market in the spring and purified during the early summer month.

2.2. Enzyme purification Diced potato tubers (5 kg) were homogenized in 2-l isolation buffer (50 mM NaAc, 0.1 M NaCl, 0.5% (w/v) sodium ascorbate, pH= 6.0) using a blender. In order to precipitate low molecular mass phenolic compounds 200 g polyvinylpolypyrrolidone (PVP, Sigma, P 6755) was added. The homogenate was squeezed through cheese cloth. All following precipitation steps were performed at 4 °C. Solid (NH4)2SO4 (25 g/100 ml solution) was added to the extract to 35% saturation. After 30 min the precipitate was removed by centrifugation (10 000 rpm, 4 °C, 90 min) and (NH4)2SO4 was added to the supernatant to 85% saturation. The precipitate was collected by centrifugation (10000 rpm, 4 °C, 90 min) after 60 min and redissolved in 2 l of isolation buffer. (NH4)2SO4 was added to the solution up to 28% saturation prior to lowering the pH to 4.0 by adding 2 N HCl. After stirring for 10 min, the precipitate was removed by centrifugation (14 000 rpm, 4 °C, 20 min). The resulting supernatant was adjusted to pH 6.0 by 0.2 M NaOH and brought to 85% saturation by the stepwise addition of (NH4)2SO4. After 60 min a browncolored pellet was collected by centrifugation and redissolved in 50 mM NaAc buffer, pH 6.0, containing 0.5 M NaCl. This solution was loaded on a sephacryl S-200 HR column (5 × 100 cm, flow rate 5 ml/min) equilibrated with the same buffer. Column eluants were monitored at 280 nm and 405 nm. The fractions showing peroxidase activity were pooled and concentrated with an ultrafiltration cell. The active fractions from sephacryl S-200 were equilibrated with 0.02 M NaAc pH 4.5 and loaded onto a sepharose fast flow (S-FF) pre-equilibrated with 0.02 M NaAc pH 4.5, and eluted with a salt gradient of 0–1 M NaCl in 0.02 M NaAc pH 4.5. Two separate protein fractions showing peroxidase activity with different matrix-binding characteristics were collected and purified separately using the following procedure.

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Each fraction was desalted using 0.02 M TEA/HCl pH 9.0 and applied to a pre-equilibrated DEAE-FF sepharose column (0.02 M TEA/HCl pH 9.0). Elution was initially performed using the equilibration buffer followed by a salt gradient of 0–1 M NaCl. After concentration by ultrafiltration, the pooled fractions were further purified under similar conditions (0.02 M TEA/HCl pH 9.0 with a salt gradient of 0–1 M NaCl) using a mono Q column. The final purification step was achieved using a concanavalin A column equilibrated with 0.5 M NaCl, 0.01 M BisTris, pH 7.0. The bound proteins were eluted with a linear gradient of 0–0.3 M methyl-α-D-mannopyranosid. Fractions containing peroxidase activity were pooled, dialyzed against 0.5 M NaCl, 0.05 M NaAc, pH 6.0 and concentrated by ultrafiltration with membrane cones (Amicon CF 25) and stored at 4 °C for several months without considerable loss of activity.

2.3. Enzyme assays Peroxidase activity was measured at a substrate concentration of 5 mM guaiacol and 0.5 mM H2O2 in 0.05 M sodium phosphate pH = 6.5 at 20 °C by monitoring the increase of absorbance at 470 nm [21]. One unit (U) of enzyme activity was defined as the amount, which increased the absorbance by 1 per minute and corresponds to 1.9 × 10− 4 mg/ml protein. To determine the pH optimum of ibPrx15, activity was expressed using 0.36 mM ABTS and 5.88 mM H2O2 at 25 °C in 50 mM sodium citrate buffer. The following buffers were used: 50 mM sodium citrate (pH = 2.75–4.75), 50 mM potassium phosphate (pH = 5.0–9.0). An absorption coefficient at 405 nm of 36.8 mM− 1 cm− 1 was used [22]. The substrate specificity of ibPrx15 was compared to the one of tobacco peroxidase and horseradish peroxidase [23] with the following substrates: 2.25 mM guaiacol and 5.88 mM H2O2 in 0.1 M potassium phosphate, pH 6.0, absorption coefficient at 470 nm, 5.57 mM− 1 cm− 1 [24]; 0.127 mM o-dianisidine and 5.88 mM H2O2 in 0.1 M potassium phosphate, pH 7.0, absorption coefficient at 460 nm, 30.0 mM− 1 cm− 1 [25]; 1.68 mM potassium iodide and 5.88 mM H2O2 in 0.1 M sodium acetate, pH 5.0, absorption coefficient at 353 nm, 25.5 mM− 1 cm− 1 [26]. The concentration and the extinction coefficients at 280 nm of the pure proteins were determined using the Bradford method [27]. The Warburg and Christian method [28] was used for the estimation of protein concentrations in partially purified protein solutions.

2.4. Analytical and spectroscopic methods SDS-PAGE (12%, polyacrylamide) was performed according to a modification of the Laemmli method [29]. Molecular mass markers ranging from 14 400 to 94 000 Da (Pharmacia Biotech, Sweden) and Coomassie staining were used. The isoelectric point was determined by analytical IEF Phast System electrophoresis (Pharmacia Biotech, Sweden) using the pI calibration kit from Pharmacia as marker. Staining of the IEF was performed using Coomassie Brilliant Blue R 250. MALDI-MS was performed on a reflectron-type time-of-flight mass spectrometer (Lamma 1000, Leybold Heraeus) according to Hillenkamp et al. [30,31] by S. Hahner, Institut für Medizinische Physik, Universität Münster. The concentration of the protein was 3 × 10− 5 M in double-distilled water. 0.01 M 2,5-dihydroxybenzoic acid was used as ultraviolet-absorbing matrix. The laser emission wavelength was 337 nm. The NH2-terminal sequences of desalted native protein samples were determined using automated Edman degradation (B. Schedding, Inst. für Physiologische Chemie, Universität Münster). Absorption spectra of native ibPrx15 as well as spectra obtained after the addition of H2O2 were monitored at room temperature in 50 mM sodium citrate pH 3.6 using a Shimadzu UV-1601PC spectrophotometer interfaced to a personal computer.

2.5. Primary structure analysis of ibPOD isoforms 2.5.1. BrCN cleavage 1 mg of highly purified and salt-free enzyme in 500 μl formic acid (70%) was incubated with 0.5 mg BrCN (∼ 50 times excess to methionine residues) for 24 h in the dark [32]. The sample was taken up in 500 μl water and lyophilized. This step was repeated for total extraction of excess BrCN. Disulfide bonds were not reduced before or after cleavage.

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Fig. 1. MALDI-MS of ibPrx15, exhibiting the (M + H)+, (M + 2H)++ and (M + 3H)+++ mass peak. 2.5.2. Glu-C cleavage 1 mg of highly purified and salt-free enzyme in 500 μl ammonium carbonate buffer (pH 8.0) was incubated with 7 U endoproteinase Glu-C from Staphylococcus aureus for 48 h at 37 °C [24]. The sample was taken up in 500 μl water and dithiothreitol to cleave disulfide bonds. 2.5.3. Peptide mapping The cleavage products were separated by reversed-phase HPLC (Kontron) using a rp-C8 column (10 μm Aquapore RP 300; 4.6 × 100 mm). The sample volume was 250 μl and peptides were eluted at a flow rate of 1 ml/min while a gradient of solvent B (0.1% TFA in acetonitrile) 0% for 5 min, 0–20% for 5 min 20–50 for 45 min and 50–100% for 5 min, respectively in solvent A (0.1% TFA in water) was applied. Peptides obtained were rechromatographed under identical conditions. 2.5.4. Sequencing of the peptides Peptide sequences were determined by automated Edman degradation using an Applied Biosystems Model 476A protein sequencer. 2.5.5. Molecular mass determination MALDI-MS was performed as described under 2.4. Theoretical masses of BrCN cleavage fragments and of native proteins were calculated using the PEPTIDE MASS tool [33] and the PROTPARAM tool of the ExPASy proteomic tools program package.

2.6. Preparation of mRNA and cDNA

applied: (1) 94 °C for 3 min, (2) 50–60 °C for 30 s, (3) 72 °C for 1.5 min, (4) 94 °C for 30 s, steps 2–4 were repeated 20–40 times, and the final step (5) was 72 °C for 10 min. Reaction products were analyzed by agarose gel electrophoresis, amplicons purified with Easy Pure DNA Purification Kit (Biozym) and ligated into pGEM T-Easy vector (Promega) by TA cloning. After transformation of E. coli DH5α or E. coli TOP10 host with this vector, insertcontaining clones were identified by X-Gal staining and restriction analysis. Plasmid DNA was purified after bulk growth using the S.N.A.P. plasmid isolation kit (Invitrogen) and sequenced externally (TOPLAB, Martinsried).

2.8. Sequence data analysis Evaluation of the sequence data, database searches and alignment of nuclear and amino acid sequences were carried out using the ‘GCG’ program package of HUSAR and UK Human Genome Project and the ‘MULTALIN’ program [36].

3. Results and discussion 3.1. Enzyme purification Sweetpotato peroxidase ibPrx15 was purified to homogeneity through a combination of precipitation, size exclusion, anion exchange and affinity chromatography. The enzyme was detected

Total RNA from 3 g sweetpotato root tissue was isolated as described by others [34,35]. The Oligotex mRNA-Kit (Qiagen) was used to isolate mRNA by affinity chromatography on oligo (dT)-cellulose from about 5 μg total RNA under conditions given by the manufacturer. About 150 ng mRNA was used for the synthesis of single-stranded cDNA using the reverse transcriptase Superscript II (GIBCO) or MMLV (Boehringer Mannheim) according to the manufacturer's instructions. A random primer mix and NotI-dT18 primer (Table 2) were applied according to instructions for the first-strand kit of Pharmacia.

2.7. Amplification and cloning of PCR products All primers used are shown in Table 2. Names of the primers derive from the amino acid translation of the primer region. Directions of the primers (fw, bw) are given in brackets. cDNA in concentrations of one-tenth of the first-strand solutions served as templates for amplification by PCR, with degenerated primers designed based on the amino acid information. The DNA fragments obtained furnished the blueprint to design specific primers. 3′RACE was performed for both isozymes with adapter-primed first-strand. PCR was carried out with a standard reaction mixture, which included 0.2 mM dNTPs and 0.5 to 1 U DNA polymerase per 50 μl reaction volume. DNA polymerases Biotherm (Genecraft) and Expand (Boehringer) were applied with buffers furnished by the manufacturer plus 1.5 mM MgCl2. The following temperature program was

Fig. 2. Isoelectric focusing of ibPrx15. Lane 1: ibPrx15, lane M: marker.

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Table 1 Molecular properties and substrate specificity of sweetpotato peroxidase (ibPrx15), tobacco anionic peroxidase (TOP) and horseradish peroxidase (HRP) according to Gazaryan and Lagrimini [23] Property

ibPrx15

TOP

HRP

Mr (kDa) pI RZ Specific activity ABTS (U/mg) Relative activity (%) ABTS Relative activity (%) Guaiacol Relative activity (%) o-Dianisidine Relative activity (%) Iodide

42 4.9 3.9 4545 ± 200 100 74 ± 6 90 ± 20 15 ± 5

36 3.5 3.4 4600 ± 200 100 85 ± 2 130 ± 5 0.07 ± 0.005

44 8.9 2.0 2700 ± 100 100 170 ± 10 145 ± 5 10 ± 0.5

by heme absorbance at 408 nm and the enzyme activity using guaiacol as the substrate was used as the basis of selection on each step. The final product represented approximately 10% of the original (total) activity. No general purification scheme is shown for two reasons: no enzyme activity could be measured during the first purification steps due to the presence of sodium ascorbate. Secondly, very different ratios of the isozymes and strongly varying total amounts of ibPrx15 were found depending on the season in which the potato tubers were harvested. During the first column (sephacryl, S 200 chromatography), several smaller and larger proteins among those a purple acid phosphatase [37,38] could be separated. ibPrx15 does not bind to the sepharose FF column. This second column is very important, since a second peroxidase ibPOD2 (not further investigated) could be separated. The third column, DEAE sepharose, separated ibPrx15 from foreign proteins. Further purification on the strong ion exchange resin mono Q resulted to the separation of a third isozyme (ibPOD3), which was not further investigated. IbPrx15 was purified to homogeneity using a concanavalin A column. The purification procedure led to electrophoretically homogeneous preparations of the isozyme ibPrx15, as stated by MALDI-MS (Fig. 1). On average more than 50 mg of pure ibPrx15 was obtained in total from 100 kg mature tubers. 3.2. Molecular mass, subunit composition, and isoelectric focusing

Fig. 4. UV/Vis spectra of the native ibPrx15 (A) and its compound I (B) and compound II (C) derivatives in 0.05 M sodium citrate buffer, pH 3.6, in the presence of (B) 1- and (C) 10-fold excess of hydrogen peroxide.

around 40 kDa. MALDI-MS analysis however reveals a difference in molecular mass. Singly charged molecule with Mr of 42 029 Da ibPrx15is detected (Fig. 1). Molecular mass investigations on HRP showed a molecular mass range from 42 200 to 44 000 Da [39]. Furthermore a MALDI-MS spectrum of ibPrx15 was taken from the low molecular weight region. A peak with Mr of 617 Da was obtained characteristic for the iron protoporphyrin IX molecule (data not shown). The IEF of ibPrx15 (Fig. 2) exhibits three bands at pI = 4.0, 4.3 and 4.9. The appearance of these three bands can be explained due to different phosphorylation and glycolysation of ibPrx15. According to amino acid composition, the theoretical pI is 5.07. 3.3. Substrate specificity, and pH dependence of enzymatic activity Comparative data on substrate specificity are reported in Table 1 and compared to tobacco anionic peroxidase and horseradish peroxidase [15,23]. ibPrx15 shows a comparably high specific activity towards ABTS as tobacco peroxidase, but the former is much more active towards iodide, which is a two-electron donor interacting directly with compound I in the catalytic cycle. Previous

Denaturing SDS-PAGE (12%) of reduced and heated (100 °C, 5 min) samples showed a single band for ibPrx15

Fig. 3. The UV/Vis spectrum of native ibPrx15.

Fig. 5. Stability of the ibPrx15 compound III: (A) UV/Vis spectrum of the native ibPrx15; (B) compound III spectrum obtained in the presence of 500-fold excess of hydrogen peroxide; (C–F) the spectral changes observed 5, 10, 20 and 40 min later, respectively; incubation at 25 °C.

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Fig. 6. Nucleotide and deduced amino acid sequences of the cDNA of ibPrx15. Positive numbering of amino acids starts at the N-terminal sequence determined by Edman degradation with Ser and overlaps with the deduced amino acid sequence of cDNA of ibPOD from position five. Amino acid sequences directly determined are bold. The stop codon is indicated by ⁎⁎⁎. Potential N-linked glycosylation sites are underlined. The region 504 to 535 is documented by two overlapping primers (“anqgga.fw” and “nqggantslp.bw”).

studies also report a comparably high catalytic activity of horseradish peroxidase towards iodide [15]. The low specific activity of tobacco peroxidase towards iodide explains a higher stability of its compound I [23] than those of HRP and ibPrx15. IbPrx15 has its pH optimum at 5.5 exhibiting a bell-shaped curve. More than 50% of activity is present in the pH range between 4 and 7. The specific activity of ibPrx15 towards ABTS in 0.05 M sodium citrate buffer at pH 3.6, which has been shown to be

optimal for ibPrx15, was 4600 U mg− 1 of protein (see also Table 1). IbPrx15 is stable in 0.1 M phosphate buffer at pH = 5.5 at 4 °C for months without loss of activity. 3.4. Spectroscopic properties The UV/Vis spectrum of ibPrx15 is presented in Fig. 3. The absorption spectrum of native ibPrx15 is characterized by a Soret maximum at 408 nm and absorption maxima in the visible

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region at 497 and 635 nm also called Q-bands. A Reinheitszahl value (ratio of heme A405 to protein A280) of 3.9 was obtained. Typical values reported for purified peroxidases range from 1.6 to 4.1 with most in the 2.6 to 3.3 range [40]. The value 3.9 again supports the high purity of the isolated peroxidase. The absorption spectra of ibPrx15 (Fig. 4, curve A) after treatment with stoichiometric amounts of H2O2 convert the enzyme predominantly to its compound I form, as shown in Fig. 4, curve B. The intensity of the Soret band decreases to 40% and the maximum of the band is shifted to 400 nm. In addition a shoulder at 350 nm and absorbances at 531 nm, 556 nm, and 663 nm are seen in the visible region. The presence of the twin peaks at 531 nm and 556 nm after peroxide treatment suggests that some amount of compound II is also present. For anionic tobacco peroxidase hydrogen peroxide addition at 3- to 300-fold excess leads to a characteristic 350-nm shoulder in the Soret peak and absorbances at 554 and 655 nm. HRP reacts with equimolar amounts of hydrogen peroxide to form a mixture of compounds I and II independent of the peroxide concentration [41]. A mixture of compounds I (85%) and II (15%) is also formed by petunia Prx [42]. After the addition of 10-fold excess of hydrogen peroxide at pH 3.55, the intensity of the Soret band was only about 35% of that of the native ibPrx15 (Fig. 4, curve C). Its Soret region peak occurred at 407 nm and the traditional twin peaks at 531 nm and 556 nm, based on the formation of compound II. For petunia Prx [42] the twin peaks are found at 528 nm and 552 nm. At high ratios of hydrogen peroxide to enzyme, (500-fold) compound III of ibPrx15 is formed (see Fig. 5, curve B). Compound III of ibPrx15 reveals a Soret band at 415.5 nm and two characteristic peaks at 531 nm and 556 nm in the visible region after a 5-min incubation period. Inactivation occurs via a decrease of the Soret band (Fig. 5 curves B to F) and an appearance of an absorption of the so-called compound P-670 nm characteristic for HRP compound III decomposition. Within 40 min the RZ dropped to 1.0. A similar spectroscopic behavior has been observed for the formation and decomposition of compound III of tobacco anionic peroxidase [23].

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3.4.1. Sequencing of cleavage fragments ibPrx15 was cleaved with BrCN and the resulting fragments were chromatographed using HPLC. The only fragment eluted as a distinct peak could be detected and collected after 15.6 min. This fragment was sequenced using Edman degradation: (M) GNISPLTGSNGEIRSN(C)RRPN. The mass of the fragment (2244.1 Da) was measured using MALDI-MS. The calculated mass of 2242.4 Da derived by the sequence data fitted well to the measured value. As there was no homoserine detected at the C-terminus of the peptide the fragment seems to be the C-terminus of the enzyme. Additional amino acid data were derived by two Glu-C fragments (detected after 26.4 min and 20.6 min). The sequence of the 26.4-min fragment could be derived as SSVSLAGGPSWNVLLGRRDGRTANQGGA(N)TSLP. The 20.6-min fragment showed two sequences parallelly sequenced: MINMGNISPL and KDALP(N)TNST. Amino acid sequence of the purified cleavage products is also presented bold in Fig. 6. No differences between the peptide sequences and the cDNA derived sequence could be observed. 3.4.2. Amplification of ibPrx15 encoding cDNA cDNA of ibPrx15 was amplified by PCR of two internal fragments. The first fragment was amplified using “anqgga” (fw) and “mgnisp” (bw) (Table 2) as degenerated primers and the anchor-primed pool as template. The PCR product could be amplified with 40 cycles at an annealing temperature of 43 °C and had the expected size of 500 bp. The second fragment could be amplified with the gene-specific bw-primer “vglnvn” synthesised on the basis of the sequence data derived from the first fragment. The fw-primer was the highly degenerated primer “fysttcp” synthesised on the basis of a region conserved in known peroxidases of higher plants, as amino acid sequence data were not available for the N-terminal region of ibPrx15. The 500-bp PCR product was amplified with an annealing temperature of 45 °C and after 50 cycles. 3′RACE was performed using the genespecific primer “vglnvn” (fw) and the anchor primer. The product

Table 2 Primers used in PCR reactions Primer

Sequence

Degeneration

Position in ibPrx15 a

anqgga.fw fysttcp.fw mgnisp.bw gesamt.bw gesamt.fw niktav.bw nqggantslp.bw sanqta.fw vglnvn.bw vglnvn.fw Adaptor 5′-CDS Adaptor-dT18 NUP SMART II UPM-MIX

5′-GCNAAYCARGGNGGNGC-3′ 5′-TTYTAYGAYRVNACNTGYCC-3′ 5′-GGRCTDATRTTNCCCAT-3′ 5′-TTCCATTAAACAAAT TACATATTGAAAATG-3′ 5′-ATCACCCACTTAGG CATTAATTATTAC-3′ 5′-AACCGCGGTCTTAATGTT-3′ 5′-GGAAGACTAGTGT TAGCTCCTCCCTGG-3′ 5′-CAGTGCAAATCAAACTGCC-3′ 5′-CATTAACATTCAATCCAACG-3′ 5′-CGTTGGATTGAATGTTAATG-3′ 5′-AACTGGAAGAATTCGCGG-3′ 5′-(T)25VN-3′ 5′-AACTGGAAGAATTCG CGGCCGCAGGAA(T)18-3′ 5′-AAGCAGTGGTA ACAACGCAGAGT-3′ 5′-AAGCAGTGGTAACAA CGCAGAGTACGCGGG-3′ 5′-CTAATACGACTCACTATAGGGC AAGCAGTGGTAAC AACGCAGAGT-3′ and 5′-CTAATACGACTC ACTATAGGGC-3′

256 1536 48

504–520 132–151 963–979 1250–1279 1–27 363–380 509–535 911–919 581–600 581–600

a

Positions relate to the ibPrx15 sequence shown in Fig. 6.

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Fig. 7. Comparison of the amino acid sequences of ibPrx15 with another sweetpotato sequence Ipomoea batatas ibPrx05, Populus trichocarpa (PtPrx72) horseradish (Armoracia rusticana, AruPrx01a), peanut (Arachis hypogaea, AhPrx04), cress (Arabidopsis thaliana, AtPrx22), tobacco (Nicotiana tabacum; NtPrx09). The numbering starts with the predicted N-terminal amino acids of ibPrx15. Identical residues of the Prxs are presented in bold.

was amplified at an annealing temperature of 49 °C and with 35 cycles. The product was used as a template for the semi-nested PCR reaction. This was reaction was performed at 49 °C with 35 cycles using the gene-specific primer “sanqta” (fw) and the anchor primer. A PCR product of about 350 bp was detected. Finally, 5′RACE reaction was performed using the ‘SMART RACE’ kit (Clontech) as described by the manufacturer using 40 cycles and an annealing temperature of 60 °C. Primers used were “nqggantslp” (bw) as gene-specific bw primer and the “UPM” anchor primer mix supplied by the manufacturer. The PCR product was used as template for the semi-nested PCR

using “niktav” (bw) as gene-specific primer and the supplied “NUP” primer as anchor primer. The PCR program included 28 cycles and an annealing temperature of 61 °C. A 5′RACE product of 380 bp was amplified, cloned and sequenced. The sequence data of the mRNA of ibPrx15 are presented in Fig. 6 and published in the EMBL database (accession number AJ242742). 3′RACE with the primers revealed a second ibPOD sequence not identical to the one described here nor to any of the Prxs of sweetpotato cell culture published before. This partial sequence is available in the EMBL database under accession number ACCNR2.

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3.4.3. Comparison to other Prxs from higher plants The sequence alignment of ibPrx15 with the sequences of other class III Prxs is presented in Fig. 7. IbPrx15 has 86% identity to another Ipomoea sequence ibPrx05 and 72% identity with a sequence from Populus trichocarpa (PtPrx72) using (http://peroxidase.isb-sib.ch/listing.php?action=view&id=296) [43]. Sequence identity to anionic Prx from horseradish (Armoracia rusticana; perb_armru, AruPrx01a) (53%), tobacco (Nicotiana tabacum; perx_tobac, NtPrx09) (50.5%) and cationic Prx from peanut (Arachis hypogaea; per1_arahy, AhPrx04) (49%) is high. 3.5. The possible role of a Glu residue Gazaryan and Lagrimini [23] proposed a possible role of a glutamate residue (Glu 141 in tobacco peroxidase) for the enzyme reactivity towards donor substrates. According to that, Glu would affect both the accessibility of the active center for electron donors and the stability of compound I. The substrate to reveal the enzymatic difference between horseradish Prx and tobacco Prx most clearly is iodide according to Gazaryan and Lagramini [23]. Glu 141 would reduce enzymatic activity of Prx towards iodide. Therefore only low activity towards iodide could be detected for tobacco Prx. In contrast, horseradish Prx shows increased activity towards this substrate due to a phenylalanine residue (Phe 143) instead of Glu on this position. However, this proposed role of Glu 141 in catalysis cannot be confirmed by the results derived for ibPrx15. IbPrx15 shows an activity towards iodide comparable to horseradish Prx (if not even stronger), thus a Phe residue or a comparable residue would be expected at the respective sequence position to confirm the abovementioned proposal. Instead the primary structure of ibPrx15 shows a glutamate residue at this position as in the case with tobacco Prx. Therefore Glu 141 is unlikely to be responsible for the low activity of tobacco Prx towards iodide, since all three Prxs can be expected to be similarly folded. Acknowledgements Financial support by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie is gratefully acknowledged. We thank B. Schedding (Inst. für Physiologische Chemie, Universität Münster) for amino acid sequencing and S. Hahner (Inst. für Medizinische Physik, Universität Münster) for performing MALDI measurements. References [1] R.H. Kenten, P.J.G. Mann, Simple method for the preparation of horseradish peroxidase, Biochem. J. 57 (1954) 347–348. [2] M. Morrison, H.B. Hamilton, E. Stotz, The isolation and purification of lactoperoxidase by ion exchange chromatography, J. Biol. Chem. 228 (1957) 767–776. [3] K.G. Paul, Peroxidases, in: P.D. Boyer, H. Lardy, K. Myrbäck (Eds.), 2nd ed., The Enzymes, vol. 8, Academic Press, New York, 1963, pp. 227–274. [4] K. Tagawa, M. Shin, K. Okunuki, Peroxidases from wheat germ, Nature (Lond.) 183 (1959) 111. [5] K.G. Welinder, Superfamily of plant, fungal and bacterial peroxidases, Curr. Opin. Struct. Biol. 2 (1992) 388–393.

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