Biochemical characterization of the major sorghum grain peroxidase Mamoudou H. Dicko 1,2,3 , Harry Gruppen 2 , Riet Hilhorst 1, *, Alphons G. J. Voragen 2 and Willem J. H. van Berkel 1 1 Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, The Netherlands 2 Laboratory of Food Chemistry, Department of Agrotechnology and Food Sciences, Wageningen University, The Netherlands 3 Laboratoire de Biochimie, CRSBAN, UFR-SVT, Universite ´ de Ouagadougou, Burkina Faso Keywords glycoform; hemeprotein; isoenzyme; peroxidase; sorghum Correspondence M. H. Dicko, Laboratoire de Biochimie, CRSBAN, UFR-SVT, Universite de Ouagadougou, 03 BP. 7021, Ouagadougou 03, Burkina Faso Fax: +226 50337373 Tel: +226 70272643 E-mail: mdicko@univ-ouaga.bf W. J. H. van Berkel, Laboratory of Biochemistry, Department of Agrotechnology and Food Sciences, Wageningen University, PO Box 8128, 6700 ET Wageningen, The Netherlands Fax: +31 317484801 Tel: +31 317482861 E-mail: willem.vanberkel@wur.nl *Present address PamGene, PO Box 1345, 5200 BJ’s-Hertogenbosch, The Netherlands Database Sequence data for sorghum peroxidase described here has been submitted to the UnitProt knowledgebase under the accession number P84516 (Received 2 February 2006, revised 18 March 2006, accepted 22 March 2006) doi:10.1111/j.1742-4658.2006.05243.x The major cationic peroxidase in sorghum grain (SPC4) , which is ubiqui- tously present in all sorghum varieties was purified to apparent homogen- eity, and found to be a highly basic protein (pI $ 11). MS analysis showed that SPC4 consists of two glycoforms with molecular masses of 34227 and 35629 Da and it contains a type-b heme. Chemical deglycosylation allowed to estimate sugar contents of 3.0% and 6.7% (w ⁄ w) in glycoform I and II, respectively, and a mass of the apoprotein of 33 246 Da. High performance anion exchange chromatography allowed to determine the carbohydrate constituents of the polysaccharide chains. The N-terminal sequence of SPC4 is not blocked by pyroglutamate. MS analysis showed that six pep- tides, including the N-terminal sequence of SPC4 matched with the predic- ted tryptic peptides of gene indice TC102191 of sorghum chromosome 1, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4, including a signal peptide of 31 amino acids. The N-terminal frag- ment of SPC4 (213 amino acids) has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%) and maize peroxidase (58%), indicative for a common ancestor. SPC4 is activated by calcium ions. Ca 2+ binding increased the protein conformational stability by rais- ing the melting temperature (T m ) from 67 to 82 °C. SPC4 catalyzed the oxidation of a wide range of aromatic substrates, being catalytically more efficient with hydroxycinnamates than with tyrosine derivatives. In spite of the conserved active sites, SPC4 differs from BP1 in being active with aro- matic compounds above pH 5. Abbreviations ABTS, 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic acid); BP1, barley peroxidase isoenzyme-1; HPAEC, high performance anion exchange chromatography; HRP C, horseradish peroxidase isoenzyme C; GlcNAc, N-acetyl-glucosamine; SPC4, major sorghum cationic peroxidase; TFMS, trifluoromethanesulfonic acid. FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS 2293 Plant secretory peroxidases (donor: hydrogen peroxide oxidoreductase, EC 1.11.1.7) are class III peroxidases that contain a Fe III –protoporphyrin-IX as the pros- thetic group linked to a proximal His residue. They catalyze the conversion of a large number of sub- strates, notably phenolic compounds for biosynthetic and catabolic functions. In general, they use hydrogen peroxide as electron acceptor [1]. Multigene families of peroxidases exist, and in the genomes of rice (Oryza sativa) and thale cress (Arabidopsis thaliania) up to 138 and 73 of peroxidase genes, respectively, were discov- ered [2,3]. Moreover, the ongoing project of sorghum genome sequencing has allowed us to currently iden- tify 160 stretches of sorghum peroxidase genes (http:// peroxidase.isb-sib.ch/index.php). The physiological functions of peroxidases are associated with defense mechanisms, auxin metabolism and the biosynthesis of cell-wall polymers such as lignin and suberin [1,4,5]. Most peroxidases are glycoproteins occurring in dif- ferent glycoforms, which may contain different glycan chains [4]. For instance, barley peroxidase (BP1) con- sists of two forms; one glycosylated at Asn300 (BP1a) and the other (BP1b) nonglycosylated [6,7]. The major glycan chain in BP1a represents 70% of the total carbo- hydrate content and has as structure Mana1–6(Xylb1– 2)Manb1–4GlcNAcb1–4(Fuca1–3)GlcNAc [6]. Next to iron, Ca 2+ is an important metal cofactor of heme per- oxidases. Class III peroxidases are known to contain two distinct Ca 2+ -binding sites, one localized on the proximal side and the other on the distal side of the heme. Ca 2+ both modulates the enzyme activity and stability [8]. Cereal peroxidases hitherto characterized are from barley [6], wheat [9], rice [10], and maize [11]. All these enzymes are monomers with molecular masses ranging from 35 to 40 kDa. The crystal structure of BP1, with two helical domains and four disulfide bridges (C18- C99, C51-C56, C106-C301 and C186-C213) is highly similar to the structure of the archetypical horseradish peroxidase (HRP C). Although BP1 shares structural similarities and catalytic properties with HRP C, its behavior is atypical, as it is unable to form compound I at pH values greater than 5 [7]. Relatively little is known about the structure and properties of sorghum peroxidase [Sorghum bicolor (L) Moench]. Sorghum is the fifth most important cereal crop in the world after wheat, rice, maize, and barley. Properties of a crude sorghum peroxidase preparation such as pI (9–10) and molecular mass (43 kDa) have been reported [12]. However, until now no sorghum grain peroxidase has been purified to homogeneity and characterized. When screening for peroxidase activity in the seeds of 50 sorghum varieties originating from different parts of the world, the cationic peroxidase was ubiquitously present in all varieties [13,14]. It was also the most abundant isoenzyme in both ungerminated and germinated sorghum grains [14]. In other cereals, the cationic isoenzymes are also the most abundant enzymes and account for more than 80% of total activity [6,15]. In recent years, it has been shown that cationic per- oxidases are more active with phenolic compounds than anionic peroxidases and laccases [16]. Thus, cationic peroxidases may be of interest for biocatalytic applica- tions such as the production of useful polymers, the treatment of waste water streams polluted with toxic aromatic compounds, and various other clinical and biotechnological applications [17]. Cationic peroxidases may also find interest in food biotechnology by modifi- cation of functional properties of food proteins and carbohydrates [18,19]. The other reason to characterize the peroxidase from sorghum is the fact that during food preparation, the peroxidase present could have a large effect on the properties of the prepared foods (beer, porridge, couscous, etc.) [14,18,19]. The resulting oxidation products have effects on human health. Therefore, knowledge of biochemical properties of the major peroxidase can help on sorghum processing. In this study, we have purified and characterized the cationic peroxidase isoenzyme from sorghum grain. Results and discussion Purification of major peroxidase from sorghum seed At least four sorghum peroxidase cationic isoenzymes, denoted SPC1, SPC2, SPC3 and SPC4, according to their order of elution, could be distinguished and separ- ated by the Mono-S cation exchange chromatographic step (Fig. 1A). SPC4 was by far the most abundant iso- enzyme. Zymography (Fig. 2A) showed that this enzyme has an experimental pI value > 9. Three inde- pendent repetitions of all purification steps were per- formed to confirm the profile and abundance of isoenzymes within sorghum grain. The purification by three chromatographic steps resulted in a final enrich- ment of SPC4 by 105-fold, with an activity yield of 28% (Table 1). The purity of SPC4 was assessed by the single protein band obtained by SDS ⁄ PAGE (Fig. 2B) and the high RZ value (4.0). The purification of SPC4 is sum- marized in Table 1. The final specific activity of SPC4 for the H 2 O 2 -dependent oxidation of ABTS was 1071 UÆmg )1 . The purified enzyme was soluble in aque- ous acetone, methanol and ethanol up to proportions of 40% (v ⁄ v) of organic solvent. The enzyme eluted from a Superdex G 75 column in one symmetrical peak with an Characterization of sorghum peroxidase M. H. Dicko et al. 2294 FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS apparent mass of 32 kDa (Fig. 1B). Together with the molecular masses obtained by SDS ⁄ PAGE (38 kDa, Fig. 2B) and MALDI-TOF-MS (34283–35631 Da, Fig. 3A), this shows that SPC4 is a monomer. Carbohydrate composition MALDI-TOF-MS analysis revealed that SPC4 consists of two species with masses of 34 283 and 35 631 Da, respectively (Fig. 3A). Chemical deglycosylation of the enzyme yielded a single protein peak with a mass of 33 449 Da (Fig. 3B). This indicates that the hetero- geneity of the enzyme is exclusively related to its glycan composition and that SPC4 has two glycoforms. For convenience, the species with a mass of 34 283 Da is further referred to as glycoform I and the species with a mass of 35 631 Da as glycoform II. The chemical deglycosylation was not complete because it leaves one unit of GlcNAc (203 Da) remaining on the polypeptide chain at each attachment site [20]. Thus, the molecular mass of fully deglycosylated SPC4 is at most 33 246 Da. The sugar contents estimated by MALDI-TOF-MS are 3.0% and 6.7% in glycoform I and II, respectively. Carbohydrate analysis of SPC4 by HPAEC showed an average carbohydrate content of approximately 5.4% (Table 2). From the overall sugar content (HPAEC) and the estimated sugar contents of the indi- vidual glycoforms (MALDI-TOF-MS), the proportions of glycoforms I and II can be calculated to be 35 and 65%, respectively. HPAEC analysis showed that the main sugar constituents of the glycan chains are fucose, mannose, xylose, and N-acetylglucosamine (Table 2). MALDI-TOF-MS analysis of HRP C as positive control showed masses of the native and deglycosylat- ed form of 43 663 Da and 35 505 Da, respectively (Fig. 3C,D). Since HRP C has eight glycan chains [21], at least 8 GlcNAc residues will remain after chemical deglycosylation. Thus, the fully deglycosylated HRP C Fig. 1. Purification of cationic isoforms of sorghum peroxidase. (A) Mono-S cation exchange chromatography: peroxidase activity (o), absorbance at 280 nm (—), absorbance at 403 nm (- - -), and 0–1 M NaCl gradient (—). (B) Elution profile of Mono S purified SPC4 on Superdex 75 PG. Fig. 2. Zymogram and SDS ⁄ PAGE of major cationic sorghum peroxidase. (A) Zymogra- phy: lane 1, crude extract and lane 2, purif- ied SPC4. (B) SDS ⁄ PAGE of purification steps of SPC4: lane M, marker proteins; lane 1, crude extract; lane 2, acetone precip- itate; lane 3, preparative Superdex 75 frac- tion; lane 4, unbound Resource-Q fraction; lane 5, Mono-S fraction; lane 6, analytical Superdex 75 fraction. Table 1. Purification of the major sorghum peroxidase. Step Total activity (U) Total protein (mg) Specific activity (UÆmg )1 ) Yield (%) Crude extract 10 710 1050 10 100 Acetone fraction 7497 407 18 70 Superdex 75 5890 200 29 55 Resource-Q 4820 12.7 379 45 Mono-S 2998 2.8 1071 28 M. H. Dicko et al. Characterization of sorghum peroxidase FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS 2295 would have a mass of 33 881 Da (35 505–203 · 8 Da), which is in good agreement with data obtained by electrospray ionization mass spectrometry [22], and also with the calculated mass based on the primary structure (Table 2). The mass of the sugar moiety in HRP C is therefore 9782 Da, corresponding to 22.4% (w ⁄ w). HPAEC analysis of the HRP C sugar composi- tion revealed a carbohydrate content of 22.1% (w ⁄ w). The comparison of sugar composition between SPC4 and HRP C is illustrated in Table 2. The sugar content of SPC4 is much lower than that observed with HRP C as well as from other cationic peroxidases except for BP1, which also has a low sugar content (Table 3). Spectral properties The UV-visible spectrum of native SPC4 (Fig. 4A) is interpreted in terms of the spin and coordination state Fig. 3. MALDI-TOF-MS analysis of native and deglycosylated forms of SPC4 and HRP C. (A) Native SPC4, (B) deglycosylated SPC4, (C) native HRP C, and (D) deglycosylated HRP C. Table 2. Molecular mass and sugar composition of SPC4 and HRP. Mass of intact protein (Da) Mass of carbohydrate moiety (Da) Proportion of carbohydrate (%, w ⁄ w) Number of residues (mol ⁄ mol) determined by HPAEC MS a MS HPAEC b MS HPAEC Fucose Mannose Xylose NGlc SPC4 c I: 35631 I:1037 1903 I: 3.0 5.4 1.4 5.6 1.7 2.7 II: 34283 II: 2385 II: 6.7 HRP d (present study) 43663 9782 9689 22.4 22.1 9.5 26.8 8.0 14.2 HRP e 42200–44000 ⁄⁄22–27 ⁄ 824 816 a MS, mass spectrometry analysis of the two glycoforms I and II; b HPAEC, high performance anion exchange chromatography analysis of both glycoforms; c SPC4, sorghum cationic peroxidase (the average molecular mass and sugar composition of the two glycoforms was con- sidered). d Horseradish peroxidase according to the present study. e Horseradish peroxidase according to theoretical prediction [21]. Characterization of sorghum peroxidase M. H. Dicko et al. 2296 FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS of the resting enzyme. The absorption spectrum of native SPC4 showed characteristics typical of high-spin iron(III) heme proteins, with a maximum in the Soret region at 403 nm and a b-band at 497 nm [23] (Fig. 4A). There is also a charge-transfer band (por- phyrin to iron) [1] in the spectrum between 630 and 640 nm. Moreover, with the spectrum of the extracted heme, a Q 0v band (vibrational transition of the iron p electrons) [1] at 532 nm and a porphyrin to iron charge transfer band at 637 nm were clearly observed. The Q 0v band at 532 nm was not visible in the native per- oxidase because it is obscured by b-band and charge transfer bands [1]. These spectral properties are charac- teristic for an iron(III)-containing protoporphyrin-IX. The molar absorption coefficient of SPC4 at 403 nm was determined to be approximately 104 mm )1 Æcm )1 . Figure 4(B) shows the mass spectral analysis of the extracted heme cofactor of SPC4. The mass of 616 Da corresponds to the mass of iron(III)–protoporphyrin- IX, confirming that SPC4 contains a type-b heme. The peak with a mass of 563 Da is ascribed to the partial loss of iron by the protoporphyrin-IX. The MALDI- TOF-MS spectrum (Fig. 4B) also shows an intense peak with a mass of 650, which is assigned to a heme-H 2 O 2 adduct. Thus, SPC4 is a type-b heme-con- taining peroxidase, which shares similar molecular properties with cereal peroxidases [1,6,15]. Far UV-circular dichroism spectroscopy indicated that SPC4 contains 42 ± 6% a-helix, 35 ± 7% b-sheet and 24 ± 7% b-turns (not shown). These val- ues should be taken with caution as in peroxidase structures predicted from CD spectra the a-helix con- tent can be underestimated. Nevertheless, this secon- dary structure content is similar to that of other plant peroxidases [24]. Amino acid composition and N-terminal sequence analysis The amino acid composition of SPC4 together with those of other cationic peroxidases is given in Table 3. The average amino acid calculated mass of cationic peroxidases is 106.7 Da (Table 3), allowing estimation of 311 amino acid residues in SPC4. From this amino acid composition, a theoretical pI value of 11 was cal- culated, assuming that all eight cysteines are involved in disulfide bridges [1,7,25,26]. The low ratio (Asx + Glx) ⁄ (Arg + Lys) of SPC4 and its pI value Table 3. Amino acid composition of SPC4 and other cationic plant peroxidases. Amino acid SPC4 a RP b WP c BP1 d CC e HRPC f PNC21 g SB1 h TP7 i Ala 31(10.0)5039223723 27 29 32 Arg 23 (7.4) 15 12 30 21 21 19 22 17 Asp+Asn 35(11.1)3238343148 35 35 39 Cys 8(2.5)89898 8 9 8 Glu + Gln 12 (3.9) 13 15 26 21 20 22 27 14 Gly 29 (9.3) 22 24 25 26 17 28 26 24 His 5(1.6)54453 5 4 3 Ile 8 (2.6) 14 11 11 13 13 13 12 15 Leu 29 (9.4) 32 31 30 28 35 25 30 21 Lys 14 (4.5) 7 10 6 4 6 12 8 10 Met 3(1.0)78284 3 6 6 Phe 16 (5.2) 11 12 17 13 20 18 17 14 Pro 15 (4.8) 12 10 21 17 17 11 15 11 Ser 27 (8.7) 36 37 26 31 25 29 30 42 Thr 25 (8.1) 26 27 16 19 25 22 16 16 Trp 2(0.6)11111 2 2 1 Tyr 6(1.9)64465 4 9 4 Val 23 (7.4) 17 20 26 17 17 24 25 19 (Asx + Glx) ⁄ (Arg + Lys) 1.27 2.05 2.41 1.67 2.08 2.52 1.84 2.07 1.96 Sum 311 (100) 314 312 309 307 308 307 322 296 Apoprotein MW j 33 226 32 437 32 382 33 825 32 508 33 918 32 954 35 029 31 086 Carbohydrate proportion 3–6% ⁄ k ⁄ 0–3% ⁄ 22–27% 12–19% ⁄ 7% Accession code l P84516 O22440 Q05855 Q40069 Q43416 P00433 P22196 Q9SSZ9 POO434 a Results of SPC4 are presented in number of amino acid ⁄ protein and in mole percentage (mol ⁄ mol) in brackets. b Rice [10], c wheat [9], d bar- ley [6], e Cenchrus ciliaris [53], f horseradish [25], g peanut [54], h Scutellaria baicalensis [55], i turnip [56]. j Calculated molecular weights using software to compute pI ⁄ MW ⁄ titration curve, available at http://expasy.ch/tools/#primary. ⁄ k , sugar composition not given. l UniProtKB ⁄ TrEMBL accession number. M. H. Dicko et al. Characterization of sorghum peroxidase FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS 2297 indicate that in comparison to other cationic peroxid- ases, SCP4 is highly basic (Table 3). Like BP1 [6], the N-terminal sequence of SPC4 is not blocked by pyroglutamate, in contrast to most other peroxidases [25]. The first 20 amino acid residues are shown in Fig. 5. A TBLASTN search at the Gram- ene website (http://www.gramene.org) indicated that the SPC4 gene is localized in the sorghum chromosome 1. At the Institute for Genomic Research (http:// www.tigr.org), the best match with 100% identity was found with gene indice TC102191 (213 amino acids). MALDI-TOF-MS analysis (Fig. 6) showed that six peptides, including the N-terminal sequence of SPC4 matched with the predicted tryptic peptides of TC102191, indicating that TC102191 codes for the N-terminal part of the sequence of SPC4. SPC4 has a signal peptide of 31 amino acids (Fig. 5). Since the expected full length of SPC4 is about 311 amino acid residues, the C-terminal sequence of about 129 amino acids is unknown. Among the currently 160 stretches of sorghum peroxidase genes that are identified (http:// peroxidase.isb-sib.ch/index.php), SPC4 corresponds to SbPrx50. With the currently ongoing sorghum genome project (http://fungen.botany.uga.edu), the full seq- uence of this gene will be available soon. The sequence of the N-terminal part of SPC4 was analyzed by searching for domain database (RPS- BLAST at NCBI: http://www.ncbi.nlm.nih.gov/blast), protein families database (Pfam9Sanger Institute: http://www.sanger.ac.uk/software/Pfam) and for speci- fic protein motifs, domains and families (InterProScan at EBI: http://www.ebi.ac.uk/InterProScan). The RPS- BLAST and Pfam searches indicated with expect val- ues of 4e-59 and 1.1e-50, respectively, that SPC4 belongs to the Class III of plant secretory peroxidases like HRP C. Furthermore, the InterProScan software, which integrates several tools for the analysis of domain and family of proteins, clearly showed that SPC4 contains all the fundamental motifs characteris- tic of Class III plant peroxidases. The TBLASTN search against the nonredundant database at NCBI (http://www.ncbi.nlm.nih.gov/blast) indicated that SPC4 is most closely related to cereal peroxidases (Fig. 5). Because the N-terminal sequences of the mature peroxidases from rice, wheat and maize are as yet unknown, the alignment of sequences in Fig. 5 is made by including the signal peptides of peroxidases (precursors). The N-terminal fragment of SPC4 has a high sequence identity with barley BP1 (85%), rice Prx23 (90%), wheat WSP1 (82%), and maize (58%), indicative for a common ancestor [27]. SPC4 consists of two domains and has an N-ter- minal extension of one and eight residues, compared to BP1 [6] and HRP C [25], respectively. The key cata- lytic residues (Arg46, Phe49, His50, Asn78, Pro150 and His180) and cysteines involved in intramolecular disulfide bridges (Cys19-Cys100; Cys52-Cys57; Cys107) are all conserved (Fig. 5). The structural motif -P-X-P- is found at sequence positions 150–152. This region is involved in the substrate binding of plant peroxidases [26]. In particular, Pro150, which is completely con- served in the plant peroxidase superfamily (class III), is crucially involved in substrate binding and oxidation [7,26]. Another important residue of SPC4 concerns Thr68, which is equivalent to Thr67 of BP1. This resi- due is conserved in most cereal peroxidases (Fig. 5), but not in HRP C. Structural studies have shown that the distal heme pocket of BP1 is significantly different to that of other plant peroxidases. In BP1, at pH above 5, the distal His makes a hydrogen bond with Thr67 and not with the distal Asn70 as in HRP C. As a result, the orientation of the distal His residue is altered and located too far from the heme iron atom to be able to catalyze the formation of compound I. In Fig. 4. Heme analysis of SPC4. (A) Spectral properties of SPC4. The absorption spectrum of purified SPC4 was recorded in 50 m M sodium acetate pH 5. The inset shows the spectrum of the extract- ed heme. (B) MALDI-TOF-MS analysis of SPC4 heme. Characterization of sorghum peroxidase M. H. Dicko et al. 2298 FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS addition, Phe48 (equivalent to Ph49 in SPC4) moves toward the heme iron, and in doing so, the accessibility of the heme iron is diminished [7]. Given the high sequence identity with BP1 it may be conceivable that a similar situation applies in SPC4. The only putative glycosylation site present in the sequenced N-terminal fragment of SPC4 is Asn78. However, Asn78 is an active site residue of class III peroxidases that is not glycosylated [6]. Thus, as found for most peroxidases, the glycosylation sites of SPC4 are localized in the C-terminus part of the enzyme. Catalytic properties SPC4 was stable between pH 3 and pH 7 for 2 h at 25 °C. The enzyme showed optimal activity with Fig. 5. Multiple sequence alignment of major cationic sorghum peroxidase (P84516) with other cereal peroxidases. The N-term- inal fragment of SPC4 is aligned with barley BP1 (Q40069), rice Prx23 (Q94D M0), wheat WSP1 (Q8LK23), and maize peroxidase (O04710). The codes under brackets are UniProtKB ⁄ TrEMBL entries. The highly con- served catalytic residues among all class III peroxidases are marked with asterisks. The N-terminal sequence of SPC4 obtained by Edman sequencing is underlined. The signal peptides of SPC4 and BP1 are shown in the boxes. Fig. 6. MALDI-TOF-MS peptide mass fingerprint of SPC4. M. H. Dicko et al. Characterization of sorghum peroxidase FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS 2299 ABTS, ferulic acid and N-acetyl-l-tyrosine at pH 3.8, 5.5 and 6.5, respectively (Fig. 7). These different pH optima are in line with reported properties of other peroxidases [28,29]. For instance the pH optima found for the activity of lettuce (Lactuca sativa) peroxidase were 4.5, 6.0, 5.5–6.0, and 6.0–6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and chlo- rogenic acid, respectively [28]. It is known that there is no correlation between the pH optima of peroxidase activity and their pI values because both anionic (pI 3.5) and cationic (pI 8.8) horseradish peroxidases display for instance the same optimum pH for the oxi- dation of p-coumaric acid [29]. The substrates oxidized at low pH (ABTS and ferulic acid) have higher cata- lytic efficiencies (Table 4) than those oxidized at higher pH values (N-acetyl-l-tyrosine) maybe because of the higher oxidation potential of the reaction intermediates compound I and II at low pH [29]. The difference in the optimum pH of peroxidase activity between sub- strates may also reflect the pH-dependence of their ionization potentials. A pH-dependence of peroxidase activity as a function of substrate could be explained by several reasons. A change in pH would affect the extent to which each functional groups of the amino acid involved in substrate binding, or catalytic residues ionizes, and thus the conformation of the peroxidase molecule. A change in the structural conformation will obviously affect the shape of the active site, and thus either increase or decrease the enzyme’s affinity for substrate molecules [1]. This hypothesis is further sup- ported by the fact that different amino acids can be involved for plant peroxidases binding to physiologi- cally relevant substrates [29]. The pH-dependence of the contribution from electrostatic repulsion or attrac- tion during substrate binding and release can also be considered. The better and maybe faster binding of electron donors have been suggested to justify the dif- ference in the oxidation of phenolic substrates by plant peroxidases [29]. Furthermore, some substrates are oxidized in a single-electron reaction (ABTS) and oth- ers in a two-electron reaction (phenolic compounds), and some products undergo nonenzymatic polymeriza- tion reactions after peroxidase oxidation of substrates from which they derived [1]. Such kinetic differences might alter the overall pH-activity profile. Nevertheless, SPC4 remarkably differs from BP1 [23] in being active with aromatic compounds above pH 5. This activity, which is also apparent from the zymography analysis (Fig. 2A), is intriguing in view of the structural relationship mentioned above. Stafford and Brown [30] reported an oxidative dimerization of ferulic acid by sorghum grain extracts. Furthermore, using a crude extract from sorghum variety NK300, a high peroxidase activity on ferulic acid and no activities on tyrosine and other phenolics were observed [31]. Here we found that purified SPC4 has a high preference for hydroxycinnamates, including ferulic acid and p-cou- maric acid, which are among the most abundant phenolic compounds in sorghum [32]. Kinetic studies performed at pH 5.5 showed that the catalytic effi- ciency of SPC4 with phenolic compounds decreased in the following order: ferulic acid > p-coumaric acid > N-acetyl tyrosine methyl ester > N-acetyl tyrosine > tyrosine > catechol > G ly-Tyr-Gly (Table 4). Fig. 7. Dependence of SPC4 activity on pH. The enzyme (10 nM) was incubated with 10 m M ABTS (d), 125 lM ferulic acid (s), or 250 l M N-acetyl tyrosine (m) in the presence of 5 mM H 2 O 2 , in dif- ferent 50 m M McIlvaine buffers (pH 2.5–8), at 20 °C. Enzymes activities were monitored as described in the Experimental proced- ures. Vertical bars indicate the standard error of each experiment. Table 4. Substrate specificity a of sorghum peroxidase. Substrate Substrate k max (nm) Substrate molar absorption coefficient (mM )1 Æcm )1 ) Product k max (nm) Apparent V max ⁄ K m (M )1 Æs )1 ) ABTS 340 34 414 c 1.16 Ferulic acid 310 b 14.9 348 d 0.92 p-coumaric acid 287 b 19.7 290 e 0.23 Indole-3-acetic acid 280 b 5.0 261 f 0.08 N-acetyl tyrosine methyl ester 275 1.4 318 c,d 0.07 N-acetyl tyrosine 275 1.4 293 c,g 0.05 Tyrosine 276 2.8 318 c,d 0.03 catechol 276 2.3 398 c,h 0.01 Gly-Tyr-Gly 275 1.3 318 c,d 0.01 a The substrates are ranked by order of preference. The reaction was followed by b substrate disappearance or c product formation according to d [18], e [29], f [34], g [51], and h [52]. Characterization of sorghum peroxidase M. H. Dicko et al. 2300 FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS The relatively high reactivity with hydroxycinnamic acid derivatives suggests that the enzyme may be involved in the formation of diferulate linkages in the plant cell wall. On the other hand, the rather low catalytic efficiency of SPC4 with tyrosine and tyrosine-containing peptides suggests that the enzyme is less involved in protein cross-linking through di- tyrosine formation. SPC4 also displayed auxin (3-indole acetic acid) activity. This activity, which takes place in the absence of added hydrogen peroxide, is mechanistically differ- ent for cationic and anionic peroxidases [33] and not a property of all plant peroxidase isoforms [34]. The physiological significance of auxin metabolism by plant peroxidases is still an area of debate. Some peroxidases regulate the level of auxin either by direct degradation or by oxidizing endogenous flavonoids, which are inhibitors of auxin transport [35]. The activity of SPC4 on auxin might be related to the presence of His48 (His40 in HRP C) in the distal domain near the heme, which is believed to play a role in auxin recognition based on sequence similarity with auxin binding pro- teins [33]. The activity of SPC4 was stimulated in the presence of CaCl 2 . The maximum increase of activity of the purified enzyme was two-fold with an apparent semi- maximal activation at 0.7 mm CaCl 2 . A similar, but somewhat stronger activation, was observed for BP1 for which the calcium binding sites are not fully occu- pied [23]. The Ca 2+ activation of SPC4 is of interest because not all peroxidases are activated by Ca 2+ [15]. HRP C for instance contains two structural calcium ions (proximal and distal) that are also of functional significance [26]. Binding of Ca 2+ decreased the intrin- sic tryptophan fluorescence intensity of SPC4. From the binding curve, a dissociation constant for the SPC4–Ca 2+ ion complex, K d ¼ 2.4 ± 0.3 mm, was determined. The affinity of SPC4 for Ca 2+ was some- what higher than that of BP1 (K d ¼ 4mm) [15]. The calcium status of BP1 is anomalous, with the distal calcium-binding site substituted by sodium [7]. Based on the sequence alignments, the distal binding site in SPC4 is formed by Asp51, Asp58, Ser60 (side chains) and Asp51, Val54, Gly56 (main chain carbonyls). The entire sequence of SPC4 is needed to establish the proximal calcium binding site. The binding of Ca 2+ has been proposed to change the electronic properties of the heme iron or the topology of the heme vicinity and might improve substrate binding [7,8,15,23]. With SPC4, such structural perturbations must be small because circular dichroism analysis revealed that Ca 2+ binding does not change the secondary structure of the enzyme (not shown). Thermal stability In the absence of added CaCl 2 , SPC4 readily lost activity when incubated at temperatures above 55 °C (Fig. 8A). However, in the presence of excess Ca 2+ ions, the enzyme kept its full activity at up to 65 °C for 90-min incubation (Fig. 8B). Arrhenius plots (Fig. 8C) of the thermoinactivation data revealed straight lines and showed that Ca 2+ binding only slightly increases the activation energy of heat inactiva- tion of SPC4 from 157 ± 12–170 ± 14 kJÆmol )1 . The increased stability of SPC4 in the presence of Ca 2+ ions was confirmed by fluorescence experiments. Upon Fig. 8. Thermoinactivation of SPC4. The enzyme (270 nM) was incubated at different temperatures in 50 m M sodium acetate pH 5, either in the absence (A) or presence (B) of 5 m M CaCl 2 :55°C(d), 60 °C(s), 65 °C(m), 70 °C(n), 75 °C(n), 80 °C(h), 85 °C(X); 90 °C(r), 95 °C(e). (C) Arrhenius plot for heat inactivation of SPC4 in the absence (r) or presence (e) of calcium. Vertical bars indicate the standard error of each experiment. M. H. Dicko et al. Characterization of sorghum peroxidase FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS 2301 heating, both in the absence and presence of Ca 2+ ions, a strong increase in protein tryptophan fluores- cence was observed (Fig. 9A,B). Independent of the presence of Ca 2+ ions, and treating the data according to van Mierlo et al. [36], SPC4 followed a simple two- state mechanism of heat-induced unfolding. This is in agreement with other plant peroxidases [37]. Thermal unfolding of SPC4 induced not only an increase of fluorescence intensity but also a bathochro- mic shift of the fluorescence maximum from 338 to 348 nm (not shown). T m values of 67 °C and 82 °C for the free and calcium-bound form, respectively, were found. In the absence of Ca 2+ , the melting tempera- ture of SPC4 was between that of HRP C (T m ¼ 60 °C) and the African palm tree peroxidase (T m ¼ 74 °C) [37]. In the presence of Ca 2+ , the T m of SPC4 is near that of soybean peroxidase, which is one of the most stable plant peroxidases with a T m of 90 °Cin the presence of calcium [38]. In conclusion, the major isoenzyme in sorghum grain (SPC4) was shown to be a cationic peroxidase having two glycoforms with unusual basic character and a high heat stability in the presence of calcium. The enzyme has relatively low carbohydrate content. It shares similar molecular properties with other cereal peroxidases such as barley peroxidase 1 but has dis- tinct catalytic properties in being active on aromatic compounds above pH 5. Therefore, the enzyme may develop as an alternative peroxidase for biochemical and clinical assays, and biocatalysis. Experimental procedures Chemicals Horseradish peroxidase [HRP, EC 1.11.1.7] (grade II, lot N°16H9522), p-coumaric acid, ferulic acid, l-tyrosine, tri- fluoromethanesulfonic acid, and indole-3-acetic acid were from Sigma-Aldrich (Zwijndrecht, the Netherlands). N-ace- tyl tyrosine, N-acetyl tyrosine methyl ester and Gly-Tyr-Gly were from Bachem, Bubendorf, Switzerland. Hydrogen per- oxide was from Merck (Darmstadt, Germany). Modified trypsin (EC 3.4.21.4) sequencing grade was from Roche Diagnostics GmbH (Mannheim, Germany). Electrophoresis gels (IEF, pH 3–9) were purchased from Amersham Bio- sciences. SDS ⁄ PAGE gradient gels (10–18%) were from Biorad (Richmond, CA, USA). Immobilon-P transfer mem- brane was from Millipore Corporation (Bedford, MA, USA). Maltodextrin MD05 standards were obtained from Spreda (Burghof, Switzerland). Low molecular weight standard proteins were from Amersham Pharmacia Biotech (Uppsala, Sweden). All other chemicals were of analytical grade. Enzyme purification The grains of sorghum variety [Sorghum bicolor (L) Moench var. Cauga 108–15] grown in 1998 were used [13]. Peroxidase isoenzymes were extracted from flour as des- cribed previously [13,14]. Protein precipitation was per- formed with slow addition of acetone ()30 °C) to the crude extract, followed by centrifugation (10 000 g, 30 min). The precipitate obtained between 40 and 80% (v ⁄ v) acetone was resuspended in the extraction buffer and dialyzed overnight against 20 mm Bis-Tris-Cl, pH 7.0, containing 1 mm CaCl 2 (starting buffer), at 4 °C. Insoluble material was removed by centrifugation (15 000 g, 45 min, 4 °C). Subsequent chromatography steps were performed at room temperature (20–22 °C). Protein eluates were monit- ored at wavelengths of 280 and 403 nm. Reinheitszahl (RZ) values (A 403 ⁄ A 280 ) were calculated directly from the chro- matograms [4]. The supernatant (150 mL) obtained after acetone precipitation and subsequent dialysis was loaded onto a Superdex 75-PG gel filtration column (65 · 15 cm, Amersham Pharmacia Biotech, Uppsala, Sweden) equili- brated with starting buffer. Proteins were eluted at a flow rate of 25 mLÆmin )1 . Fractions containing peroxidase A B Fluorescence intensity (AU)Fluorescence intensity (AU) Fig. 9. Thermal unfolding of SPC4 as followed by intrinsic trypto- phan fluorescence. The enzyme (2.22 l M) was heated in 10 mM sodium acetate pH 5, either in the absence (A) or presence (B) of 5m M CaCl 2 at a rate of 0.5 °CÆmin )1 . The excitation wavelength was 295 nm. The emission at 342 nm was monitored at 0.5-min intervals. Solid lines are the best fit of the two states unfolding equation (Eqn 1) [36]. Characterization of sorghum peroxidase M. H. Dicko et al. 2302 FEBS Journal 273 (2006) 2293–2307 ª 2006 The Authors Journal compilation ª 2006 FEBS [...]... between 2.5 and 8.0, using the standard assay The effect of pH on enzyme stability was determined by preincubating the enzyme in various pH buffers [46] and determining the residual activity with the standard assay The effect of Ca2+ ions on peroxidase activity was analyzed by measuring peroxidase activity in the presence of varying (010 mm) concentrations of CaCl2 Prior to the assay, the enzyme was desalted... in degặcmặ dmol)1 The secondary structure of the enzyme was analyzed as described by Venyaminor et al [50] The effect of Ca2+ binding on the protein secondary structure was deter- 2304 mined by recording CD spectra in the presence of increasing concentrations of CaCl2 (010 mm) 1ị where Yobs is the measured uorescence, R is the gas constant, and a and b are the intercept and slope of the preand postunfolding... concentrations of CaCl2 for 10 min, at 25 C The reaction was then started by adding H2O2 The nal concentration of the enzyme in the reaction medium was 10 nm Substrate specicity Steady-state kinetics of SPC4 were performed by measuring the initial rate of enzyme activity in 50 mm sodium acetate pH 5.5, containing 1 mm CaCl2, in the presence of 2.5 mm H2O2 and varying concentrations of hydrogen donor, at 25 C The. .. correct for IAA autooxidation Acknowledgements The organization for the prohibition of chemical weapons (OPCW) via the International Foundation for Characterization of sorghum peroxidase Science (Sweden), and the Stichting voor Sociale en Culturele Solidariteit, Zeist, the Netherlands are acknowledged for supporting the research carried out by Dr M H Dicko The authors wish to thank Dr Henk Schols for... 2.0 with acetic acid The UV-spectrum of the heme was recorded in the extraction solution Fluorescence emission spectra were recorded on a Varian Cary Eclipse Fluorescence Spectrophotometer (Bergen op Zoom, the Netherlands) Ca2+ binding of the enzyme was studied at Yobs ẳ aU ỵ bU Tị ỵ Thermal stability Thermal stability was studied by incubating SPC4 in the absence or presence of 5 mm CaCl2 at temperatures... spectrometry MALDI-TOF-MS was performed with a Voyager-DE-RP Biospectrometry Workstation elite reectron time of ight mass spectrometer (PerSeptive Biosystems, Inc., Framingham, Manchester, England) with a delayed extraction MALDI ion source Between 100 and 256 scans were averaged for each of the spectra shown Samples were deposited in nonwelled gold plates The mass of the heme cofactor of sorghum peroxidase. .. unfolding of SPC4 was studied by monitoring the intrinsic tryptophan uorescence of the enzyme (2.2 lm) in 10 mm sodium acetate pH 5.0 in the absence or presence of 5 mm CaCl2 The temperature of the continuously stirred protein solution, as measured with a digital sonde with a reading precision of 0.01 C, was increased from 25 C to 95 C at a speed of 0.5 C per min [37] The uorescence emission at 342 nm was... 2006 FEBS 2305 Characterization of sorghum peroxidase M H Dicko et al 14 Dicko MH, Gruppen H, Traore AS, Zouzouho OC, van Berkel WJH & Voragen AGJ (2006) Effects of germination on the activities of amylases and phenolic enzymes in sorghum varieties J Sci Food Agric 86, 953963 15 Converso DA & Fernandez ME (1996) Ca2+ activation of wheat peroxidase: a possible physiological mechanism of control Arch... Horseradish peroxidase oxidation of tyrosine-containing peptides and their subsequent polymerization: a kinetic study Biochemistry 36, 85048513 52 Liu W, Fang J, Zhu WM & Gao PJ (1999) Isolation, purication and properties of the peroxidase from the hull of Glycine max var HH2 J Sci Food Agric 79, 779785 53 Ross AH (1994) Investigation of peroxidase genes and genetic transformation in buffel grass PhD Thesis,... respectively DH is the enthalpy change for unfolding measured at Tm, Tm the melting temperature and T the absolute temperature The standard errors in Tm values were Ê 0.2 K Data were tted by nonlinear, least-squares analysis using the general curve t option of the Prot program (Quantum Soft, Zurich, Switzerland) Enzyme activity Peroxidase activity was measured spectrophotometrically by monitoring the H2O2-dependent . characterized the cationic peroxidase isoenzyme from sorghum grain. Results and discussion Purification of major peroxidase from sorghum seed At least four sorghum peroxidase. which is one of the most stable plant peroxidases with a T m of 90 °Cin the presence of calcium [38]. In conclusion, the major isoenzyme in sorghum grain (SPC4)