Purification and Characterization of a Nove1 Class 111 ...ing to current classification, the plant...

Plant Physiol. (1 997) 114: 1237-1 245

Purification and Characterization of a Nove1 Class 111 Peroxidase lsoenzyme from Tea Leaves'

Mamuka Kvaratskhelia, Chris Winkel, and Roger N.F. Thorneley*

Nitrogen Fixation Laboratory, John lnnes Centre, Norwich, United Kingdom NR4 7UH (M.K., R.N.F.T.); and Unilever Research Laboratorium Vlaardingen, Olivier Noortlaan 120, 31 33 AT Vlaardingen,

The Netherlands (C.W.)

A novel, basic (isoelectric point > 10), heme peroxidase isoenzyme (TP; relative molecular weight = 34,660 & 10, mean 2 SE) that can account for a significant part of the ascorbate peroxidase activity in tea (Camellia sinensis) leaves has been purified to homogeneity. The ultraviolet/visible absorption spectrum is typical of heme-containing plant peroxidases, with a Soret peak at 406 nm ( E = 115 mM-' cm-') and an A40dA280 value of 3.4. The enzyme has a high specific activity for ascorbate oxidation (151 pmol min-' mg-'), with a pH optimum in the range of 4.5 to 5.0. Substrate- specificity studies have revealed significant differences between TP and other class 111 peroxidases, as well as similarities with class I ascorbate peroxidases. TP, like ascorbate peroxidase, exhibits a preference for ascorbate over guaiacol, whereas other class III isoenzymes are characterized by 2-orders-of-magnitude higher activity for guaiacol than for ascorbate. TP also forms an unstable porphyrin rr cation radical-type compound I, which is converted to compound II within approximately 2 min in the absence of added reductant. Amino acid sequence data show TP to be the first example, to our knowledge, of a class III peroxidase with a high specificity for ascorbate as an electron donor.

~ ~ ~ _ _ ~ ~~ ~~ ~~

Peroxidase (EC 1.11.1.7) is an ubiquitous plant enzyme that catalyzes the oxidation of cellular components by either H,O, or organic hydroperoxides. Each plant usually encodes an estimated 8 to 15 peroxidase families. Accord- ing to current classification, the plant peroxidases are di- vided into two types (Welinder, 1992), APX, which is from the plant chloroplast and cytosol, is distinguished from classical plant peroxidase isoenzymes by significant differences in primary structure. However, APX closely resem- bles CCP (38% amino acid sequence identity) (Mittler and Zilinskas, 1991a; Chen et al., 1992). Therefore, these enzymes are classified as members of the class I family of heme peroxidases, of which CCP is the archetype that is part of the evolutionary linkage to prokaryotic peroxidases (Welinder, 1992).

Classical, secretory plant peroxidases that share 50 to 95% sequence homology within the superfamily of peroxidases are assigned to class 111 (Welinder, 1992). Since guaiacol (2-methoxyphenol) is commonly used as a reducing substrate, these enzymes are also referred to as guaiacol-

~

' This work was supported by a grant from Unilever Research,

* Corresponding author; e-mail [email protected]; Vlaardingen, The Netherlands.

fax 44 - 01- 603-454970.

type peroxidases. Detailed structural studies of plant peroxidases have revealed other important differences between ascorbate and guaiacol-type peroxidases. APX has no Cys-disulfide bridges, structural Ca2+ ions, carbohydrate, or ER signal sequences (Mittler and Zilinskas, 1991a; Patterson and Poulos, 1995), whereas horseradish and peanut peroxidases, archetypal class I11 isoenzymes, have four conserved disulfide bridges and two structural Ca2+ ions (Welinder, 1979; Schuller et al., 1996). In addition, most secretory plant peroxidases are glycosylated (Lagrimini et al., 1987; Johansson et al., 1992). A11 enzymes of this family have an ER signal peptide (Welinder, 1992).

There are significant reactivity differences between APX and guaiacol-type peroxidase. APX has a marked preference for ascorbic acid as a reducing substrate, whereas classical plant peroxidases oxidize phenolic compounds at a much higher rate (Asada, 1992). APX compound I is extremely unstable and is rapidly converted to compound I1 without the addition of reducing substrate (Patterson et al., 1995), whereas guaiacol peroxidases are characterized by a very stable compound I. On the basis of these and other structure-function differences, it has generally been accepted that APX and guaiacol-type peroxidase also differ with respect to their physiological roles. APX with ascorbate as its reducing substrate is believed to scavenge excess H,O, formed in plant cells under normal and stress conditions, as do glutathione peroxidases in mammals, CCP, and NAD(P)H peroxidase in bacteria (Asada, 1992). The oxidation products (monodehydroascorbic acid radicals) do not seem to play a physiological role, since they are merely reduced back to ascorbate by monodehydroascorbic acid reductase with NAD(P)H as the electron donor (Asada et al., 1996) or they disproportionate to ascorbate and dehydroascorbate. In contrast, guaiacol peroxidases oxidize a wide range of organic substrates and the oxidation products are involved in important biosynthetic processes. Guaiacol peroxidases have been proposed to participate in lignification of the cell wall, degradation of IAA, biosynthe- sis of ethylene, wound healing, and defense against patho- gens (Gazaryan et al., 1996; Kobayashi et al., 1996).

Abbreviations: APX, ascorbate peroxidase; CCP, yeast Cyt c peroxidase; AE, difference in E values between substrate and prod- uct; E, extinction coefficient; HRP, horseradish peroxidase isoenzyme C; HRP4B, horseradish peroxidase isoenzyme 4B; TobP, tobacco peroxidase; TP, tea peroxidase.

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1238 Kvaratskhelia et al. Plant Physiol. Vol. 11 4, 1997

Recent studies have shown that during plant develop- ment APX activity is regulated by ascorbate content (Gara et al., 1996). An increase in APX activity as a response to several stress conditions is well documented (Gara et al., 1996); however, it is still not clear whether only class I peroxidase isoenzymes are responsible for scavenging H,O, in plant cells. It has recently been suggested that ascorbate may be the natural substrate for even nonspecific peroxidases (Mehlhorn et al., 1996). Ascorbate-dependent detoxification of H,O, by HRP is considerably increased in the presence of 3,4-dihydroxyphenolic compounds (Mehl- horn et al., 1996); however, the guaiacol peroxidases studied to date exhibit very low ascorbate-oxidation activity (Chen and Asada, 1989).

In an attempt to better understand the structure-function relationship of the enzymes that catalyze ascorbate- dependent detoxification of H,O, in plant cells, we have extended our studies to the purification and characterization of TP isoenzymes with high ascorbate activity. Three tea (Camellia sinensis) APXs have previously been isolated and studied, two of which are acidic APX isoenzymes (Chen and Asada, 1989). We recently found that tea leaves also contain several basic APX isoenzymes. We report the purification, biochemical properties, and amino acid sequence data of a nove1 basic isoenzyme, TP, that accounts for a significant percentage of APX activity in tea leaves. The purified enzyme has a remarkably high ascorbate- oxidation activity (150 pmol mg-') and functionally resem- bles cytosolic APXs. However, in complete contrast, the sequence data of the tryptic digestion peptide fragments of the enzyme have provided strong evidence that this TP is a class I11 rather than a class I peroxidase. This isoenzyme is therefore the first example, to our knowledge, of a guaiacol-type peroxidase with a high ascorbate-oxidation activity.

MATERIALS A N D METHODS

Plant Material and Chemicals

Leaves of tea (Camellia sinensis var Sinensis clone BBK35) were supplied by Unilever (Colworth House, Bedford, UK) from plants grown in Kenya. Freshly plucked leaves (approximately 1100 shoots / kg, averaging two to three leaves and a bud) were received freshly frozen in dry ice. A11 chemicals were purchased from Sigma or Aldrich unless otherwise stated. HRP4B (EC 1.11.1.7) with an ASoret peak/ A,,, value of 3.33 was from Biozyme (Gwent, UK). Reagent grade H,O, (30%) was obtained from BDH Laboratory Supplies (Lutterworth, Leicestershire, UK) and its concentration was calculated by iodide titration in the presence of HRP4B (Cotton and Dunford, 1973).

Purification of the Class 111 lsoenzyme

Tissue homogenization, centrifugation, and dialysis were carried out at 4 to 8°C. Fast protein liquid chromatography purification steps were performed at room temperature. One kilogram of tea leaves was ground with 2.5 L of extraction buffer comprising 50 mM Mes (pH 5.5), 30

mM ascorbic acid, 1% (v/v) Triton X-100, 1 M NaC1, 1 mM EDTA, and 2% (w / v) soluble PVP for 3 min with a blender (model HR2845 / AM, Philips, Eindhoven, The Nether- lands) operated at maximum speed. After centrifugation of the homogenate at 9000 rpm for 30 min, the supernatant was treated with 10% (w/v) insoluble PVP, and the mixture was reblended for 1 min at maximum speed. The solid was removed by centrifugation as described above and the supernatant was then dialyzed against 25 mM malonate, pH 5.5, containing 10 mM ascorbic acid. The pellet formed during dialysis was removed by centrifugation as described above and the supernatant was applied to an SP- Sepharose high-performance 26/ 10 column (Hi-Load, Pharmacia) that had previously been equilibrated with 50 mM malonate, pH 5.5. The column was prewashed with 150 mL of equilibrating buffer, and then 1.5 L of a linear gradient comprising O to 150 mM NaCl was used to separate the different peroxidase isoenzymes.

The fractions that contained the peroxidase activity peak (range 120-140 mM NaC1) were separated and concentrated by ultrafiltration through a PM-10 membrane (Amicon, Beverly, MA). The concentrated enzyme (2 mL) was loaded onto a 26/60 gel-filtration column (Superdex 200 Hi-Load, Pharmacia), equilibrated with 150 mM NaCl in 50 mM malonate, pH 5.5, and eluted with the same buffer. The active fractions were dialyzed against 50 mM malonate, pH 5.5, and loaded onto a Mono-S column (Pharmacia) equilibrated with the same buffer. The enzyme was eluted with a linear gradient of NaCl (100 mL, 0-150 mM). The active fractions were collected and dialyzed against 10 mM sodium phosphate, pH 7.0. The dialysate was loaded onto a hydroxyapatite column (Bio-Scale CHT2-1, Bio-Rad) equilibrated with 10 mM phosphate, pH 7.0. A linear gradient of 80 mL of 10 to 400 mM sodium phosphate, pH 7.0, was used for the final purification of the enzyme. The active fractions were pooled and either used immediately or stored at -80°C.

Electrophoresis

SDS-PAGE was performed in a vertical gel apparatus (Mini-Protean, Bio-Rad) with 10% precast Tris-Gly gel (Bio- Rad), as described by Laemmli (1970). Samples containing approximately 10 pg of protein were applied to the gel and electrophoresed at 200 V for 45 min. Proteins were stained with 0.2% (w/v) Coomassie brilliant blue in 50% (v/v) methanol and 7.5% (v/v) acetic acid for 60 min.

IEF was performed using the Pharmacia Fast Gel system on prepoured gels (pH 3-9) with the following standards: trypsinogen (pI 9.3), lentil lectin-basic (pI 8.65), middle (pI 8.45) and acidic (pI 8.15) bands, and myoglobin-basic band (pI 7.35). The pIs of the standards were assumed to be as designated by the supplier (Pharmacia).

Cel Filtration

A Superdex-200 HR 10/30 column (Pharmacia) was equilibrated at room temperature with 50 mM phosphate buffer, pH 7.0, containing 150 mM NaCl. Purified TP and calibration proteins (P-amylase [200,000], alcohol dehydro-

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A Nove1 Class I I I Peroxidase from Tea Leaves 1239

genase [ 150,0001, BSA [66,000], carbonic anhydrase [29,000], and Cyt c [12,400]; 1 mg each; supplied by Sigma) were applied on the column and eluted at a flow rate of 0.5 mL/min. An estimate of the M , of the native TP was calculated from a semilogarithmic plot of the M, values for the calibration proteins against elution volume.

M, Determination

A precise M, value of the apoenzyme was determined by an electrospray mass spectrophotometer (V.G. platform quadrupole spectrophotometer, School of Biological Sci- ences, University of East Anglia, Norwich, UK). A sample of 5 p~ TP in 5 mM Tris, pH 8.0, was diluted 1:l with a 1% formic acid solution in 1:l (v/v) water:acetonitrile. Enzyme samples (20 pL, approximately 500 pmol) were injected into the carrier solvent stream (1:I [v/v] water:acetonitrile) of the device a t a flow rate of 10 pL/min. Scans were taken over a range of 700 to 1700 mlz. Horse heart myoglobin was used as a standard for the calibration. The results were analyzed using the software provided by the instrument manufacturer.

Determination of Protein Concentration and Enzyme Assay

Protein concentrations were determined using the Bio- Rad protein assay procedure with BSA as a standard. The assay kit comprising the dye reagent and BSA was purchased from Bio-Rad. The heme content and molar extinction coefficient of the enzyme was determined by the pyridine hemochromogen assay (Paul et al., 1953). The peroxidase activity with ascorbic acid as the reducing substrate was determined in a reaction mixture (1 mL) containing 50 mM sodium citrate (pH 4.5), 1 mM ascorbate, and 0.5 mM H,O,. Oxidation of ascorbate was followed by the decrease in A,, (A€ = 2.8 mM-' cm-I). One unit of activity is defined as the amount of enzyme that oxidizes 1 pmol ascorbate/min at room temperature (20°C) under the above assay conditions. The rates of oxidation of alterna- tive electron donors were measured using the same reaction mixture but with ascorbate replaced by 20 mM pyrogallol (A43o; A E = 2.47 mM-' cm-') or 10 mM guaiacol (A47o; A E = 26.6 mM-* cm-I), with the concentration of H,O, increased to 2 mM.

Kinetic Data Analysis and Spectrophotometry

The values of apparent K , and V,,, for TP on varying the concentration of H,O, at a fixed saturating concentration of ascorbic acid were calculated by triplicate measure- ments of initial velocity at each H,O, concentration. The same procedure was used to determine these kinetic con- stants for reducing substrates using a saturating concentration of H,O,. The reciprocals of the variances of initial velocity were used as weighting factors in the nonlinear regression fitting of initial velocity versus [substrate] data to the Michaelis-Menten equation. The fitting was carried out using a Gauss-Newton algorithm implemented in a BASIC program. Initial estimates of apparent K , and V,,,

were obtained from the Lineweaver-Burk equation. The apparent K , values were determined only for comparison with other peroxidases and no mechanistic significance in terms of Michaelis-Menten kinetic analysis is implied.

Rapid scans were recorded with a stopped-flow spectrophotometer (model SF-61, Hi-Tech Scientific, Salisbury, UK) equipped with a diode array system (MG 6000, Hi- Tech). UV/ visible absorption spectra were recorded in quartz cuvettes (1 cm) on a spectrophotometer (model UV-2101PC, Shimadzu, Columbia, MD) with a spectral bandwidth of 1 nm and a scan speed of 120 nm min-'.

Tryptic Digestion of Tea Apoperoxidase and Amino Acid Sequence Analysis

Apoenzyme was obtained by methylethylketone extraction of heme at pH 1.7 from 500 pg of native TP (Teale, 1959). The apoenzyme-containing aqueous phase was dialyzed against 10 mM sodium phosphate buffer to yield a pH of 7.6. Tryptic digestion was performed as described by Stone and Williams (1993), with the following modifica- tions. Concentrated stock solutions of urea, NH,HCO,, and DTT were added to the dialyzed apoenzyme solution to final concentrations of 6 M, 0.3 M, and 7.5 mM, respectively. This mixture was incubated at 50°C for 30 min. No iodoac- etamide treatment was carried out. The solution was diluted 3-fold with distilled water. ~-1-Tosylamide-2- phenylethyl-chloromethyl-ketone-treated trypsin (Sigma) was added to give a 1:lOO (w/w) enzyme:apoprotein ratio. Digestion was performed at 37°C for 90 min and stopped by boiling the mixture for 5 min. The peptides were separated on a reverse-phase C,, HPLC column (Phenomenex, Torrance, CA) using a linear gradient of O to 20% acetonitrile in 1% aqueous trifluoroacetate. The sequence analysis of purified peptides was performed by automated Edman degradation using one protein sequencer (model 491, Ap- plied Biosystems) for peptide 1 and a different protein sequencer (model LF 3000, Beckman) for peptides 2 to 5.

RESULTS

Purification of a Class 111 Peroxidase from Tea Leaves

Extractions using different buffers over the pH range 5.5 to 8.0 showed that for the initial extraction step Mes, pH 5.5, is optimal (data not shown). The presence of 30 mM ascorbic acid was found to be essential during the extraction and the first dialysis step, because it prevents the rapid formation of polymeric pigments, which are known to be inhibitors of peroxidase activity (Pruidze, 1987). Treatment of the extract with an insoluble PVP removed phenolic impurities and resulted in a slight increase in peroxidase activity. Cation-exchange chromatography of the tea extract showed the presence of severa1 basic isoenzymes. In this paper we are concerned with the purification and characterization of the second most abundant isoenzyme with APX activity. This peak in activity was eluted from an SP-Sepharose column with an NaCl gradient of 120 to 140 mM and was found to contain a single peroxidase isoenzyme (data not shown). Three subsequent column chroma-



1240 Kvaratskhelia et al. Plant Physiol. Vol. 114, 1997

tography steps were necessary to obtain the homogeneousenzyme. As a result, up to a 28-fold purification of theisoenzyme, with an overall yield of 1.8%, has beenachieved (Table I). The isoenzyme had a specific activity of150 jiimol min"1 mg'1 protein with ascorbic acid as thereducing substrate. The purified enzyme gave a singleband on SDS-PAGE (Fig. 1). The ASort,t pt,ak/A2go value,which is a good criterion of purity and heme content, was3.4. Silver IEF and activity staining of purified TP resultedin a single band with a pi value greater than 10 (data notshown).

Mr

The native Mr of the enzyme estimated by the gel-filtration method (Mr = 34,500 ± 1,000, mean ± SE) wasconsistent with the SDS-PAGE result (Fig. 1), indicatingthat the enzyme is monomeric. The precise Mr of theapoenzyme is 34,660 ± 10 (triple determination by electro-spray MS; data not shown). These data also provided fur-ther evidence for the homogeneity of the enzyme sample.

Substrate Specificity

One of the ways of distinguishing ascorbate and class IIIperoxidases is to compare the relative enzymatic activitiesfor guaiacol, ascorbate, and pyrogallol oxidation. The ratesof oxidation of these electron donors by TP were compa-rable, but the specific activity for ascorbate was slightlyhigher than for guaiacol (Table II). These relative activitiesappear to be characteristic of cytosolic APXs (Table II).However, the reactivity differences between TP and othertype III enzymes were striking, with HRP catalyzing theoxidation of guaiacol at 5-fold and pyrogallol at 25-foldhigher rates than ascorbate (Table II).

Stability and Inhibitor Studies

Although it has been reported that APX was not stable inan ascorbate-depleted medium (Chen and Asada, 1989),later studies revealed that inactivation in the absence ofascorbate was taking place only in ruptured chloroplasts;no inactivation was observed after purification to homoge-neity of both chloroplastic and cytosolic isoenzymes (Mi-yake and Asada, 1996). We have confirmed that the pres-ence of ascorbate is important but only at the initial isola-tion step. It is thought to scavenge radical intermediatesformed by enzymatic oxidation of phenolic compounds,thereby protecting the enzyme from inhibition by phe-

MW

66000—45000—36000—

29000—24000—

Figure 1. SDS-PAGE of TP. Lane 1, Purified TP; lane 2, HRP. Thearrows indicate the positions of the molecular weight markers BSA(66,000), egg albumin (45,000), glyceraldehyde-3-phosphate dehy-drogenase (36,000), carbonic anhydrase (29,000), and trypsinogen(24,000). The difference between TP and HRP is presumably due toglycosylation. MW, M,.

nolic oxidation products (Pruidze, 1987). However, afterthe first column purification step the enzyme was stablein the absence of ascorbate, suggesting that potential in-hibitors present in the plant extract were removed bycation-exchange chromatography.

Sodium azide and sodium cyanide, both potent inhibi-tors of many hemoprotein-catalyzed reactions, markedlyinhibited ascorbic acid peroxidase activity catalyzed by TP,suggesting the heme nature of the active group (data notshown). Specific inhibitors of APX, such as p-chloro-mercuribenzoate, hydroxyurea, and p-aminophenol (Chenand Asada, 1990; Amako et al., 1994), had only a slighteffect on the ascorbate-dependent peroxidase activity of TP(data not shown). However, APX from red algae has beenshown to remain 80% active after incubation withp-chloromercuribenzoate (Sano et al., 1996).

pH Optimum and Kinetic Studies

A universal buffer composed of 50 mM citrate and 50 mMphosphate was used to determine the pH dependence ofascorbic acid-oxidation activity by TP. The enzyme exhib-ited a pH optimum in the range of 4.5 to 5.0 (data notshown). The values of apparent Km for ascorbate and H2O2were 470 p,M (at 2 mM H2O2) and 188 JU.M (at 2 mM ascor-bate), respectively, at the saturating concentrations of thesecond substrate shown in the parentheses.

UV/Visible Absorption Spectra

The UV/visible absorption spectrum of TP was typical ofa heme-containing plant peroxidase, with a Soret peak at

Table 1. Purification of a class III peroxidase isoenzyme from 7 kg of tea leaves

Purification Step

ExtractSP-SepharoseGel filtrationMono-SHydroxyapatite

Protein

mg2400

123.62.41.5

Activity

units12,900

502360288226

SpecificActivity

[Lmol mg" '5.3

42101120151

Yield

°/

1004.02.82.21.8

Purification

-fold18

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Table I I . Effect of different reductants on the relative activities of class 111 peroxidases form tea (present work), horseradish (present work), and spinach (Chen and Asada, 1989) and cytosolic APX from tea (Chen and Asada, 1989) and pea (Mittler and Zilinskas, 1991a)

Relative Activity" Electron Donor

TP H R P Sninach Tea APX Pea APX

0%

Ascorbate 1 O 0 (1 50)b 1 O 0 (6) 1 O 0 NC 1 O 0 (1 00) 1 O0 (600) Cuaiacol 82 556 1,660 25.2 4 0 Pyrogallol 150 2475 21,080 497 174

r' The average of at least triple determination. The values in parentheses are the specific ascorbate oxidase activity (pmol min- ' mg-'). -, Not reported.

406 nm ( E = 115 mM-' cm-l; Table 111). In addition to this band, the oxidized enzyme showed a and /3 absorption bands at 500 and 640 nm (Fig. 2), respectively. These two bands disappeared and the Soret peak shifted to 438 nm when the enzyme was reduced by sodium dithionite (Fig. 2A). The CO complex of the ferrous enzyme exhibited a Soret peak at 423 nm and characteristic bands at 540 and 572 nm (Fig. 2B). Compound I was generated after the addition of a stoichiometric amount of H,O, to the native ferric enzyme in 50 mM sodium phosphate buffer, pH 7.0 (Fig. 3A). The conversion of the ferric enzyme to compound I was followed by stopped-flow rapid scan spectrophotometry, which showed that the Soret intensity decreased by about 35% at 407 nm, with the maintenance of an isobestic point at 426 nm. This clearly suggests a m-cation radical form for compound I. However, this catalytic intermediate was unstable and rapidly converted to compound I1 (half- time of approximately 60 s at 23°C). A single isobestic point at 395 nm between compound I and compound I1 was observed (Fig. 3B). A compound I1 spectrum was recorded after the addition of 2 M equivalents of H,O, to the native ferric enzyme (Fig. 4A). The Soret peak at 420 nm (91% intensity of that of the native enzyme; Table 111) and peaks characteristic for this intermediate at 531 and 552 nm were observed. Compound I1 slowly converted to native ferric enzyme within about 90 min, displaying a single isobestic point at 418 nm (data not shown). Compound I11 was formed after the addition of a 100-fold molar excess of H,O, to the native enzyme (Fig. 4B). This species showed a Soret peak at 417 nm and two characteristic peaks at 547 and 581 nm (Table 111).

Table 111. Absorption maxima for native and intermediate oxidation states of TP

Enzyme Species Absorption Maximum

nm Ferric enzyme 406 (1 15)" 500 (9.8) 640 (2.1) Compound I 407 (75) 553 (6.6) 650 (4.9) Compound II 420 (91) 531 (9.2) 552 (9.5) Compound I I I 41 7 (94) 547 (1 1) 581 (9.5) Ferrous enzyme 438 (91) 560 (14) CO complex of ferrous enzyme 423 (147) 540 (13.5) 572 (14)

a The values in parentheses are the extinction coefficient (units of mM-' Cm-').

Sequence of Trypsin-Digested Fragments of TP

Native purified TI' was not accessible to N-terminal sequencing, suggesting that, like other classical plant peroxidases, it is blocked by pyroglutamate (Welinder, 1979). This contrasts with the N termini of a11 native AI'X isoenzymes sequenced to date (Mittler and Zilinskas, 1991a; Chen et al., 1992; Koshiba, 1993). Since native TI' was highly resistant to proteolysis, the heme cofactor was first removed and the apoenzyme was subsequently digested

- (x 4)

0.30 :

-

.-. ...

0.20 : 0.10 " 0.00 c , E I I I I I I I I I

350 400 450 500 550 600 650 700 Wavelength (nm)

Figure 2. Optical spectroscopy of reduced (A) and Fe(ll)-CO complex (B) of TP. Dashed lines, Spectra of the native enzyme. The sample cuvette (0.5 mL, 1 cm) contained 3.5 p~ purified enzyme in 50 mM sodium phosphate buffer, pH 7.0. Ferric enzyme was reduced by the addition of approximately 300 pg of Na,S,O,. After the spectrum of reduced enzyme was recorded the sealed cuvette was flushed with CO and the spectrum of the Fe(ll)-CO complex was determined. The reference cuvette contained only buffer.



1242

O24

0.:

0.16

3 0.12

0.08

0.04

<

Kvaratskhelia et al.

A B

I “’I I

Plant Physiol. Vol. 114, 1997

d I & Sbo & 410 lp 430 4b 150 n0 500 UO 410 CB OP 440 450

Wavelength (nm) Wavelength (nm)

Figure 3. Stopped-flow rapid scan spectrophotometric studies of the reaction of TP (4 FM) with stoichiometric H202 (4 FM)

in 50 mM sodium phosphate, pH 7.0, 23°C. A, Scans taken 0.3, 0.5, 0.7, 1 .O, and 1.4 s after mixing. 6, Scans taken 4, 12, 22, 45, and 65 s after mixing. The arrows show the direction of the absorbance change with time. Abs, Absorbance.

with trypsin as described above (see ”Materials and Meth- ods”). Reverse-phase C,, column chromatography yielded approximately 30 fragments (Fig. 5).

The N-terminal sequences of five purified fragments containing a sufficient number of residues were determined (Fig. 5). The sequencing of peptide 1 resulted in a blank in the sixth cycle (Fig. S), indicating that this residue is either Cys or a glycosylated residue. The sequence comparison with other classical plant peroxidases suggests that this residue is probably Cys located in a conserved loop (Fig. 6). In contrast, APXs have neither Cys at this position nor a glycosylated residue (Mittler and Zilinskas, 1991a). It was pos- sible to assign peptides 1 and 3 of TP by the alignment of their sequences to those of known classical plant peroxidases and APXs (Fig. 6). The comparison shows that the Trp residue that is conserved in the distal heme pocket of APX (Mittler and Zilinskas, 1991a; Welinder, 1992) is absent in TP. This residue in TP, like other classical peroxidases, is replaced by nonpolar Phe (Fig. 6). Unlike APX, TP does contain the conserved Asp ligand that in class I11 peroxidases is coordinated to a Ca2+ ion (Welinder, 1992). There is 75% identity between peptides 1 and 3 of TP and the heme distal pocket regions of HRP (Fig. 6). There is also 30% homology between peptide fragment 5 and the C-terminal region (helixes I and J) of classical plant peroxidases (Fig. 6). However, there is very little sequence similarity between the five peptide fragments of TP and APX. These results clearly suggest that TP is a class I11 rather than a class I peroxidase.

DlSCUSSlON

Biochemical Properties

APX is distinguished from classical plant peroxidases by its marked preference for ascorbic acid as an electron do- nor. The biochemical properties of a large number of APXs isolated from diverse plant sources (tea leaves [Chen and Asada, 19891, pea [Mittler and Zilinskas, 1991b], red algae [Sano et al., 19961, oilseeds [Bunkelmann and Trelease, 19961, root nodules [Dalton et al., 19871, potato tubers [Elia et al., 19921, seedlings and leaves of maize [Koshiba, 19931, and spinach [Miyake et al., 19931) have confirmed the high specificity of these class I peroxidases for ascorbate. In contrast, class I11 plant peroxidases are known to preferentially oxidize organic phenolic compounds and exhibit a very poor activity for ascorbate (Chen and Asada, 1989). TP appears to be the first example of a class I11 peroxidase with a remarkably high APX activity. The catalytic properties of TP show the striking functional differences between this isoenzyme and other classical plant peroxidases. The rate of oxidation of ascorbate by TP is 24 times higher than that of HRP and is comparable to those of cytosolic APXs. It is particularly noteworthy that this enzyme exhibits 1.5-fold higher specific APX activity than cytosolic APX isoenzyme from tea (Chen and Asada, 1989). Moreover, TP catalyzes the oxidation of ascorbate at a higher rate than guaiacol, whereas other type I11 peroxidases are characterized by




a, 0.50

5 0.40 e $ 0.30

O

2 0.20 0.10

0.00

Wavelength (nm)

Figure 4. Optical spectroscopy of TP compound II (A) and compound 111 (B). Dashed lines, Spectra of the native enzyme. The sample cuvette (0.5 mL, 1 cm) contained 6 y~ purified enzyme in 50 mM sodium phosphate, pH 7.0. The compound I1 spectrum was recorded after the addition of 2 equivalents of H,Oz to the native ferric enzyme. Compound 111 formed after the addition of a 100-fold molar excess of H,O, to the native enzyme.

2-orders-of-magnitude higher specific activity for guaiacol relative to ascorbate.

The spectroscopic studies of peroxidase cycle intermedi- ates of TP and HRP have shown another significant functional difference between these two enzymes. Compound I of HRP is very stable and is only very slowly converted to compound I1 (half-time typically 20 min). In contrast, the addition of stoichiometric H,O, to TI' results in a very unstable compound I, which is converted to compound I1 within approximately 2 min in the absence of added reductant (Fig. 3). The formation of an extremely unstable compound I is a characteristic of APX (Patterson et al., 1995). Compound I of pea APX, formed with an equimolar mixture of the native enzyme and H,O,, was converted to compound I1 within 60 s (Marquez et al., 1996). Lignin peroxidase compound I has also been reported to have a very short half-time ( approximately 30 s; Renganathan and Gold, 1986). The electron donors for the reduction of compound I to compound I1 in the absence of an exogenous reducing substrate are still not well established. Specula- tions include reducing impurities in the enzyme preparation or the endogenous conversion by reducing groups within the enzyme (Harvey et al., 1989; Tuisel et al., 1990).

Although H,O, has also been suggested as an electron donor (Andrawis et al., 1988), it is less likely in this study,

O00 1 ' ' ' I ' ' ' ' ' ' ' ' ' ' ' ' ' I ' ' ' ' ' ' ' ' 1 10 15 20 25 30 35 40

Time (min)

Figure 5. HPLC purification and sequence data of trypsin digestion fragments of TP. The tryptic-digested peptide fragments of TP were separated on a reverse-phase C,, HPLC column using a 45-mL linear gradient of O to 20% acetonitrile (solid line) in 1% aqueous trifluoroacetate at flow rate of 1 mL/min. 1, Leu-His-Phe-His-Asp-*-Phe- Val-Lys; 2, Ser-Thr-Val-l le-Thr-G I y-G ly-Pro-Ser-Ser-C lu-Val-Pro- Leu-Gly-Gly; 3, Met-Ala-Ala-Ser-Leu-Leu-Arg; 4, Tyr-Ala-Glu-Asn- Ser-G lu-Leu-Phe-Phe-G I u-G I n; and 5, G ly-Asp-G I n-Asn-Leu-Phe- Phe-Leu-Ala-Phe-Val. Abs, Absorbance.

because 1 equivalent of H,O, was used for compound I formation. To remove any impurities from the enzyme solution, we added 2 equivalents of H,O, to TP, assuming that any exogenous reductants would be quickly oxidized. After rapid formation of compound I1 this intermediate was converted back to the native ferric form within approximately 90 min. The subsequent addition of stoichiometric H,O, to the enzyme still resulted in the formation of an unstable compound I species. The rate of

Dista1 Haem Pocket

( Helix B )

Tea P Y K

HRP C I N

Tob P A N

Pea APX C A P L I F

C-Terminal Region

( Helixes I and J )

Tea P

HRP C

Tob P

Pea APX

Figure 6. Amino acid sequence alignment of TP with tobacco (La- griminr et al., 1987), horseradish (Welinder, 1992), and pea APXs (Mittler and Zilinskas, 1991a). *, Residue not determined.



1244 Kvaratskhelia et al. Plant Physiol. Vol. 114, 1997

reduction of this species to compound I1 was the same as for the untreated enzyme. Intensive dialysis of the homo- geneous enzyme against Milli-Q water (Millipore) also had no effect on the stability of compound I. These results suggest that the presence of reducing groups within the enzyme are more likely to be the cause of the rapid reduction of compound I.

Amino Acid Sequence

Although TP exhibits striking functional diversity from classical plant peroxidases, the TP amino acid sequence data suggest that it is a class I11 peroxidase (see “Results”). Although TP and APX have similar catalytic properties, there is very little similarity between the sequences of these two enzymes. The presence of Trp residues in APX at positions analogous to Trp-51 and Trp-191 in CCP (which are replaced by nonpolar Phe in class I11 peroxidases) appears to be an important structural difference at the active sites of these enzymes (Patterson and Poulos, 1995). There- fore, it was anticipated that proximal and distal Trp residues in APX might be responsible for the distinct catalytic properties of ascorbate-specific peroxidases (Pappa et al., 1996). However, our sequence data show that there is no Trp residue in the distal pocket of TP. As in other classical plant peroxidases, the distal Trp is substituted by Phe (Welinder, 1979; Schuller et al., 1996). Despite this, TP, like APX, exhibits a remarkably high APX specific activity and forms a very unstable compound I. Lignin peroxidase, which, like type I11 peroxidases, has a Phe residue instead of a distal Trp (Poulos et al., 1993), is also characterized by an unstable compound I.

The sequence of TP on the proximal side of the heme has not been determined. However, it was recently demon- strated that the proximal Trp is not essential for APX activity (Pappa et al., 1996). In APX the indole ring nitrogen of the proximal Trp is hydrogen-bonded with an Asp residue, which itself hydrogen bonds with the His proximal heme ligand (Patterson and Poulos, 1995). This His-Asp- Trp H-bonded triad is not conserved in classical plant peroxidases, because these enzymes contain a nonpolar Phe at the position homologous to the proximal Trp in APX (Welinder, 1979; Schuller et al., 1996). Pappa et al. (1996) mutated a proximal Trp to Phe in pea APX. This mutation disrupted the H-bonding network between these three proximal residues, thereby making the proximal region of the enzyme resemble that of a classical plant peroxidase. This structural change did not induce a change in ascorbate-dependent activity but did cause the characteristic rapid endogenous reduction of the porphyrin radical cation (compound I) to be increased by a factor of 2. Thus, neither distal nor proximal Trp residues are responsible for APX activity. The complete sequencing of TP, which is in progress, will hopefully provide further insights into structure-function relationships for plant peroxidases.

Physiological Role of TP

The sequence alignment (Fig. 6) suggests that, like HRP and TobP, TP is an extracellular enzyme exported via the

ER. The presumed Cys could be in the conserved cystine loop, with the preceding Asp providing a carboxylate ligand at the Ca2+-binding site adjacent to the distal heme pocket of TP. In addition, the blocking of the N terminus, presumably due to a pyroglutamate residue by analogy with other extracellular peroxidases, would occur by post- translational modification in the ER. Despite this homology, TP and TobP exhibit distinctly different catalytic properties from HRP (Gazaryan and Lagrimini, 1996). TP and TobP share not only sequence homology but also similar substrate specificities and pH optima for ascorbate oxidation (M. Kvaratskhelia, I.G. Gazaryan, and R.N.F. Thorne- ley, unpublished data). The preparation of antibodies to TP should provide a means of confirming its cellular location.

In conclusion, this work clearly demonstrates that tea plants contain a class I11 peroxidase isoenzyme with a remarkably high specific activity for ascorbate in addition to the well-characterized APX enzyme. This suggests that the ascorbate-dependent detoxification of H,O, in plants may not be exclusively carried out by APXs but could also be effected by this type I11 peroxidase that we have isolated from tea. Moreover, the striking functional differences between TP and conventional classical plant peroxidases (e.g. HRP, spinach peroxidase) indicate that class I11 peroxidases should be assigned to two subclasses depending on their physiological role. The enzymes that exhibit a remark- able preference for ascorbate as an electron donor (e.g. TP and TobP) probably function primarily as scavengers of O,-activated species to minimize O,-induced stress. The major role for enzymes preferentially oxidizing phenolic compounds ( e g HRP and spinach peroxidase) would seem to be in the lignification of plant cell walls and in related wound-healing processes.

ACKNOWLEDCMENTS

We thank Dr. Andrew Parry (Unilever, Colworth Laboratory) for helpful discussions and facilitating the supply of freshly frozen tea leaves from Kenya. We are grateful to Dr. Mike Naldrett (John Innes Centre) and Dr. Dick van Wassenaar (Unilever Research Laboratorium, Vlaardingen) for amino acid sequencing.

Received February 18, 1997; accepted April 16, 1997. Copyright Clearance Center: 0032-0889/97/ 114/ 1237/09.

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