ARCHIVES

Vol.

OF BIOCHEMISTRY

284, No. 1, January,

AND

BIOPHYSICS

pp. 151-157,

1991

Purification and Characterization of p-Coumaroyb glucose Hydroxylase of Sweet Potato (lpomoea batatas) Roots Miho

Tanaka

and Mineo

Institute

for Biochemical

Received

June

11,

Kojima’

Regulation,

1990, and in revised

Faculty of Agriculture,

form

August

Nagoya University,

INTRODUCTION

Higher plants produce various kinds of phenolic compounds containing caffeic acid (3,4-dihydroxycinnamic acid) or its derivatives. Chlorogenic acid (5-0-caffeoyl correspondence

should

0003.9861/91 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form

be addressed.

Nagoya 464-01, Japan

27, 1990

p-Coumaroyl-D-glucose hydroxylase in sweet potato (Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination of anion- and cation-exchange, hydrophobic and gel filtration chromatography. The purified enzyme was a monomer with a molecular weight of 33,000 and pI of 8.3. The purified enzyme showed not only hydroxylase activity but also polyphenol oxidase activity. L-Ascorbic acid was the best electron donor for the hydroxylation reaction, which had an optimum pH of 7.0. The enzyme hydroxylated p-coumaroyl-D-glucose, p-coumaric acid, and pcresol but did not act on o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, p-hydroxybenzoic acid or L-tyrosine. While the enzyme utilized pcoumaroyl-D-glucose and p-coumaric acid equally at pH 7.0, it hydroxylated only p-COUmarOyl-D-glucose at pH 5.5. The enzyme oxidized diphenols such as D,L-(3,4-dihydroxyphenyl) alanine and caffeic acid, but exhibited no clear pH optimum in this reaction characteristic of polyphenol oxidase. Both the hydroxylase and the polyphenol oxidase activities were strongly inhibited by @-mercaptoethanol, diethyldithiocarbamate, KCN, andpcoumaric acid (in concentrations higher than 5 mM). Ammonium sulfate and sodium chloride activated the hydroxylase activity but not the polyphenol oxidase activity of the enzyme. The enzyme activity and L-ascorbic acid contents changed in a manner suggesting their involvements in chlorogenic acid biosynthesis during incubation of sliced sweet potato root tissues. 8 1991 Academic PMS. h.

’ To whom

Chikusa-ku,

quinic acid) is one of these derivatives and is ubiquitous in the plant kingdom (1) but its biosynthetic pathway has not yet been established. From studies on sweet potato roots in which a marked synthesis of chlorogenic acid is induced by wounding (2-g)) we have proposed a pathway with hydroxycinnamoyl-D-glucoses as intermediates. On the other hand, Stijckigt and Zenk proposed a pathway with hydroxycinnamoyl-CoA as an intermediate (9). Our proposed pathway is as follows; t-cinnamic acid, produced from L-phenylalanine by the action of L-phenylalanine ammonia lyase (lo), is converted to t-cinnamoyl-D-glucose in the first step of the pathway and then converted to caffeoyl-D-glucose via p-coumaroyl-D-glucose. Finally, a trans-esterification between the D-glucose moiety of caffeoyl-D-glucose and D-quinic acid produces 50caffeoyl quinic acid (chlorogenic acid). We have previously isolated and characterized two enzymes of the proposed pathway (5,6). However, two enzymes in the pathway remained to be detected and studied; one is t-cinnamoyl-D-glucose hydroxylase, which hydroxylates t-cinnamoyl-D-glucose to form p-coumaroyl-D-glucase, and the other is p-coumaroyl-D-hydroxylase, which hydroxylatesp-coumaroyl-D-glucose to caffeoyl-D-glucose. We tried to detect the hydroxylase activity of p-coumaroyl-D-glucose or p-coumaric acid in sweet potato roots by following the reported methods for various related hydroxylases (11-13). However, we were not able to detect any analogous enzyme. Instead, we found that a crude enzyme preparation from wounded sweet potato roots hydroxylated p-coumaroyl-D-glucose and p-coumaric acid efficiently in the presence of L-ascorbic acid. The enzyme preparations also exhibited polyphenol oxidase activity. It, however, is reported that polyphenol oxidase in sweet potato roots showed no hydroxylase (cresolase) activity in vitro (14, 15). Here, we describe the purification and characterization of a p-coumaroyl-D-glucose hydroxylase from wounded 151

Inc. reserved.

152

TANAKA

AND

sweet potato roots. The enzyme appears to be involved in chlorogenic acid biosynthesis in the tissue. EXPERIMENTAL

PROCEDURES

Materials Chemicals. p-Coumaroyl-D-glucose was chemically synthesized by a method described previously (5). The following materials were obtained from commercial sources: catalase (Sigma), DEAE-Sephacel (Pharmacia), butyl-Toyopearl 650M (Toso), Diaflo YM-IO filter (Amicon), Ampholine PAG plate, pH 3.5-9.5 (Pharmacia), protein molecular weight standards (calibration protein II, Boehringer), protein assay kit (BioRed), silver stain kit (Wake), and Centricon 10 (Amicon). Roots of sweet potato (Ipomoea batatas Lam., cv, Norin 1) were harvested in October and stored at 13’C until used.

Enzyme Assay Hydroxylase activity. Because of the limited availability of p-coumaroyl-D-glucose, the enzyme was assayed using p-coumaric acid as a substrate, unless otherwise stated. The reaction mixture contained the enzyme, 3 pmol p-coumaric acid, 12 wmol sodium L-ascorbate and 125 units of catalase in 800 ~1 of 50 mM Tris-HCl (pH 7.5). Catalase was included to prevent the peroxidase-related hydroxylation ofp-coumaric acid (16). The mixture was allowed to incubate at 30°C for 30 min. Reactions were stopped by adding 60 ~1 of 12 N HCl and 0.5 ml ammonium sulfate (4.1 M). The mixture was extracted twice with 3 ml of ethylacetate. The combined ethylacetate extracts were concentrated in cacao and applied to a paper strip (3 X 45 cm) as a band. The paper then was developed with the upper phase of a mixture of benzene-acetic acid-water (40:10:1, v/v). A fluorescent band of caffeic acid (reaction product) at R, 0.3 was cut out and eluted with a mixture of ethanol and water (3:1, v/v). The absorbance of the eluate was measured at 310 nm and the value was interpolated on a standard curve prepared with authentic caffeic acid. One unit of activity was defined as the amount of enzyme which produced 167 nmol of caffeic acid per minute under the assay conditions. Polyphenol oxidase actiuity. During the enzyme purification, polyphenol oxidase was also monitored using o,L-(3,4-dihydroxyphenyl) alanine (D,L-dopa)’ as a substrate. The activity was assayed according to the method of Duke and Vaughn (16) with minor modifications. The assay mixture contained 25 pmol D,L-dopa, 125 units catalase, and the enzyme in 1.25 ml of 80 mM Tris-HCl (pH 7.5). Catalase was added to prevent peroxidation of the substrate. The assay was initiated by addition of the enzyme and the activity was measured in terms of the increase in absorbance at 490 nm at 25OC. One unit of activity was defined as the amount of enzyme causing an increase of 0.01 in the absorbance at 490 nm per minute multiplied by a factor of 1000. This multiplication factor was used to make the data more manageable for presentation. D,L-Dopa was used in the assay of polyphenol oxidase in this report, unless otherwise mentioned. Enzyme pur$ication. It has been shown that a marked production of chlorogenic acid takes place in sweet potato root tissue in response to wounding and that induction of some enzymes involved in its synthesis also occurs (4). We therefore used sweet potato root slices (2 mm thick) which had been incubated at 25°C for 24 h as a source of the enzyme. Three kilograms of slices was homogenized with 3 liters of 50 mM TrisHCl (pH 7.5) containing 30 g sodium isoascorbate and 300 g polyvinylpolypyrrolidone. The homogenate was squeezed through a cheesecloth and centrifuged at 5800g for 50 min. The supernatant was fractionated with ammonium sulfate and the precipitate obtained between 30 and

Z Abbreviations used: D,L-dopa, SDS, sodium dodecyl sulfate.

D,L-(3,4-dihydroxyphenyl)alanine;

KOJIMA 60% saturation was collected by centrifugation at 58OOg for 50 min. The precipitate was dissolved in 250 ml of 50 mM Tris-HCl (pH 7.5) and applied to a column of Sephadex G-25 (6.5 X 30 cm) preequilibrated with the same buffer. The void volume fraction (250 ml) from the column was collected and applied to a column of DEAE-Sephacel (5 X 16 cm) which had been equilibrated with 50 mM Tris-HCl (pH 7.5). The column was washed with 2 bed volumes of the same buffer and then eluted by a linear gradient of NaCl between 0 and 0.5 M in 50 mM Tris-HCl (pH 7.5, 1 liter). The unabsorbed fraction (420 ml) was collected and condensed to 22 ml by filtration through DIAFLO YM-10. Solid ammonium sulfate was then added to the condensed solution to give 30% saturation and centrifuged at 14,lOOg for 20 min after standing for 1 h. The resulting supernatant was applied to a butyl-Toyopearl column (2.6 X 13 cm) which had been equilibrated with 50 mM Tris-HCI (pH 7.5) containing 30% ammonium sulfate. The column was washed with 2 bed volumes of the same buffer and subsequently eluted by decreasing a linear gradient of ammonium sulfate between 30 and 0% in 50 mM Tris-HCl (pH 7.5, 350 ml). The active fractions were pooled (120 ml) and condensed to 9 ml using a DIAFLO membrane filter. The condensed solution was dialyzed against 50 mM phosphate buffer (pH 6.5) and then applied to a CM-Toyopearl650 M column (0.9 X 16 cm) preequilibrated with 50 mM phosphate buffer (pH 6.5). The column was washed with 2 bed volumes of 50 mM phosphate buffer (pH 6.5) and eluted by a linear gradient of NaCl between 0 and 0.2 M in 50 mM phosphate buffer (pH 6.5,50 ml). The active fractions were pooled (22 ml), condensed to 4 ml by filtration, and applied to a column of Ultrogel AcA 44 (1.6 X 75 cm) that had been equilibrated with 50 mM Tris-HCI (pH 7.5). The sample was eluted from the column with the same buffer solution and the active fractions were pooled.

Protein Determination Protein was determined either by the Lowry Bio-Rad protein assay kit. Bovine serum albumin

method (17) or with a was used as a standard.

Molecular Weight Estimation The molecular weight of native enzyme was estimated by calibrated Ultrogel AcA 44 gel filt.ration under the conditions described above. The reference proteins and their molecular weights were aldolase (158,000), bovine serum albumin (68,000), ovalbumin (45,000), chymotrypsinogen A (25,000), and cytochrome c (12,500). The subunit molecular weight of the enzyme was estimated by comparing the mobility of protein during polyacrylamide gel (10%) electrophoresis (0.1% SDS) with the mobility of other proteins of known molecular weight. Proteins were visualized by silver staining. The reference proteins used and their subunit molecular weights were phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), trypsin inhibitor (ZO,lOO), and n-lactoalbumin (14,400).

Determination of Isoelectric Point The purified enzyme was isoelectrically focused on Ampholine PAG plate (pH 3.5-9.5) using a flat bed apparatus (Pharmacia) for 1.5 h at 1500 V. The enzymes were visualized by silver staining. The reference proteins and their pI were: amyloglucosidase (3.50), soybean trypsin inhibitor (4.55), fi-lactoglobulin A (5.20), bovine carbonic anhydrase B (5.85), human carbonic anhydrase B (6.55), horse myoglobin (6.85, 7.35), lentil lectin (8.15, 8.45, 8.65), and trypsinogen (9.30).

Substrate Specificity of the Enzyme in the Hydroxylation Reaction Various compounds (4 mM) were incubated in the standard hydroxylase assay mixture for 1 h at 30°C. After incubation, the assay mixture was extracted with ethylacetate three times. The ethylacetate fractions were concentrated and the residues were spotted on Avicel plates which

p-COUMAROYL-D-GLUCOSE

HYDROXYLASE

were developed with the upper phase of benzene-acetic acid-water (40: lO:l, v/v). The developed plates were examined under uv lamps (302 nm, 260 nm) or by spraying with 2% ferric chloride in ethanol. When L-tyrosine was tested, the incubated reaction mixture was directly spotted on the plate after addition of 12 N HCl (50 ~1) since the anticipated product, L-dopa, is not extracted into the ethylacetate layer. The spotted plate was developed with methylisobutylketone-formic acid-water (14: 3:2, v/v) and sprayed with ninhydrin reagent for detection of spots.

Time-Course Analysis of Changes in Enzyme Activity and in the Content of L-Ascorbic Acid and Polyphenols in Sweet Potato Root Disks During Incubation Disks (2.0 X 0.2 cm) were prepared from sweet potato roots and incubated at 25°C in a moist chamber. After incubation for various periods, disks were harvested and assayed for p-coumaroyl-D-glucose hydroxylase, L-ascorbic acid, and polyphenol contents. Enzyme preparation and assay. The DEAE-Sephacel unabsorbed fraction (4 ml) was prepared as described under Enzyme purification from 30 incubated disks and condensed to 1.0 ml using a Centricon 10. p-Coumaroyl-D-glucose hydroxylase activity was assayed using the condensed enzyme solution under standard conditions. L-Ascorbic acid contents. The five incubated disks were homogenized in 15 ml of 2% m&a-phosphoric acid. The homogenate was centrifuged at 12,000g for 10 min. L-Ascorbic acid content in the supernatant was determined by the method of Tono and Fujita (18). The five incubated disks were extracted with Polyphenol contents. 50 ml of 80% ethanol. Polyphenol contents in the extracts were determined according to the method of Zucker and Ahrens (19). Since the major polyphenolic compounds in wounded sweet potato roots are chlorogenic and isochlorogenic acids, the standard curve was made using authentic chlorogenic acid. RESULTS

Purification

of the Enzyme

The results of a typical purification procedure are shown in Table I. In the step involving DEAE-Sephacel chromatography, activity was observed in both unabsorbed and absorbed fractions (data not shown). The enzymes in the unabsorbed and absorbed fractions differed from each other in the ratio of hydroxylase activity to

IN

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polyphenol oxidase activity; the enzyme in the unabsorbed fraction showed high hydroxylase activity and low polyphenol oxidase activity, while the one in the absorbed fraction exhibited high hydroxylase activity as well as high polyphenol oxidase activity. Since the aim of the present study was the detection and characterization of the enzyme which is responsible for hydroxylation reaction in chlorogenic acid biosynthesis in sweet potato roots, we used the enzyme in the unabsorbed fraction for subsequent purification steps. After gel filtration through Ultrogel AcA 44, the eluant protein profile showed only one peak which corresponded with the peaks of the hydroxylase and polyphenol oxidase activities (data not shown). This procedure resulted in 5250-fold purification of the hydroxylase enzyme with an 18% yield (Table I). The SDS-polyacrylamide gel electrophoregrams of the enzyme preparation in various purification steps are given in Fig. 1. Properties of the Enzyme Identification of hydroxylase reaction products. The products of the hydroxylase reactions were identified using p-coumaroyl-D-glucose andp-coumaric acid as substrates and L-ascorbic acid as an electron donor. The product formed from p-coumaroyl-D-glucose was chromatographed on a Avicel plate, where it gave the sameR, values as authentic CaffeOyl-D-glucose in three solvent systems; RI 0.6 in n-butanol-acetic acid-water (20:5:11, v/v); R, 0.20 in methylisobutylketone-formic acid-water, (14:3:2, v/v; upper layer); and R, 0.58 in ethylacetate-acetic acidformic acid-water (18:3:1:4, v/v). The product formed fromp-coumaric acid was extracted into ethylacetate and chromatographed on Avicel plates using three solvent systems. It showed the sameRfvalues as authentic caffeic acid: Rf 0.76 in n-butanol-pyridine-water (14:3:3, v/v); Rf 0.29 in benzene-acetic acid-water (125:72:3, v/v); and R, 0.8 in n-butanol-ethanol-water (4:1:2, v/v).

TABLE

I

Purification of the Enzyme from Sweet Potato Roots Specific

Total Fraction Crude extract Ammonium sulfate (30-60%) Sephadex G-25 DEAE-Sephacel Butyl-Toyopearl CM-Toyopear Ultrogel AcA44 Note.

Three

kilograms

protein (md

Hydroxylase (A) (units/mg)

8800 3350 23.8 7.7 0.82 0.33

1.2 1.7 120 300 1710 2690 sweet potato

root

Ratio of activities (A/B)

Yield (4 (%)

0.50

1.0

100

0.51 0.90 41.0 75.0 590 400

2.4 1.9 2.9 3.9 2.9 6.7

210 150 56 45 28 18

Oxidase (B) (units/mg)

0.51

9890

of wounded

Activity

slices were used. Unit

of enzyme

activity

is defined

in the text.

Purification (A) (-fold) 1 2.4 3.4 230 580 3350 5250

154

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AND

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TABLE I1

123456

Electron Donor Specificity in Hydroxylase Reaction by the Enzyme kDa

Electron

94

+

67

+

43

+

30

+

20

+

FIG. 1. SDS-polyacrylamide gel (10%) electrophoresis of enzyme preparations in various purification steps. The protein bands were visualized by silver staining. 1, molecular weight markers; 2, void volume fraction after Sephadex G-25 chromatography; 3, unabsorbed fraction after DEAE-Sephacel chromatography; 4, active fraction after butylToyopearl650M chromatography; 5, active fraction after CM-Toyopearl 650M chromatography; 6, the purified enzyme after Ultrogel AcA 44 chromatography.

Molecular weight. The relative molecular weight of the purified native enzyme was estimated to be 33,000 by means of gel filtration on a calibrated Ultrogel AcA 44 column (data not shown). When subjected to SDS-polyacrylamide gel electrophoresis, the purified enzyme yielded one silver-stained band with a molecular weight of 40,000 (Fig. 1). Thus, the enzyme appeared to be a monomeric protein with an approximate molecular weight of 33,000. The isoelectric point of the enzyme Isoelectric point. was determined to be 8.3 by electrofocusing (data not shown). This was consistent with its behavior in DEAESephacel chromatography; it was eluted in the unabsorbed fraction at pH 7.5. Electron donor specificity. Various reducing agents were assayed at 7.5 mM for activity as electron donors in the hydroxylation ofp-coumaric acid (Table II). L-Ascorbic acid showed the highest activity and was utilized for further studies in the present work. pH optimum of the enzyme. The pH dependency of the hydroxylase reactions was determined using p-coumaric acid and p-coumaroyl-D-glucose as substrates (data not shown). Although the optimum pH of the enzyme for the two substrates was the same, i.e., pH 7.0, the dependency on pH was significantly different. Withp-coumaric acid, there was no activity below pH 5.5, while a broad peak was obtained with p-coumaroyl-D-glucose. The pH

donor

Relative

(7.5mM) L-Ascorbate NADH NADPH Dimethyltetrahydropterine Glutathione

activity” (%) 100 69 64 21 4

’ The activity was measured by monitoring the hydroxylation coumaric acid in the standard assay mixture containing various donors (7.5 mM).

of pelectron

dependency of the polyphenol oxidase reaction was determined using D,L-dopa as a substrate. The enzyme showed no distinct dependency on pH; it showed almost the same activity between pH 5.5 and 8.5 (data not shown). Substrate specificity. The substrate specificity of the enzyme in the hydroxylation reaction was examined using p-coumaroyl-D-glucose, p-coumaric acid, o-coumaric acid, m-coumaric acid, 4-hydroxy-3-methoxycinnamic acid, phydroxybenzoic acid, p-cresol, and L-tyrosine as substrates. It acted only on p-coumaroyl-D-glucose, p-coumaric acid, and p-cresol. p-Coumaroyl-D-glucose and p-coumaric acid are present in sweet potato roots, while p-cresol is not (8). We compared the activity of the enzyme on p-coumaroyl-D-glucose and p-coumaric acid at pH 7.0 and at pH 5.5 (Table III). The enzyme hydroxylated pcoumaroyl-D-glucose and p-coumaric acid with approximately equal activity at pH 7.0. In contrast, the enzyme hydroxylated only p-coumaroyl-D-glucose at pH 5.5. The K, value of p-coumaroyl-D-glucose was determined to be 1.5 mM at pH 7.0. The enzyme oxidized diphenols such as D,L-dopa (K,, 49 mM), caffeic acid (K,, 24 mM), and pyrocatechol (K,, 1.4

mM).

TABLE III pH

Dependency

of Substrate

Specificity

in Hydroxylase Reaction Amount of product (nmol/min) Substrate p-Coumaroyl-D-glucose p-Coumaric acid

at

pH 7.0 190 150

at pH 5.5 180 0

Note. The reaction mixture contained the purified enzyme (0.5 pg), 2.9 pmol of either p-coumaroyl-D-glucose or p-coumaric acid, 12 ymol sodium L-ascorbate and 125 units catalase in 800 ~1 of either pH 7.0 or pH 5.5 citrate-NaOH buffer (25 mM). The assay was carried out under the standard assay conditions.

p-COUMAROYL-D-GLUCOSE

HYDROXYLASE

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We also examined the effect Of p-COUmarOyl-D-glUCOSe on the enzyme activity. The maximum solubility of pCOUmarOyl-D-ghXOSe,however,was 4.0mM.Atsuchconcentration, inhibition was not evident in the hydroxylation reaction, but was detected in the polyphenol oxidase reaction. Changes in p-Coumaroyl-D-Glucose Hydroxylase Activity and in Contents of L-Ascorbic Acid and Polyphenols during Incubation of Sweet Potato Root Disks

p-Coumaric

Acid

Concentration

(mM)

FIG. 2. Effect of substrate (p-coumaric acid) concentration on the hydroxylase reaction. The enzyme assay was performed in the reaction mixtures including various concentrations of p-coumaric acid as a substrate.

Effect of Various Inhibitors

on Enzyme Activity

The hydroxylase activity of the enzyme was inhibited by P-mercaptoethanol (noncompetitive, Ki, 1.4 X 1O-4 M), diethyldithiocarbamate (competitive, Ki, 9.2 X lop6 M) and KCN (uncompetitive, Ki, 5.1 X 10e5 M). The oxidase activity of the enzyme was also inhibited by these inhibitors, although the Ki value for each inhibitor differed from the hydroxylase reaction; P-mercaptoethanol (noncompetitive, Ki, 2.0 X lop4 M), diethyldithiocarbamate (competitive, K,, 1.4 X 10m4 M), and KCN (uncompetitive, Ki, 1.2 X 10m4M). These results suggested the involvement of a disulfide bond and metals such as Cu2+ in both enzyme reactions. Effect of Ammonium Enzyme Activity

To evaluate the possible role of p-coumaroyl-D-glucose hydroxylase in vivo, its activity in sliced disks incubated over a period of 2$ days was examined in relation to contents of L-ascorbic acid and polyphenols in the disks (Fig. 3). L-Ascorbic acid was included in the assays in this experiment since it appeared to be an electron donor in vivo (Table II). The activity of p-coumaroyl-D-glucose hydroxylase and the amount of L-ascorbic acid increased during the incubation period, as did the polyphenol content, suggesting their involvement in the biosynthesis of polyphenols such as chlorogenic acid in sweet potato roots. The increase in L-ascorbic acid in incubated sweet potato roots had previously been reported by Oba (22). DISCUSSION Several hydroxylases involved in the formation of caffeic acid or its derivatives have been already detected and characterized. t-Cinnamic acid 4-hydroxylase is known to be responsible for the conversion of t-cinnamic acid to p-coumaric acid (4-hydroxycinnamic acid) in the biosynthesis of phenylpropanoids such as lignin and flavonoids (23). This enzyme is a cytochrome P450 enzyme and is

Sulfate and Sodium Chloride on

Since Vaughan and Butt reported that ammonium sulfate and sodium chloride activated both hydroxylase and oxidase activities of polyphenol oxidase from spinach beet (20), we also tested the effect of these compounds on the purified enzyme (data not shown). Both compounds activated hydroxylase activity considerably, but showed an inhibitory effect instead on polyphenol oxidase activity. This was in contrast to results obtained with spinach beet polyphenol oxidase (20). Effect of p-Coumaric

Acid on Enzyme Activity

As shown in Fig. 2, a marked inhibition of the hydroxylation of p-coumaric acid was observed in assay mixtures containing p-coumaric acid in concentrations higher than 5 mM. p-Coumaric acid also noncompetitively inhibited polyphenol oxidase (Ki, 5.7 X 10m3 M). Inhibitions by pcoumaric acid have previously been reported with polyphenol oxidases from sweet potato (15) and pear (21).

0

I Incubation

2 Time (day)

FIG. 3. Time course of changes in p-coumaroyl-D-glucose hydroxylase activity(a) and contents of L-ascorbic acid(b) and polyphenols(c) in sweet potato root disks during incubation. The experimental conditions are given under Experimental Procedures.

156

TANAKA

localized in microsomal membranes (24). On the other hand, the enzyme which catalyzes the hydroxylation of p-coumaric acid has not been well characterized. Kamsteeg et al. reported the occurrence of a mixed function oxidase requiring both NADPH and FAD in petals of Silene diocia that catalyzed the hydroxylation ofp-coumaroyl-CoA to caffeoyl-CoA (11). An analogous enzyme was demonstrated in potato by Boniwell and Butt (12). The enzyme catalyzed the hydroxylation of 4-hydroxyphenylpropanoid carboxylic acids such as p-coumaric acid and L-tyrosine in the presence of NADH as well as FAD. Recently, a novel hydroxylase catalyzing the formation of caffeoyl-CoA from p-coumaroyl-CoA was demonstrated in crude extracts from cultured parsley cells (25). The enzyme required both L-ascorbic acid and Zn’+. We detected p-coumaric acid hydroxylase in mung bean seedlings treated with tentoxin, a fungal toxin, in which polyphenol oxidase was completely eliminated (13). The enzyme showed a pH optimum of 5.0 and hydroxylated only p-coumaric acid in the presence of NADPH. Polyphenol oxidase (phenolase or tyrosinase) is also able to hydroxylate 4-hydroxy-substituted aromatic compounds including p-coumaric acid in the presence of an electron donor such as L-ascorbic acid, in vitro (26,27). There are, however, some arguments against the participation of polyphenol oxidase in the biosynthesis of phenolic compounds in Go, as discussed below. A microsomal50-(4-coumaroyl) shikimate 3-hydroxylase was first detected in parsley cell suspension cultures (28) and subsequently an analogous enzyme, 5-0-(4-coumaroyl)-D-quinatelshikimate 3-hydroxylase was detected in the microsomal preparations from carrot cell suspension culture (29). The authors of the paper (29) claimed that 5-o-(4-coumaroyI)-D-quinate is the final intermediate of chlorogenic acid biosynthesis and is hydroxylated to chlorogenic acid by the enzyme in carrot cells. We, however, were unable to detect 5-O-(4-coumaroyl)-Dquinate in extracts from sweet potato root disks supplied with trans-[3-14C]cinnamic acid, a precursor of chlorogenie acid (8). Instead, we detected t-cinnamoyl-D-glucose and p-coumaroyl-D-glucose as possible intermediates of chlorogenic acid biosynthesis in this tissue. In this study, we have purified and characterized a hydroxylase from wounded sweet potato roots which catalazes the conversion of p-coumaroyl-D-glucose to caffeoylD-glucose; as such, it carries out an essential reaction in our pathway proposed for chlorogenic acid biosynthesis. The substrate specificity of the enzyme was rather strict; the enzyme hydroxylated only p-coumaroyl-D-glucose, pcoumaric acid, and p-cresol among eight compounds tested. Among these monophenols, p-coumaroyl-D-glucose and p-coumaric acid occur in sweet potato roots, while pcresol is not present (8). The enzyme hydroxylated pcoumaroyl-D-glucose and p-coumaric acid equally well at pH 7.0 (Table III), but utilized only p-CoumaroyI-D-glucase at pH 5.5 (Table III). At present, we have no infor-

AND

KOJIMA

mation on the cellular localization of the enzyme of the pH at that site. In this context, however, it should be noted that the optimum pH of the other enzymes in our proposed pathway are all near pH 5.5. These are: UDPG: t-cinnamate glucosyltransferase, pH 5.8 (5); hydroxycinnmoyl-D-glucose:quinate hydroxycinnamoyl transferase, pH 6.0 (6); chlorogenic acid:chlorogenate caffeoyl transferase, pH 5.0 (7). p-Coumaric acid was also hydroxylated by the enzyme, although strong inhibition of both hydroxylase and polyphenol oxidase activities was observed in assay mixtures containing p-coumaric acid at concentrations higher than 5 mM, which appears to be the physiological concentration. The inhibitory effect ofp-coumaric acid in vitro suggests a possible regulatory role for this compound in phenylpropanoid metabolism in uivo. The purified enzyme did exhibit both hydroxylase activity and polyphenol oxidase activity. The enzyme, therefore, appears to be one of the isozymes of polyphenol oxidase in sweet potato roots. In fact, the enzyme resembles polyphenol oxidase in the following two respects: first, both enzyme activities are strongly inhibited by diethyldithiocarbamate, a Cu-chelating agent (30). Second, the enzymes require L-ascorbic acid as an electron donor in the hydroxylation reaction (20). Seven isozymes of polyphenol oxidase have already been purified from wounded sweet potato roots and characterized by Hyodo and Uritani (15). Those isozymes differ from the enzyme purified in this study because they showed no hydroxylase (cresolase) activity. Polyphenol oxidase is found in most higher plants. Its role, however, has not yet been clearly established, although the following functions have been proposed: involvement in defense reaction of plants against pathogens (31), oxygen scavenger in photosynthesis (31), involvement in the formation of hard seed coats (32), and hydroxylation of monophenols in secondary metabolism (26). It seems unlikely that all such diverse functions should be ascribed to a single polyphenol oxidase. Presumably, different isozymes of polyphenol oxidase are responsible for the different functions. It has been a matter of dispute whether or not polyphenol oxidase is involved in the biosynthesis of phenolic compounds. The arguments against such a role are based on the following experimental evidence: first, polyphenol oxidase has a broad substrate specificity (33). Second, polyphenol oxidase requires electron donors to reduce the quinone produced by the action of polyphenol oxidase activity as well as being required in the hydroxylation reaction. Some investigators doubt whether plants are able to supply such a large amount of electron donors (34). Third, tentoxin, a fungal toxin, had no effect on the polyphenol contents of mung bean seedlings despite the fact that polyphenol oxidase activity was completely eliminated in the toxin-treated seedlings (16). However,

p-COUMAROYL-D-GLUCOSE

HYI

these facts are not enough to rule out the involvement of polyphenol oxidase in biosynthesis of polyphenols in uiuo. Generally, the substrate specificity of enzymes of secondary metabolism is rather broad. The broad substrate specificity of these enzymes is considered to be one cause of network pathways in secondary metabolism in higher plants. As for the second argument, it is difficult to assess the capacity of plants to supply electron donors at the sites of polyphenol biosynthesis. As for the third objection, we found that p-coumaric acid hydroxylase still existed in tentoxin-treated mung bean seedlings which lacked polyphenol oxidase activity (13). On the other hand, there is evidence supporting the involvement of polyphenol oxidase in polyphenol biosynthesis. Polyphenol oxidase changes in concert with changes in the polyphenol contents of some plants (35). Furthermore, polyphenol oxidase isozymes which showed high hydroxylase activity and low polyphenol oxidase activity have been isolated from sorghum (30) and spinach beet (36). These isozymes may function as hydroxylases in Go. p-Goumaroyl-D-glucose hydroxylase isolated in this paper showed high hydroxylase activity and low polyphenol oxidase activity. The enzyme activity changed in a manner consistent with its involvement in chlorogenic acid biosynthesis during the incubation of root disks (Fig. 3). In addition, L-ascorbic acid, which is a possible electron donor for the hydroxylation reaction by the enzyme in uiuo, also changed in a similar manner. In summary, the p-coumaroyl-D-glucose hydroxylase described in this paper appears to be the enzyme that catalyzes the conversion of p-coumaroyl-D-glucose to caffeoyl-D-glucose in our proposed pathway for chlorogenic acid biosynthesis in sweet potato roots. REFERENCES 1. Bradfield, A. F., Flood, (1952) Nature (London) M.,

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Purification and characterization of p-coumaroyl-D-glucose hydroxylase of sweet potato (Ipomoea batatas) roots.

p-Coumaroyl-D-glucose hydroxylase in sweet potato (Ipomoea batatas Lam.) has been purified to apparent electrophoretic homogeneity using a combination...
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