- Email: [email protected]
Purification and some properties of UDP-glucose:o-dihydroxycoumarin7-O-glucosyltransferase from tobacco cell cultures
Plant Science Letters, 18 (1980) 177--184
177
© Elsevier/North-HollandScientific Publishers Ltd.
PURIFICATION AND SOME PROPERTIES OF UDPA]LUCOSE :o -DIHYDROXYCOUMARIN 7-O-GLUCOSYLTRANSFERASE FROM TOBACCO CELL CULTURES RAGAI K. IBRAHIMand BERNARD BOULAY Department of Biological Sciences, Concordia University, Sir George Williams Campus, Montreal, Quebec (Canada)
(Received September 13th, 1979) (Accepted December 13th, 1979)
SUMMARY An enzyme, catalysing the glucosylation of o-dihydroxycoumarins and utilising UDPG as the glucosyl donor, ~ been isolated and partially purified (80-fold) from tobacco cell suspension culture. The enzyme exhibited strict position specificity towards the 7-hydroxyl group of both daphnetin (Kin 95 ~M)and esculetin (Kin 111 ~M). It could also glucosylate scopoletin (Kin 1.43 raM), umbelliferone and caffeic acid, but to a lesser extent. With esculetin as substrate, the apparent Km for UDPG was 50 ~M. The physiological role of this enzyme would be to glucozylate daphnetin, esculetin and possibly scopoletin to their corresponding 7~)~lucosides: daphnin, chichoriin and scopolin. It is suggested that the formation of two isomeric glucosides, esculin (esculetin-6-O-glucoside) and daphnetin-8-O-giucoalde), is catalysed by a different enzyme with a high affin!ty towards the 6- and 8-hydroxyl groups, respectively.
INTRODUCTION Phenolic glucosides are of widespread occurrence in plants [ 1] and their in vitro synthesis has been shown to be catalysed by a number of glucosyltransferases [2]. These enzymes mediate the transfer of the glucose moiety of a nucleotide diphosphate glucose to such acceptors as simple hydroxyphenols [3- 7], phenolic acids [ 7 - 9 ] and flavonoids [10--18]. Whereas giucosyltransferases were believed to possess a broad specificity [19], it was only recently that few enzymes were reported to exhibit high affinity towards certain phenolic compounds such as lignin monomers [20,21], flavone or flavonols [14--16] and anthocyanidins [17,18]. It is interesting to note that, among the large variety of giucosides based
178 on simple phenolic and fiavonoid compounds [1], only few coumarin glucosides are known [1,22] ; nonetheless little information is available on their glucosylation. Preliminary work in this laboratory [23] indicated the presence in tobacco cell culture of a glucosyltransferase which catalysed the transfer of glucose from UDPG to scopoletin (6-methoxy-7-hydroxycoumarin) to form scopolin; though the enzyme exhibited highest affinity towards the o-dihydroxycoumarins, daphnetin and esculetin. In view of the lack of information on the glucosylation of phenylpropanoid compounds, we report in this paper the partial purification and some properties of this enzyme, especially with reference to its position specificity.
MATERIALS AND METHODS Tobacco (Nicotiana tabacum L. cv. Wisconsin No. 38) cell culture was initiated from the stem pith and its growth was maintained on a saltnutrient medium [24] containing 3% sucrose, 2 uM IAA and 0.1 uM kinetin. The conditions for culture growth and the determination of growth parameters were as previously described by Tsang and Ibrahim [23].
Extraction and purification of tobacco glucosyltransferase. Seven-day-old cells were homogenized with 0.2 M Tris--HC1 buffer (pH 7.5) (1 : 2, w/v) in the presence of Polyclar AT and fine sand. The homogenate was centrifuged at 15 000 g for 15 rain and the supernatant was stirred with Dowex 1X2 which had previously been equilibrated with the same buffer. The filtered buffer extract was fractionated with solid a m m o n i u m sulphate and the protein which precipitated between 60--80% saturation was collected by centrifugation and resuspended in the minimal a m o u n t of 25 mM Tris--HC1 buffer (pH 7.5). The latter was desalted on Sephadex G-25 column using the same buffer. The protein ex~act was chromatographed on a DEAE-Sephadex A-50 column which had previously been equilibrated with 25 mM Tris-~HC1 buffer (pH 7.5). After washing the column with two volumes of the same buffer, the protein was eluted with 4 column volumes of a sait~gradient between 0--300 mM KC1 in the same buffer, and fractions (2.5 ml) were collected at a rate of 20 ml/h and assayed for enzyme activity. The protein content was determined by the m e t h o d of Lowry et al. [25]. Glucosyltransferase a m y . The standard assay mixture consisted of 20--40 nmol of the phenolic substrate (in 1 0 , 1 of DMSO), 0.4 nmol of UDP-[U-14C]glucose containing 0.05 ~Ci, 1.4 ~mol ~-mercaptoethanol and 50--100 ~1 enzyme protein in a total volume of 150 ~1. The assay mixture was incubated for 60 rain at 30°C and the reaction was stopped by the addition of 20/~1 of glacial acetic acid. The mixture was chzomatographed on Whatman No. 3 paper or on cellulose-silica gel G (1 : 1) plates using nrbutanol/acetic acid/water (4 : 1 : 2.2) as solvent.
179
12° ~k
/ ," •
ooo°°°
,
'~.. . . . ,J" 1 "7
d
,/
i ~,..
O 0
•
i
i
i
i
I
4
41
•
!0
I ~'
"time l d a y s l
Fig. 1. Time course changesin glucosyltransferaseactivity against esculetin (=-- -- --t ) and scopoletin (~------[]) as substrates and the accumulation of scopolin (A. • . . , ) during growth of tobacco cell culture. The latter was determined by cell flruh weight (e =) and soluble protein content (o v).
Identification of reaction products. The glucosylated products were identified by cochromatography with reference compounds, autoradiography on X-ray films, hydrolysis with ~-glucosidase and identification of the hydrolysis products. The radioactive spots were cut off the paper chromatograms or scraped off the TLC plates and counted for radioactivity by liquid scintillation. During the process of enzyme purification, the assay mixture was extracted twice with 0.5 ml of ethyl acetate and an aliquot of the organic layer was counted for activity determination. RESULTS
Growth and glucosyltransferase activity of tobacco cell culture. Figure 1 shows that the exponential growth of tobacco cells was associated with a sharp increase in the glucosylating activity of both esculetin and scopoletin, though the latter was 3--4 times lower than that of the former substrate. There was also a concomitant accumulation of scopolin (scopoletin-7-Oglucoside), the natural metabolite of tobacco cells, during culture growth.
Purification and properties of glucosyltransfem~. The enzyme was purified by precipitation with ammonium sulphate (60--80% saturation) which eliminated 90% of the contaminatirig protein while retaining 80% of the enzyme activity. It was further purified by ion filtration on DEAE Sephadex A-50, which resulted in an 80-fold increase in specific activity as
180 TABLE I PURIFICATION OF TOBACCO o-DIHYDROXYCOUMARIN GLUCOSYLTRANSFERASE The standard enzyme assay was used as described in the Methods section, with esculetin as substrate. Fraction
Total protein (rag)
Total activity (pkat)
Crude extract Dowex 1X2 (NH,)=8 O, 60--80% satn. a DEAE Sephadex A-50
50.8 42.5
16.26 36.72
5.2 0.75
Specific activity (pkat/mg)
Purification (-fold)
0.32' 0.86
34.28 14.78
-2.7
6.60 26.37
20.6 82.4
aAfter desalting on Sephadex G-25 column.
compared with the crude extract (Table I). The enzyme activity was eluted at approximately 0.23-0.25 M salt concentration and had a specific activity of 26.4 pkat/mg protein (Fig. 2). The products of enzymic glucosylation of daphnetin, esculetin, umbelliferone, scopoletin, hydrangetin and caffeic acid (Table II, Fig. 3) were identified as daphnin (daphnetin-7~)-glucoside), cichoriin (esculetin-7-Oglucoside), skimmin (umbelliferone-7-O-glucoside), scopolin, hydrangin and caffeic-4-O-glucoside, respectively, by cochromatography with reference
[ ~ :0. -0.3
•.' .. i-..- / •/ .
/
.0.2 ~
O 0
..~ -~
-..
--,.
.O.l
0 U
2 0
QJ
.
.
.
~ I ....'
. ZO
F r a c t i o n no. Fig. 2. Elution profile of proteins from DEAE Sephadex A-50 and activity of esculetin7-O-glucosyltransferase. ( ) A~a0; ( - - - - - - ) salt gradient (M); (, • • • - ) glucoaylt r a n s f e r u e activity (cpm X 10-4).
181 T A B L E II S U B S T R A T E SPECIFICITY O F T O B A C C O G L U C O S Y L T R A N S F E R A S E Substrate a
Spec. act. (product d p m / m g )
Esculetin Scopoletin Isoacopoletin Daphnctin Hydrangetin Caffeic acid Ferulic acid Isoferulic acid 5-Hydroxyferulic acid Sinapic acid Umbelliferone p-Coumaric acid p-Hydroxybenzoic acid Protocatechuic (3,4-dihydroxybenzoic) acid o-Pyrocatechuic (2,3-dihydroxybenzoic) acid Vanillic (3-methoxy-4-hydroxybenzoic) acid Isovanfllic (3-hydroxy-4-methoxybenzoic) acid Syringic (3,5-dimethoxy-4-hydroxybenzoic)acid
1 178 000 310 000 1 240 372 694 62
Percent activity b 95 25 0
000 000 000 000
100 30 56 5 0 4 3 52 0 0
49 000 37 200 644 000
148 000 24 800 173 000
12
124 000
10
2
14
--
0
a The assay m i t r e contained 0.2--0.3 mM of the indicated substrates. SeeFig. 3 for structural formulae. b Relative to daphnetin = 100%.
compounds, radioautography, hydrolysis with ~-glucosidase and recovery of the 14C-labelled glucose. The partially purified enzyme preparation (G-25 fraction) lost approx. 25% of its activity after storage for 24 h at 4°(3 and only 50% of the activity remained after 48 h. The purified fraction, on the other hand had a half-life
R1 R2~O Rz
R z ~ R2'~ R3
cOOH
Coumarin
Ri
R=
Rs
Cinnamic acid
RI
Rz
R3
Umbelliferone Esculetin Scopolctin Isoscopoletin Daphnetin Hydrangetin
-OH OMe OH ---
OH OH OH OMe OH OH
----OH OMe
p-Coumaric Caffeic Ferulic Isoferulic 5-OH-Ferulic Sinapic
-OH OMe OH OMe OMe
OH OH OH OMe OH OH
---OH OMe
Fig. 3. Structural formulae o f substituted coumarirm and cinnamic acids used as potential lu~trates.
182
of approx. 16 h. The loss of enzyme activity could not be prevented by the addition of bovine serum albumin (1- -4 mg/ml) or ethylene glycol (5--20%). This relative instability prevented further purification of the enzyme. The glucosylation of esculetin was proportional to protein concentration up to 200 ~g and with time up to 120 rain. The rate of reaction was n o t inhibited by substrate concentration up to 500 ~M of either daphnetin, esculetin or scopoletin. The apparent Km values for the three substrates, as determined from a Lineweaver-Burk plot, were 95 ~M, 111 ~M a n d 1.43 mM, respectively. With esculetin as substrate, the apparent Km for UDPG was 50 ~M. The pH o p t i m u m for the glucosylation of esculetin was 7.5 in both Tris-HC1 and phosphate buffers, though the latter resulted in 20--25% decrease in enzyme activity. Substrate specificity. A number of substituted coumarins, cinnamic and benzoic acids were tested for their relative glucose" acceptor ability. T h e results given in Table II indicate that the purified enzyme exhibited an expressed specificity towards o-dihydroxycoumarins. When either daphnetin or esculetin was used as substrate, the only enzymic product obtained was the corresponding 7-O-glucoside. Furthermore, opening of the lactone ring of the o-dihydroxycoumarin (as in caffeic acid) or removal of one hydroxyl group (as in umbelliferone) resulted in 50% drop of enzyme activity; whereas methylation of one hydroxyl group (as in scopoletin or hydrangetin) brought about 70--75% decrease in activity. Almost no activity was observed when the substrate underwent both opening of the lactone ring and substitution of one hydroxyl g r o u p - - a s in ferulic, 5-OH-ferulic and sinapic acids (Table II). In addition, the enzyme seems to exhibit positional specificity towards the hydroxyl group para to the side chain of the phenolic ring, since it did n o t react with either isoscopoletin, isoferulic or isovanillic acids; nor did it accept such compounds as esculin or daphnetin-8-O-glucoside, both of which have a free 7-hydroxyl group. There was some activity (10--14%) observed with the benzoic acids, protocatechuic, vanillic and syringic acids, but none at all with the m o n o h y d r o x y compounds, p-coumatic orp-hydroxybenzoic acids. The enzyme did n o t react with any of the flavonoid substrates tested: luteolin, chrysoeriol, quercetin or isorhamnetin.
DISCUSSION This study represents the first report of a glucosyltransferase with a high affinity for the o-dihydroxycoumarins, esculetin and daphnetin, and a distinct position specificity towards their 7-hydroxyl group. The enzyme requires for expression of full activity an intact coumarin ring system and an o-dihydroxy group, so that the 7-OH becomes exclusively glucosylated. No radioactivity was observed in either the 6-19oaition of esculetin or the 8-position of daphnetin. Opening of the lactone ring of esculetin or methyla-
183
tion of its 6-hydroxyl group resulted in a substantial drop o f activity; whereas the combination of both changes resulted in complete loss of the e n z y m e activity. We propose the systematic name UDP-glucose:o-dihydroxycoumarin 7-O~lucosyltransferase for this enzyme. Its role would be to glucosylate both esculetin and daphnetin to form cichoriin and daphnin, respectively, but n o t to the exclusion o f umbeUiferone scopoletin and hydrangetin, though with lower efficiency. Scopol-in represents the o n l y phenolic metabolite in tobacco cell culture [23] and the recent discovery in that culture of an O-methyltransferase catalysing the O-methylation o f esculetin to scopoletin [ 13], together with this report, provide the basis for understanding the pathways involved in the biosynthesis of coumarin glucosides [26]. Due to the limited extent of enzyme purification, it is possible to assume that two enzymes/isoenzymes m a y exist in tobacco cell culture which m a y be involved in the glucosylation of esculetin and daphnetin. However, the expressed position specificity of tobacco glucosyltransferase towards the 7-hydroxyl group o f o
1 J.B. Harborne, in J.B. Harbone (Ed.), Biochemistry of phenolic compounds, Academic Press, London, 1964, p. 129. 2 K. Hahlbrock and H. Grisebach, in J.B. Harborne, T. Mabry and H. Mahry, (Eds.), the fiavonoids, Academic Press, New York, 1975, p. 866. 3 T. Yamaha and C.E. Cardini, Arch. Biochem. Biophys., 86 (1960) 127. 4 J.B. Pridham, Phytochemistry, 3 (1964) 493. 5 H. Pilgrim, Phm~mazie,25 (1970) 568. 6 A.D~I. Glass and B.A. Bo.hm, Phytocbemistry, 9 (1970) 2197. 7 M. Tabata, F. Ikeda, N. Hiraoka and M.Konochima, Phytochemistry, 15 (1976) 1225. 8 A. Kleinhofs, F.A. Haskin and H.J. Gorz, Phytochemistry, 6 (1967) 1313. 9 J.J. Macbeix, C.R. Acad. Sci. Paris Ser. D, 284 (1977) 33. 10 G.A. Barber and E.F. Neufeld, Biochem. Blophys. Res. Commun., 6 (1961) 44. 11 G.A. Barber, Biochemistry, 1 (1962) 463. 12 R.L. Larmon,Phytochemistry, 10 (1971) 3073. 13 R.L. Larson and C.M. Lonegran, Planta, 103 (1972) 361. 14 A. Sutter, R. Ortmann and H. Grisebech, Biochim. Biophys. Acta, 281 (1972) 71.
184 15 16 17 18 19 20 21 22 23 24 25 26 27 28
A. Sutter and H. Grisebach, Biochim. Biophys. Aeta, 309 (1973) 289. J.E. Poulton and M. Kauer, Planta, 136 (1977) 53. N.AM. Saleh, H. Fritsch, P. Witkop and H. Grisebach, Planta, 133 (1976) 41. N.A.M. Saleh, J.E. Poulton and H. Grisebaeh, Phytochemistry, 15 (1976) 1865. R.A. Dedonde~, Annu. Rev. B/ochem., 30 (1961) 347. R.K. Ibrahim and H. Grisebaeh, Arch. Biochem. Biophys., 1"/6 (1976) 700. R.K. Ibrahim, Z. Pflanzenphysiol., 85 (19"/7) 258. W. Karrer, Konstitution und Verkommen der Organischen Pflanzstoffe, Birkh~/user Verlsg, Basel & Stuttgart, 1958. Y.F. 'l~sang and R.K. Ibrahim, Phytochemistry, 18 (1979) 1131. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 478. O.H. Lowry, N.J. Rosebrough, H.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. S.A. Brown, in T. Swain, J.B. Harborne and C.F. vanSumere, (Eds.), Biochemistry of Plant Phenolics, Plenum Press, New York, 1979, p. 249. J.B. Trommsdorff, Anal. Chem., 14 (1885) 189. K.A. Zirvi, Planta Medica, 31 (1977) 119.
177
© Elsevier/North-HollandScientific Publishers Ltd.
PURIFICATION AND SOME PROPERTIES OF UDPA]LUCOSE :o -DIHYDROXYCOUMARIN 7-O-GLUCOSYLTRANSFERASE FROM TOBACCO CELL CULTURES RAGAI K. IBRAHIMand BERNARD BOULAY Department of Biological Sciences, Concordia University, Sir George Williams Campus, Montreal, Quebec (Canada)
(Received September 13th, 1979) (Accepted December 13th, 1979)
SUMMARY An enzyme, catalysing the glucosylation of o-dihydroxycoumarins and utilising UDPG as the glucosyl donor, ~ been isolated and partially purified (80-fold) from tobacco cell suspension culture. The enzyme exhibited strict position specificity towards the 7-hydroxyl group of both daphnetin (Kin 95 ~M)and esculetin (Kin 111 ~M). It could also glucosylate scopoletin (Kin 1.43 raM), umbelliferone and caffeic acid, but to a lesser extent. With esculetin as substrate, the apparent Km for UDPG was 50 ~M. The physiological role of this enzyme would be to glucozylate daphnetin, esculetin and possibly scopoletin to their corresponding 7~)~lucosides: daphnin, chichoriin and scopolin. It is suggested that the formation of two isomeric glucosides, esculin (esculetin-6-O-glucoside) and daphnetin-8-O-giucoalde), is catalysed by a different enzyme with a high affin!ty towards the 6- and 8-hydroxyl groups, respectively.
INTRODUCTION Phenolic glucosides are of widespread occurrence in plants [ 1] and their in vitro synthesis has been shown to be catalysed by a number of glucosyltransferases [2]. These enzymes mediate the transfer of the glucose moiety of a nucleotide diphosphate glucose to such acceptors as simple hydroxyphenols [3- 7], phenolic acids [ 7 - 9 ] and flavonoids [10--18]. Whereas giucosyltransferases were believed to possess a broad specificity [19], it was only recently that few enzymes were reported to exhibit high affinity towards certain phenolic compounds such as lignin monomers [20,21], flavone or flavonols [14--16] and anthocyanidins [17,18]. It is interesting to note that, among the large variety of giucosides based
178 on simple phenolic and fiavonoid compounds [1], only few coumarin glucosides are known [1,22] ; nonetheless little information is available on their glucosylation. Preliminary work in this laboratory [23] indicated the presence in tobacco cell culture of a glucosyltransferase which catalysed the transfer of glucose from UDPG to scopoletin (6-methoxy-7-hydroxycoumarin) to form scopolin; though the enzyme exhibited highest affinity towards the o-dihydroxycoumarins, daphnetin and esculetin. In view of the lack of information on the glucosylation of phenylpropanoid compounds, we report in this paper the partial purification and some properties of this enzyme, especially with reference to its position specificity.
MATERIALS AND METHODS Tobacco (Nicotiana tabacum L. cv. Wisconsin No. 38) cell culture was initiated from the stem pith and its growth was maintained on a saltnutrient medium [24] containing 3% sucrose, 2 uM IAA and 0.1 uM kinetin. The conditions for culture growth and the determination of growth parameters were as previously described by Tsang and Ibrahim [23].
Extraction and purification of tobacco glucosyltransferase. Seven-day-old cells were homogenized with 0.2 M Tris--HC1 buffer (pH 7.5) (1 : 2, w/v) in the presence of Polyclar AT and fine sand. The homogenate was centrifuged at 15 000 g for 15 rain and the supernatant was stirred with Dowex 1X2 which had previously been equilibrated with the same buffer. The filtered buffer extract was fractionated with solid a m m o n i u m sulphate and the protein which precipitated between 60--80% saturation was collected by centrifugation and resuspended in the minimal a m o u n t of 25 mM Tris--HC1 buffer (pH 7.5). The latter was desalted on Sephadex G-25 column using the same buffer. The protein ex~act was chromatographed on a DEAE-Sephadex A-50 column which had previously been equilibrated with 25 mM Tris-~HC1 buffer (pH 7.5). After washing the column with two volumes of the same buffer, the protein was eluted with 4 column volumes of a sait~gradient between 0--300 mM KC1 in the same buffer, and fractions (2.5 ml) were collected at a rate of 20 ml/h and assayed for enzyme activity. The protein content was determined by the m e t h o d of Lowry et al. [25]. Glucosyltransferase a m y . The standard assay mixture consisted of 20--40 nmol of the phenolic substrate (in 1 0 , 1 of DMSO), 0.4 nmol of UDP-[U-14C]glucose containing 0.05 ~Ci, 1.4 ~mol ~-mercaptoethanol and 50--100 ~1 enzyme protein in a total volume of 150 ~1. The assay mixture was incubated for 60 rain at 30°C and the reaction was stopped by the addition of 20/~1 of glacial acetic acid. The mixture was chzomatographed on Whatman No. 3 paper or on cellulose-silica gel G (1 : 1) plates using nrbutanol/acetic acid/water (4 : 1 : 2.2) as solvent.
179
12° ~k
/ ," •
ooo°°°
,
'~.. . . . ,J" 1 "7
d
,/
i ~,..
O 0
•
i
i
i
i
I
4
41
•
!0
I ~'
"time l d a y s l
Fig. 1. Time course changesin glucosyltransferaseactivity against esculetin (=-- -- --t ) and scopoletin (~------[]) as substrates and the accumulation of scopolin (A. • . . , ) during growth of tobacco cell culture. The latter was determined by cell flruh weight (e =) and soluble protein content (o v).
Identification of reaction products. The glucosylated products were identified by cochromatography with reference compounds, autoradiography on X-ray films, hydrolysis with ~-glucosidase and identification of the hydrolysis products. The radioactive spots were cut off the paper chromatograms or scraped off the TLC plates and counted for radioactivity by liquid scintillation. During the process of enzyme purification, the assay mixture was extracted twice with 0.5 ml of ethyl acetate and an aliquot of the organic layer was counted for activity determination. RESULTS
Growth and glucosyltransferase activity of tobacco cell culture. Figure 1 shows that the exponential growth of tobacco cells was associated with a sharp increase in the glucosylating activity of both esculetin and scopoletin, though the latter was 3--4 times lower than that of the former substrate. There was also a concomitant accumulation of scopolin (scopoletin-7-Oglucoside), the natural metabolite of tobacco cells, during culture growth.
Purification and properties of glucosyltransfem~. The enzyme was purified by precipitation with ammonium sulphate (60--80% saturation) which eliminated 90% of the contaminatirig protein while retaining 80% of the enzyme activity. It was further purified by ion filtration on DEAE Sephadex A-50, which resulted in an 80-fold increase in specific activity as
180 TABLE I PURIFICATION OF TOBACCO o-DIHYDROXYCOUMARIN GLUCOSYLTRANSFERASE The standard enzyme assay was used as described in the Methods section, with esculetin as substrate. Fraction
Total protein (rag)
Total activity (pkat)
Crude extract Dowex 1X2 (NH,)=8 O, 60--80% satn. a DEAE Sephadex A-50
50.8 42.5
16.26 36.72
5.2 0.75
Specific activity (pkat/mg)
Purification (-fold)
0.32' 0.86
34.28 14.78
-2.7
6.60 26.37
20.6 82.4
aAfter desalting on Sephadex G-25 column.
compared with the crude extract (Table I). The enzyme activity was eluted at approximately 0.23-0.25 M salt concentration and had a specific activity of 26.4 pkat/mg protein (Fig. 2). The products of enzymic glucosylation of daphnetin, esculetin, umbelliferone, scopoletin, hydrangetin and caffeic acid (Table II, Fig. 3) were identified as daphnin (daphnetin-7~)-glucoside), cichoriin (esculetin-7-Oglucoside), skimmin (umbelliferone-7-O-glucoside), scopolin, hydrangin and caffeic-4-O-glucoside, respectively, by cochromatography with reference
[ ~ :0. -0.3
•.' .. i-..- / •/ .
/
.0.2 ~
O 0
..~ -~
-..
--,.
.O.l
0 U
2 0
QJ
.
.
.
~ I ....'
. ZO
F r a c t i o n no. Fig. 2. Elution profile of proteins from DEAE Sephadex A-50 and activity of esculetin7-O-glucosyltransferase. ( ) A~a0; ( - - - - - - ) salt gradient (M); (, • • • - ) glucoaylt r a n s f e r u e activity (cpm X 10-4).
181 T A B L E II S U B S T R A T E SPECIFICITY O F T O B A C C O G L U C O S Y L T R A N S F E R A S E Substrate a
Spec. act. (product d p m / m g )
Esculetin Scopoletin Isoacopoletin Daphnctin Hydrangetin Caffeic acid Ferulic acid Isoferulic acid 5-Hydroxyferulic acid Sinapic acid Umbelliferone p-Coumaric acid p-Hydroxybenzoic acid Protocatechuic (3,4-dihydroxybenzoic) acid o-Pyrocatechuic (2,3-dihydroxybenzoic) acid Vanillic (3-methoxy-4-hydroxybenzoic) acid Isovanfllic (3-hydroxy-4-methoxybenzoic) acid Syringic (3,5-dimethoxy-4-hydroxybenzoic)acid
1 178 000 310 000 1 240 372 694 62
Percent activity b 95 25 0
000 000 000 000
100 30 56 5 0 4 3 52 0 0
49 000 37 200 644 000
148 000 24 800 173 000
12
124 000
10
2
14
--
0
a The assay m i t r e contained 0.2--0.3 mM of the indicated substrates. SeeFig. 3 for structural formulae. b Relative to daphnetin = 100%.
compounds, radioautography, hydrolysis with ~-glucosidase and recovery of the 14C-labelled glucose. The partially purified enzyme preparation (G-25 fraction) lost approx. 25% of its activity after storage for 24 h at 4°(3 and only 50% of the activity remained after 48 h. The purified fraction, on the other hand had a half-life
R1 R2~O Rz
R z ~ R2'~ R3
cOOH
Coumarin
Ri
R=
Rs
Cinnamic acid
RI
Rz
R3
Umbelliferone Esculetin Scopolctin Isoscopoletin Daphnetin Hydrangetin
-OH OMe OH ---
OH OH OH OMe OH OH
----OH OMe
p-Coumaric Caffeic Ferulic Isoferulic 5-OH-Ferulic Sinapic
-OH OMe OH OMe OMe
OH OH OH OMe OH OH
---OH OMe
Fig. 3. Structural formulae o f substituted coumarirm and cinnamic acids used as potential lu~trates.
182
of approx. 16 h. The loss of enzyme activity could not be prevented by the addition of bovine serum albumin (1- -4 mg/ml) or ethylene glycol (5--20%). This relative instability prevented further purification of the enzyme. The glucosylation of esculetin was proportional to protein concentration up to 200 ~g and with time up to 120 rain. The rate of reaction was n o t inhibited by substrate concentration up to 500 ~M of either daphnetin, esculetin or scopoletin. The apparent Km values for the three substrates, as determined from a Lineweaver-Burk plot, were 95 ~M, 111 ~M a n d 1.43 mM, respectively. With esculetin as substrate, the apparent Km for UDPG was 50 ~M. The pH o p t i m u m for the glucosylation of esculetin was 7.5 in both Tris-HC1 and phosphate buffers, though the latter resulted in 20--25% decrease in enzyme activity. Substrate specificity. A number of substituted coumarins, cinnamic and benzoic acids were tested for their relative glucose" acceptor ability. T h e results given in Table II indicate that the purified enzyme exhibited an expressed specificity towards o-dihydroxycoumarins. When either daphnetin or esculetin was used as substrate, the only enzymic product obtained was the corresponding 7-O-glucoside. Furthermore, opening of the lactone ring of the o-dihydroxycoumarin (as in caffeic acid) or removal of one hydroxyl group (as in umbelliferone) resulted in 50% drop of enzyme activity; whereas methylation of one hydroxyl group (as in scopoletin or hydrangetin) brought about 70--75% decrease in activity. Almost no activity was observed when the substrate underwent both opening of the lactone ring and substitution of one hydroxyl g r o u p - - a s in ferulic, 5-OH-ferulic and sinapic acids (Table II). In addition, the enzyme seems to exhibit positional specificity towards the hydroxyl group para to the side chain of the phenolic ring, since it did n o t react with either isoscopoletin, isoferulic or isovanillic acids; nor did it accept such compounds as esculin or daphnetin-8-O-glucoside, both of which have a free 7-hydroxyl group. There was some activity (10--14%) observed with the benzoic acids, protocatechuic, vanillic and syringic acids, but none at all with the m o n o h y d r o x y compounds, p-coumatic orp-hydroxybenzoic acids. The enzyme did n o t react with any of the flavonoid substrates tested: luteolin, chrysoeriol, quercetin or isorhamnetin.
DISCUSSION This study represents the first report of a glucosyltransferase with a high affinity for the o-dihydroxycoumarins, esculetin and daphnetin, and a distinct position specificity towards their 7-hydroxyl group. The enzyme requires for expression of full activity an intact coumarin ring system and an o-dihydroxy group, so that the 7-OH becomes exclusively glucosylated. No radioactivity was observed in either the 6-19oaition of esculetin or the 8-position of daphnetin. Opening of the lactone ring of esculetin or methyla-
183
tion of its 6-hydroxyl group resulted in a substantial drop o f activity; whereas the combination of both changes resulted in complete loss of the e n z y m e activity. We propose the systematic name UDP-glucose:o-dihydroxycoumarin 7-O~lucosyltransferase for this enzyme. Its role would be to glucosylate both esculetin and daphnetin to form cichoriin and daphnin, respectively, but n o t to the exclusion o f umbeUiferone scopoletin and hydrangetin, though with lower efficiency. Scopol-in represents the o n l y phenolic metabolite in tobacco cell culture [23] and the recent discovery in that culture of an O-methyltransferase catalysing the O-methylation o f esculetin to scopoletin [ 13], together with this report, provide the basis for understanding the pathways involved in the biosynthesis of coumarin glucosides [26]. Due to the limited extent of enzyme purification, it is possible to assume that two enzymes/isoenzymes m a y exist in tobacco cell culture which m a y be involved in the glucosylation of esculetin and daphnetin. However, the expressed position specificity of tobacco glucosyltransferase towards the 7-hydroxyl group o f o
1 J.B. Harborne, in J.B. Harbone (Ed.), Biochemistry of phenolic compounds, Academic Press, London, 1964, p. 129. 2 K. Hahlbrock and H. Grisebach, in J.B. Harborne, T. Mabry and H. Mahry, (Eds.), the fiavonoids, Academic Press, New York, 1975, p. 866. 3 T. Yamaha and C.E. Cardini, Arch. Biochem. Biophys., 86 (1960) 127. 4 J.B. Pridham, Phytochemistry, 3 (1964) 493. 5 H. Pilgrim, Phm~mazie,25 (1970) 568. 6 A.D~I. Glass and B.A. Bo.hm, Phytocbemistry, 9 (1970) 2197. 7 M. Tabata, F. Ikeda, N. Hiraoka and M.Konochima, Phytochemistry, 15 (1976) 1225. 8 A. Kleinhofs, F.A. Haskin and H.J. Gorz, Phytochemistry, 6 (1967) 1313. 9 J.J. Macbeix, C.R. Acad. Sci. Paris Ser. D, 284 (1977) 33. 10 G.A. Barber and E.F. Neufeld, Biochem. Blophys. Res. Commun., 6 (1961) 44. 11 G.A. Barber, Biochemistry, 1 (1962) 463. 12 R.L. Larmon,Phytochemistry, 10 (1971) 3073. 13 R.L. Larson and C.M. Lonegran, Planta, 103 (1972) 361. 14 A. Sutter, R. Ortmann and H. Grisebech, Biochim. Biophys. Acta, 281 (1972) 71.
184 15 16 17 18 19 20 21 22 23 24 25 26 27 28
A. Sutter and H. Grisebach, Biochim. Biophys. Aeta, 309 (1973) 289. J.E. Poulton and M. Kauer, Planta, 136 (1977) 53. N.AM. Saleh, H. Fritsch, P. Witkop and H. Grisebach, Planta, 133 (1976) 41. N.A.M. Saleh, J.E. Poulton and H. Grisebaeh, Phytochemistry, 15 (1976) 1865. R.A. Dedonde~, Annu. Rev. B/ochem., 30 (1961) 347. R.K. Ibrahim and H. Grisebaeh, Arch. Biochem. Biophys., 1"/6 (1976) 700. R.K. Ibrahim, Z. Pflanzenphysiol., 85 (19"/7) 258. W. Karrer, Konstitution und Verkommen der Organischen Pflanzstoffe, Birkh~/user Verlsg, Basel & Stuttgart, 1958. Y.F. 'l~sang and R.K. Ibrahim, Phytochemistry, 18 (1979) 1131. T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 478. O.H. Lowry, N.J. Rosebrough, H.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. S.A. Brown, in T. Swain, J.B. Harborne and C.F. vanSumere, (Eds.), Biochemistry of Plant Phenolics, Plenum Press, New York, 1979, p. 249. J.B. Trommsdorff, Anal. Chem., 14 (1885) 189. K.A. Zirvi, Planta Medica, 31 (1977) 119.