ARCHIVES
OF
BIOCHEMISTRY
Oxidation
ARNE Biologisches
AND
170, 547-556
(1975)
of Flavanone to Flavone with Cell-Free from Young Parsley Leaves’
SUTTER,
Znstitut
BIOPHYSICS
JONATHAN
ZZ der Universitit
POULTON,
Freiburg,
Lehrstuhl Germany
Received
March
AND
fiir
HANS
Biochemie
Extracts
GRISEBACH
der Pflanzen,
D-78
Freiburg,
24, 1975
Cell-free extracts from very young primary leaves of parsley plants (Petroselinum hortense Hoffm.) catalyse the oxidation of naringenin (5,7,4’-trihydroxyflavanone) to apigenin (5,7,4’-trihydroxyflavone). Enzyme activity is found in the 150,000 g supernaheat stable tant. The reaction requires, in addition to oxygen and Fe 2+ ions, (anlother and dialysable nonproteinaceous cofactor(s). The reaction can be inhibited by lo-’ M ophenanthroline. It has a pH optimum of about 7.5. The enzyme preparation also catalyses the oxidation of 7,4-dihydroxyflavanone to the corresponding flavone. The isomeric chalcone (4,2’,4’-trihydroxychalcone) cannot serve as substrate. Only after cyclization to the flavanone with chalcone-flavanone isomerase is oxidation to flavone observed. The enzyme catalyzing this oxidation cannot be a peroxidase, because the reaction is not HzOz-dependent and is not inhibited by catalase, cyanide, or 1.4.10-* M mercaptoethanol.
Although considerable progress has been achieved in the enzymology of flavonoid biosynthesis, the conversion of flavanones to flavones in vitro has not yet been reported in detail. Tracer studies on the biosynthesis of apigenin (Fig. 1, III, R=OH) in parsley have shown that the corresponding flavanone naringenin (II, R=OH) is a far better precursor for apigenin than the corresponding 3-hydroxytlavanone dihydrokaempferol (1). Furthermore, the enzymatic oxidation of naringenin to apigenin has already been used during identification of naringenin, which had been formed enzymatically from pcoumaroyl-CoA and malonyl-CoA (5). In the present paper, we describe some properties of a cell-free extract from very young primary leaves of parsley plants which catalyses the oxidation of naringenin and 7,4’-dihydroxyflavanone (II, R=H) to the corresponding flavones. In the case of 7,4’-dihydroxyflavanone, it ‘This work was schungsgemeinschatt
supported (SFB 46).
by
Deutsche
could also be shown that the flavanone and not the isomeric chalcone (I, R=H) is the substrate of the reaction. MATERIALS
Substrates,
METHODS
Enzymes, and Reference Compounds
[2-‘ClNaringenin was obtained from 4,2’,4’,6’tetrahydroxy - [p - “Cl - chalcone - 2’ - glucoside (2). 4,2’4’-Trihydroxy-[&“C]-chalcone was a gift from Dr. W. Barz, Munster. (-)(2 S) 7,4’dihydroxy[2-‘Vlflavanone (13) was obtained by incubation of the chalcone with chalcone-flavanone isomerase. The flavanone was purified by paper chromatography with solvent D. Apigenin and cosmosiin were purchased from C. Roth, Karlsruhe. Prunin was from our laboratory collection, and 7,4’-dihydroxyflavone was a gift from Dr. E. Wong, Palmerston, New Zealand. Polyamide and cellulose MN 300 for thin layer chromatography were obtained from Macherey and Nagel, Duren. All other biochemicals were purchased from Boehringer, Mannheim. Catalase (EC 1.11.1.6.), chymotrypsin (EC 3.4.21.1.), horseradish peroxidase (EC 1.11.1.7.1, and xanthine oxidase (EC 1.2.3.2.) were obtained from Boehringer, Mannheim, and @glucosidase (EC 3.2.1.21.) from Serva, Heidelberg. Superoxide dismutase (EC
For547
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
548
SU’R’ER,
““flO”
_ R
POULTON
AND
. “o~ao~2,.,+
0
R
I
0
of flavanone to flavone. (R-H); II, naringenin or 7,4’-dihydroxyllavone
1.15.1.1.) from the blue alga Spir&na was a gift from Dr. E. Elstner, Bochum. Chalcone-flavanone isomerase (EC 551.6.) was purified from mung bean seedlings (Phaseolus aureus Roxb.) by a modification of the method of Hahlbrock et al. (12). The 45-60% (NH&SO, precipitate was redissolved in 20 mM Tris-HCl, pH 7.6, 14 mM &mercaptoethanol, and residual (NH&SO, was rdmoved by passage of the enzyme preparation through a Sephadex G-25 column, equilibrated in the same buffer. The eluate was then applied to a column of DEAE-cellulose and elution was carried out using a linear KC1 gradient up to 0.5 M KCl. Fractions possessing the highest isomerase activity were pooled and concentrated by (NH&SO, (O-0.9) precipitation, followed by desalting on a Sephadex G-25 column in this buffer. The purified enzyme had a specific activity of 95 nmol/min/mg protein.
Chromatographic
Methods
For descending paper chromatography on Whatman 3MM prewashed with methanol, 10% acetic acid and 10 mM EDTA, the following solvent systems were used: A, 30% acetic acid; B, 15% acetic acid; C, chloroform/acetic acid/water (50:45:5, by vol); D, formic acid/2 N HWwater (50:20:30, by vol); E, butanoll2 N HCl/acetic acid/water (6:1:1:2, by vol). Thin-layer chromatography was performed on polyamide and cellulose (15:6, by weight) with the solvent system: F, methanol/water/methylethylketone/acetylacetone (50:45:25:5, by vol).
and Cell Cultures
Parsley plants (Petroselinum hortense Hoff.) were grown from seeds (3). The cultivation of cell suspension cultures of parsley was carried out as described previously (4).
Cell-Free Extract Buds of very young primary leaves were ground for 5 min in a chilled mortar with quartz sand and 0.2 M Tris-HCl (pH 7.O)‘containing 14.2 rnM mercap*In vanone used.
“ofloH R
II
FIG. 1. Oxidation 4,2’,4’-trihydroxychalcone III, aplgenin (R-OH)
Plant Material
GRISEBACH
the later experiments with 7,4’-dihydroxyllaas substrate, the same buffer of pH 7.6 was
0
III I, 4,2’,4’,6’-tetrahydroxychalcone (R-OH) or 7,4’-dihydroxyflavanone (R-H).
(R-OH) (R-H);
or
toethanol. The ratio of fresh bud weightiuffer/ quartz sand was 1:1:0.5 by weight. The homogenate was centrifuged for 2 min at 3OOOg, and the supematant fluid was used as such, or after dilution with the same volume of the extraction buffer, for the incubations. The crude extract contained about 5 mg protein/ml and could be stored at -20°C for 24 h without considerable loss of activity.
Standard Incubation The incubation mixture consisted of 10 nmol of 12J’Clnaringenin (2 mWmmo1) dissolved in 10 ~1 of ethyleneglycol monomethyl ether, 215 nmol of FeSOl (1 rnM) dissolved in water, and crude extract in a total volume of 215 ~1. The reaction was started by addition of enzyme and incubated for 30 min at 30°C. At the end of this period, the incubation mixture was transferred into a reaction vessel containing 10 pg of apigenin and 10 pg of cosmosiin in 10 ~1 of ethyleneglycol monomethyl ether. The mixture was then immediately applied as a 5-cm long band to the chromatogram paper and developed with solvent system A for lo-11 h by letting the solvent drip off the paper. The ultraviolet absorbing zones (350 nm) of apigenin and cosmosiin were cut out and counted in a toluene scintillation fluid (1 liter toluene, 5 g PPO) with a Beckman LS 233 scintillation spectrometer (counting efficiency approx 55-60%). The sum of the radioactivity in apigenin and cosmosiin was taken as measure for enzyme activity.
Incubation
With Trihydroxychalcone Dihydroxyflavanone
or
Incubation with 10 nmol of 4,2’,4’-trihydroxyl/3-W-chalcone (2 mCi/mmol) or with c-)(2 S)-7,4’dihydroxy-[2-‘*Cl-flavanone was carried out as described above with the following modifications. Incubation time was loo-120 min. At the end of this period, 10 rl(10 Fg) of 7,4’-dihydroxyllavone in ethyleneglycol monomethyl ether were added to the reaction mixture. The paper chromatogram was developed with solvent system D. The flavone (R, = 0.49) was located by its bright blue fluorescence when the chromatogram was viewed in uv light. Using this solvent system the R, values for trihydroxychalcone and 7,4’-dihydroxyflavanone were 0.22 and 0.68, respectively.
ENZYMATIC
Dependence
of the Reaction
OXIDATION
on Oxygen
Incubations were carried out in Warburg vessels. The central well contained the absorption solution in which a folded filter paper was placed to enlarge the surface for better gas absorption. The absorption solution for the experiment in argon contained 300 ~1 of 30% KOH and 100 ~1 of 15% pyrogallol and the solution for the experiment in air contained 300 ~1 of 30% KOH and 100 ~1 of water. The crude extract was poured into the side arm and naringenin and the FeSOa solution were placed in the vessel. Before the reaction was started, the solutions were preincubated in the respective atmospheres for 10 min in the presence of the absorption solution.
Ultraviolet-Illumination A uv-lamp at a distance used.
of Crud-e Extmct
type TL-900 from Camag, of 0.5 cm from the quartz
West Berlin, cuvettes was
RESULTS
Zdentification ofProducts from Incubation of Naringenin with Cell-Free Extract When [2-‘4C]naringenin was incubated with a cell-free extract from buds of primary leaves of 21-day-old parsley plants, three radioactive products were detected on a paper chromatogram with solvent sys-
I
i
I
OF
549
FLAVANONE
tern A, which corresponded in their Rfvalues to apigenin, apigenin-i’-0-glucoside (cosmosiin) and naringenin-7-0-glucoside (prunin) (Fig. 2a). The identity of the radioactive zone with R,0.28 with apigenin was further established by cochromatography with authentic apigenin on paper with solvent systems B and C and on polyamide plates with solvent system F (Table I). When the radioactive zone with Rf 0.45 was eluted with water and incubated with @-glucosidase and the resulting mixture chromatographed on a polyamide plate with solvent system F, the total radioactivity appeared in the zone of apigenin. When 2 mM UDP-glucose was present in the incubation, radioactivity was located predominantly in the zones of cosmosiin and prunin (Fig. 2b). In incubations with strongly diluted crude extract or in incubations which contained mainly Sephadex G25 filtrate (see below) and only a small amount of crude extract, apigenin was the only detectable radioactive product. Dependence Enzyme
of Enzyme Activity of Leaves activity
on the Age
for the oxidation
of nar-
i
FIG. 2a. Radioscan of a paper chromatogram (solvent system A) from an incubation of [2-‘Qiaringenin with a cell-free extract from buds of primary leaves of parsley. Bars indicate the position of reference compounds. Api, apigenin; Cos, cosmosiin; Nar, naringenin; Pru, prunin. FIG. 2b. Same as 2a but with addition of 0.2 mM UDP-glucose to the incubation.
TABLE COMPARISON
OF
R,
VALUES
OF THE
MAIN RADIOACTIVE WITH REFERENCE
I PRODUCT FROM SUBSTANCES
Product Apigenin Cosmosiin Naringenin Prunin
0.28 0.28 0.45 0.61 0.83
OF [2-“C]NARINGENIN
Thin-layer
Paper chromatography A
INCUBATION
chromatography on polyamide
B
C
F
0.10
0.70 0.70 0.42
0.05 0.05 0.48
0.10 0.25
550
SLITTER.
POULTON
AND
GRISEBACH
*
ingenin to apigenin was found to a high extent only in primary buds. Primary leaves of 42-day-old plants contained less than 1% of the activity found in the buds. It was further shown that the dramatic decrease in enzyme activity could not be due to the presence of an inhibitor in the older leaves since a mixture of extracts from buds and older leaves showed an enzyme activity corresponding to the dilution of the young bud extract with the extract of older leaves (Table II). Some Properties of the Enzyme System for the Oxidation of Naringenin When the crude extract was centrifuged for 90 min at 15O,OOOg, all enzyme activity remained in the supernatant. The pH optimum of the reaction was about 7.5. The yield of apigenin at this pH was about twice as high in Tris-HCl buffer as in phosphate buffer (Fig. 3). The formation of apigenin in the fourfold diluted crude extract was linear with time for about 40 min. After 100 min the yield of apigenin reached about 50% of the added naringenin. Apigenin formation increased with protein concentrations up to about 250 pg protein in the standard incubation and remained fairly constant at higher protein concentrations. The dependence of product yield on protein concentration was concave at large dilution of the crude extract and convex at lower dilution probably because of the presence of inhibitors and cofactors in the crude extract (see below). The influence of mercaptoethanol on enzyme activity was investigated. Aliquots of a cell-free extract, prepared by homogenizing the tissue in Tris-HCl buffer alone, were then diluted with an equal volume of TABLE DEPENDENCE
OF.ENZYME
60
Counts
extract
(~1)
per minute
in apigenin”
70
75
6.0 PH
FIG. 3. pH optimum of the oxidation nin to apigenin. Experiments were carried standard incubation. 150 ~1 of the buffers to 50 ~1 of the crude extract. (0-O) 0.2 (e-0) 0.5 M Na-phosphate buffer.
Oxygen Dependence
of the Reaction
When the standard incubation was carried out in an argon atmosphere the oxidation of naringenin was less than 1% of that which occurred in the control in air (Table IV). Dependence
of the Reaction
on Fe’+ Ions
The activity of undiluted crude extracts could be stimulated approximately twofold by the addition of 1 mM Fez+. With diluted II ACTIVITY
ON LEAF
AGE
Primary leaves (42 day&B
A plus
200
200
100/100
215.104
200
1.1310
D sum of apigenin plus its 7-0-glucoside. Experiments were carried out in the standard
of naringeout in the was added Tris-HCl;
buffer containing various concentrations of mercaptoethanol. As it can be seen from Table III, mercaptoethanol has a stimulatory and protecting effect on enzyme activity. At -20°C and in the presence of 14 mM mercaptoethanol, 77% of enzyme activity remained after 2 days and 40% after 5 days.
Buds (21 day&A Crude
65
incubation
with
[2-“Clnaringenin.
B
ENZYMATIC
OXIDATION
OF
TABLE INFLUENCE Mercaptoethanol
in
III
OF MERCAPTOETHANOL
incubation
0
0
0 140 (351b
1 3250 132
551
FLAVANONE
ON’ ENZYME
ACTIVITY
3.5
3.5
7
7
35
0 1530 (11)
1 3306 810
0
1 3670 688
2018 497
35
blM)
Fe*+ (mM) Counts per minute” Counts per minute” after 24 h at 4°C
in apigenin in apigenin
1745 (43)
1
0
5371 2384
n Mean values from two experiments. * Figures in parentheses are too low to be significant. TABLE OXYGEN
DEPENDENCE
IV
OF THE ENZYMATIC
REACTIONS
Air Incubation Counts (1 Experiments
no. per minute were
1 in apigenin carried
2
2.104
out in Warburg
vessels
[mMl
MgCl, bM1 o-Phenantroline [mMl Counts per minute in apigenin n A lower but sianiticant stimulation merit.3 were carried out in the standard
as described
and Sephadex
2
100
200
in Methods.
V
OF NARINCENIN
OXIDATION
ON Fe*+
0
1”
0
0
1
0
0
0
0
0
0 4.3.103
0 8.5.103
0.01 3.1.103
was observed incubation.
extracts or with filtrates from very short Sephadex G-10 columns, 1 mM Fez+ stimulated the reaction more than tenfold. However, o-phenanthroline, a strong chelator for Fez+ (11) at a concentration of 0.1 mM, inhibited the reaction almost completely. This inhibition could be overcome by addition of 1 mM Fe’+, but not by 1 mM Mg2+ (Table V). Fe2+ ions consistently gave an approximately tenfold higher stimulation than Fe3+ ions (data not shown). Furthermore, the stimulation of naringenin oxidation by Fez+ ions was not affected by the presence of 5 mM ascorbate. Dialysis
1
1.85.104
TABLE DEPENDENCE FeS04
Argon
Gel-Filtration
The enzymatic activity of the crude extract was found to increase during the first 30 min of dialysis, suggesting the presence of inhibitory substances within the extract. Further evidence for the partial inhi-
with
Fez+ concentration
0.1 200
0.1 8.3.103
as low as 0.02 mM.
0 1 0.1 200 Experi-
bition of naringenin oxidation with the unpurified extract was shown by the fact that a 1:l mixture of crude extract and Sephadex G-25 filtrate gave an approximately threefold higher activity than with the crude extract alone. After gel-filtration of the crude extract through a 40-ml Sephadex G-25 column or following prolonged dialysis, the filtrate could not be reactivated by 1 mM Fe’+ alone (Table VI). Reactivation was achieved only by addition of the supernatant fraction of a heat-denatured extract or by addition of the concentrated dialysate. The nature of the cofactor(s) within the crude extract is as yet unclear. Since the supernatant fraction from the heat-denatured extract could reactivate the G-25 Cltrate, the cofactor(s) must be heat-stable. Further evidence for the nonproteinaceous nature of the cofactor(s) was indicated by the fact that 40 mU of chymotrypsin did not
552
SU’ITER,
POULTON
influence its ability for reactivation. Attempts to identify the unknown cofactors by means of reconstitution experiments with G-25 filtrates have so far been unsuccessful. Various potential cofactors, added at concentrations from 0.01 to 1 mM in various combinations with or without Fe’+ ions, did not restore enzyme activity in the G-25 filtrate (Table VII). Sephadex G-25 filtrates reactivated by addition of Fe’+ TABLE REACTIVATION Crude extract (/.~ll G-25 filtrate (~1)” Heat-denatured supernatant +lY’ Dialysate (/.~l)~ Counts per minute in apigenin
200 0 0 0 7.8.103
AND
GRISEBACH
ions and a supernatant fraction of a crude heat-denatured extract served as controls in these experiments. Heat-denatured yeast extract also did not serve as a cofactor source. Since flavoproteins are in general uvsensitive, the potential participation of such proteins in the reaction was further tested by illumination of the incubation mixture in a quartz cuvette for 5 and 30 VI EXPERIMENTS 0 200 0 0 300
0 200 50 0 7.9.103
0 200 0 100 4.6.103
0 0 200 0 0
a 2 ml of the crude extract was applied to a Sephadex G-25 (30 ml) column and the protein eluted with 0.2 Tris-HCl, pH 7.5, containing 7 mM mercaptoethanol. b The crude extract was heated for 2 min at 100°C and then cleared by centrifugation. c 2.5 ml of the extract were dialysed against 50 ml bidistilled water for 12 h. The dialysate was freezedried and the residue dissolved in 1.5 ml water. TABLE SUBSTANCES
Electron FAD FMN NADP+ NAD+
WHICH WERE TESTED FOR REACTIVATION
acceptors
Electron
Phenazinemethosulfate Methyleneblue Dichlorophenolindophenol Benzylviologen Methylviologen Triphenyltetrazolium-chloride Cytochrome c Menadione
NADPH NADPH-regenerating Tetrahydropteridin Ascorbic acid Catechol Protocatechuic Caffeic acid
TABLE REACTIVATION Tris-HCl buffer (~11 Crude extract (~1) Filtrate from 10 ml G-10 column Filtrate from 40 ml G-10 column Filtrate from 40 ml G-25 column FeSO, lmM1 Counts per minute in apigenin o b c d
5.8 3.5 2.1 1.8
mg mg mg mg
protein/ml. protein/ml. protein/ml. protein/ml.
VII
IN ADDITION TO Fez+ AS POTENTIAL OF SEPHADEX G-25 FILTRATES
(/.# (~1)’ (/.#
OF DIFFERENT 100 100 0 0 0 1 1.4.104
donors
COFACTORS
Metal
ions
Mg” cu2+ Zn2+ MO”
system
acid
VIII GEL FILTRATES 0 0 200 0 0 0 1.103
BY Fe’+
0 0 200 0 0 1 1.34.104
0 0 0 200 0 0 750
0 0 0 200 0 1 2.4.103
0 0 0 0 200 1 86
ENZYMATIC Unknown
-‘: 10
;I
I 10
OXIDATION
OF
553
FLAVANONE
FlO"a"One
075
050
0 25
I 075
1 0 50
I 0 25
kt
kt
0
I 0
FIG. 4a. Radioscan of a paper chromatogram (solvent system D) from an incubation of 4,2’,4’-trihydroxy-[P-14C1-chalcone with the cell-free extract. For condition see text. Bar indicates position of 7,4’-dihydroxyflavone. FIG. 4b. Same as 4a after preincubation with chalcone-flavanone isomerase.
min with ultraviolet light (350 nm or 254 nm) at 0 and 25°C. Ultraviolet light had no influence on the enzymatic activity of the extracts. The molecular size of the unknown cofactor(s) was however estimated by gel-filtration experiments. Although complete reactivation by 1 mM Fez+ was observed with a filtrate from a lo-ml Sephadex G-10 column, only partial reactivation of the filtrate by Fe !z+ ions was possible after the extract had been passed through a 40 ml G-10 column (Table VIII). It seems very likely therefore that Fez+ ions alone are retained on the smaller G-10 column, whereas the unknown cofactors are retained on the Sephadex G-25 column in addition to Fe’+ ions. Is the Substrate for Oxidation Chalcone or the Flavanone?
the
The important question whether the chalcone or the isomeric flavanone is the substrate for the oxidation could not be solved with compounds having a phloroglucinol substitution pattern in ring A. When the incubation was carried out with 4,2’,4’,6’-tetrahydroxy-[p-‘4C]-chalcone instead of naringenin, 90% of the chalcone cyclized to the flavanone within 10 s and the yield of apigenin was the same as when naringe-
nin was the substrate. The equilibrium of the isomerization at pH 7.0 lies to more than 99% on the flavanone side. Addition of partly purified chalcone-flavanone isomerase from parsley cell cultures (F. Kreuzaler, unpublished work) to the incubation had no influence on the yield of apigenin. In view of the greater stability of 4,2’,4’trihydroxychalcone,3 this chalcone and the corresponding flavanone liquiritigenin were incubated to determine whether they could also be oxidized by the parsley system. When 4,2’,4’-trihydroxy-[P-14Cl-chalcone was incubated with the enzyme preparation with addition of Fe*+, no radioactivity was present in the zone of 7,4’-dihydroxyflavone (Fig. 4a1, although in addition to an unknown compound, some flavanone was formed in this incubation by nonenzymic cyclization of the chalcone. The isomerase from parsley does not catalyse the cyclization of trihydroxychalcone (12). However, when the trihydroxychalcone was preincubated for 45 min with chalcone-flavanone isomerase from mung bean, subsequent incubation with the parsley extract and Fez+ gave strong radioactivity in the zone 3The equilibrium none (14).
constant
is 37 in favor
of flava-
554
SUlTER,
POULTON
of 7,4’-dihydroxyflavone (Fig. 4b). Controls with isomerase alone, with boiled isomerase, or with active isomerase but boiled parsley extract showed no radioactivity in the flavone zone. Without addition of Fe*+, the radioactivity in the flavone zone decreased by about 85%. The identity of the radioactive zone with R, = 0.48 (solvent D) with 7,4’-dihydroxyflavone was further established by cochromatography with an authentic reference sample on paper with solvents A, B, and E. The above results were confirmed with purified (--X2 S) 7,4’dihydroxy-2-‘*C-flavanone but, with this substrate, it was found that addition of the chalcone-flavanone isomerase to the incubation mixture partially inhibited 7,4’dihydroxyflavone formation since by catalyzing the formation of trihydroxychalcone, it lowered the substrate concentration. Could the Oxidation of Naringenin Apigenin be due to a Peroxidase?
to
Preincubation of the incubation mixture with 50 units of catalase had no influence on the yield of apigenin, nor was the reaction influenced by the addition of cyanide up to concentrations of 2 mM. Whereas addition of 10 PM Hz02 had no influence on naringenin oxidation, 1 mM Hz02 inhibited the reaction to about 50%. Sephadex G-25 filtrates could not be reactivated by addition of 1 mM Fez+ and H,Oz. In a peroxidase-catalysed reaction free radicals could be formed. However, the radical scavengers glutathione and hydroquinone added in 1 mM concentration had no influence on apigenin formation. Furthermore, superoxide dismutase did not inhibit the oxidation of naringenin. When [2-‘*C]naringenin was incubated with horseradish peroxidase and H202 and the reaction mixture chromatographed on paper with 30% acetic acid, a new unidentified radioactive product with R, = 0.85 was detected. No radioactivity was present in the apigenin zone. The oxidation of naringenin with horseradish peroxidase and H,Oz was completely inhibited by 5 units of catalase and by 14 mM mercaptoethanol. When [2-‘*Clnaringenin was incubated with xanthine and xanthine oxidase (from cow milk) in the presence and absence of
AND
GRISEBACH
Fe’+, no reaction tected.
products
could
be de-
Experiments with Cell Suspension Cultures of Parsley as Enzyme Source It had been shown previously that the activity of enzymes involved in flavonoid biosynthesis reaches maximum activity after about 24 h after onset of illumination of the cell cultures with white light (4). When cell-free extracts of parsley cells which had been illuminated for 18-22 h were incubated with [2-‘*Clnaringenin under the optimal conditions for the enzyme from parsley buds, no oxidation to apigenin could be observed. As a check for the enzymatic acitivity of the extracts, the apigenin-7-0-glucosyl transferase (2) activity was determined in parallel experiments. High activity of this enzyme was present. Furthermore, mixtures of cell-free extracts from parsley buds and parsley cell cultures were tested for enzyme activity. Both extracts were dialysed for 20 min against the Tris buffer before use. The same enzyme activities were found when different mixtures of extracts from buds and cell cultures or from buds and Tris buffer were incubated. Therefore extracts from cell cultures have neither a stimulatory nor an inhibitory effect on the enzyme activity of buds. The heat-denatured supernatant from buds could not activate the cell cultpre extract. Addition of various potential cofactors (flavin nucleotide, methylene-blue, dichlorophenol-indophein different nol, Mo5+, Fe’+, and NADPH) combinations to the cell culture extract was also without effect. DISCUSSION
The product formed from naringenin with a cell-free extract from buds of primary leaves of parsley was unequivocally identified as apigenin. The crude extracts apparently contain some UDP-glucose to convert part of naringenin and apigenin to their i’-0-glucosides with the corresponding glucosyltransferase present in young parsley leaves (3). In diluted extracts or in extracts dialysed for a short period of time, the only enzymatic product formed was apigenin.
ENZYMATIC
Chalcone
or Flavanone
-
OXIDATION
OF
eA'-
eA'-
mAr
0
0
of flavone
(VI)
0
Y
IF
FIG. 5. Formation intermediate (V).
555
FLAVANONE
from
PT
o-hydroxydibenzoylmethane
Apigenin formation requires oxygen, Fez+ ions and an additional heat-stable nonproteinaceous cofactor of low molecular weight. The additional cofactor(s) is (are) retained on a Sephadex G-25 column but only partly on a G-10 column (Mlim -700) of the same bed volume. It can therefore be estimated that the molecular weight of this factor(s) is in the range of the exclusion limit of Sephadex G-10. Previous attempts to clarify the question whether further transformations of the chalcone-flavanone intermediate proceed from the chalcone or flavanone gave equivocal results (6, 7). It is therefore gratifying that we have now proved unequivocally that the flavanone is the substrate for the oxidation to flavone. This is also interesting in view of the fact that a flavanone (naringenin) and not a chalcone is the primary product of the condensation of p-coumaroyl-CoA and malonyl-CoA catalysed by a “flavanone synthase” from parsley cell cultures (15). It has been suggested that peroxidases may take part in the biosynthesis of some flavonoids from chalcone-flavanone (8, 9). Our results clearly show that the enzyme responsible for the oxidation of naringenin to apigenin cannot be a peroxidase. The experiments with radical scavengers, superoxide dismutase and xanthine oxidase further show that 02- or OH- species are not involved in the reaction. Because 2,4,6-trihydroxydibenzoylmethane-4-glucoside was found together with the corresponding flavone chrysin’l-glucoside in leaves of a M&s species it was assumed that o-hydroxydibenzoylmethanes could be intermediates in flavone biosynthesis (Fig. 5) (16). Higher substituted dibenzoylmethanes cyclize very rapidly nonenzymatically to flavones. Tracer experiments with 2-hydroxy-[y-“Cldibenzoylmethane and leaves of Primula farinosa excreting the unsubstituted flavone (17) gave no incorporation of radioactivity into flavone which was higher than
(IV)
via
background activity obtained with caused by nonenzymic cyclization Grisebach, unpublished results).
the
enolate
boiled leaves and (L. Schill and H.
The following arguments strongly suggest that the enzyme activity which we describe here, is responsible for the formation of flavones in plants: (a) flavanone oxidase activity is found only in very young leaves of parsley, in which other enzymes involved in the flavone glycoside pathway also reach maximum activity (3). (b) The specificity of the reaction; only the flavanone but not the chalcone is oxidized, and no other product except flavone is formed. Since all other enzymes of the flavone glycoside pathway have been detected in crude extracts of parsley cell cultures (181, and since these cultures form appreciable amounts of flavones (lo), it is surprising that all attempts to find enzyme activity for oxidation of naringenin in these extracts have so far failed. Further characterization of the flavanone oxidase was severely hampered by the lack of a good enzyme source. It took 24 h to collect sufficient primary buds for the preparation of 1 ml crude extract. It is hoped that a better source can be found, which would allow the properties of this enzyme to be studied more closely. ACKNOWLEDGMENTS One of us (J.P.) thanks the Alexander von Humboldt Foundation for the award of a Research Fellowship. We thank Miss M. Kauer and Miss A. Striiter for skilled technical assistance. REFERENCES 1. GRISEBACH, Naturforsch. 2. SUTTER,
(1972) 3.
A.,
H.
BILHUBER, 746-751. ORTMANN, R., AND
ORTMANN,
tochemistry
W.
(1967)
Z.
GRISEBACH, H. Acta 258, 78-87. K., SUTTER, A., WELLMANN, E., R., AND GRISEBACH, H. (1971) Phy10, 109-116.
Biochim.
HAHLBROCK,
AND
22b,
Biophys.
556
SU’ITER,
POULTON
4. HAHLBROCK, K., EBEL, J., ORTMANN, R., Sum TER, A., WELLMANN, E., AND GRISEBACH, H. (1971) Biochim. Biophys. Actu 244, 7-15. 5. KREUZALER, F. AND HAHLBROCK, K. (1972) FEBS Lett. 28, 69-72. 6. WONG, E. AND GRISEBACH, H. (1969)Phytochemisty 8, 1419-1426. 7. PELTER, A., BRADSHAW, J., AND WARREN, R. F. (1971) Phytochemisty 10, 835-850. 8. RATHMELL, W. G. AND BENDALL, D. S. (1972) Biochem. J. 127, 125-132. 9. WONG, E. AND WILSON, J. M. (1972)Phytochemistry 11, 875. 10. KREUZALER, F. ANDHAHLBROCK, K. (1973)Phytochemistry 12, 1149-1152. 11. IRVING, H. AND MILLER, D. H. (1962) J. Chem.
AND
GRISEBACH
Sot. 5222-5237. 12. HAHLBROCK, K., WONG, E., SCHILL, L., ANDGRISEBACH, H. (1970) Phytochemistry 9.949-958. 13. HAHLBROCK, K., ZILG, H., AND GRISEBACH, H. (1970) Eur. J. Biochem. 15, 13-18. 14. BOLAND, M. J. AND WONG, E. (1975) Eur. J. Biochem. 50, 383-389. 15. KREUZALER, F. AND HAHLBROCK, K. (1975) Eur. J. Biochem., in press. 16. WILLIAMS, A. H. (1967) Chem. Znd., 1526-1527. 17. HARBORNE, J. B. (1968) Phytochemisty 7,12151230. 18. GRISEBACH, H. AND HAHLBROCK, K. (1974) in Recent Advances in Phytochemistry (Runeckles, V. C. and Conn, E. E., eds.), Vol. 8, pp. 21-52, Academic Press, New York.
OF
BIOCHEMISTRY
Oxidation
ARNE Biologisches
AND
170, 547-556
(1975)
of Flavanone to Flavone with Cell-Free from Young Parsley Leaves’
SUTTER,
Znstitut
BIOPHYSICS
JONATHAN
ZZ der Universitit
POULTON,
Freiburg,
Lehrstuhl Germany
Received
March
AND
fiir
HANS
Biochemie
Extracts
GRISEBACH
der Pflanzen,
D-78
Freiburg,
24, 1975
Cell-free extracts from very young primary leaves of parsley plants (Petroselinum hortense Hoffm.) catalyse the oxidation of naringenin (5,7,4’-trihydroxyflavanone) to apigenin (5,7,4’-trihydroxyflavone). Enzyme activity is found in the 150,000 g supernaheat stable tant. The reaction requires, in addition to oxygen and Fe 2+ ions, (anlother and dialysable nonproteinaceous cofactor(s). The reaction can be inhibited by lo-’ M ophenanthroline. It has a pH optimum of about 7.5. The enzyme preparation also catalyses the oxidation of 7,4-dihydroxyflavanone to the corresponding flavone. The isomeric chalcone (4,2’,4’-trihydroxychalcone) cannot serve as substrate. Only after cyclization to the flavanone with chalcone-flavanone isomerase is oxidation to flavone observed. The enzyme catalyzing this oxidation cannot be a peroxidase, because the reaction is not HzOz-dependent and is not inhibited by catalase, cyanide, or 1.4.10-* M mercaptoethanol.
Although considerable progress has been achieved in the enzymology of flavonoid biosynthesis, the conversion of flavanones to flavones in vitro has not yet been reported in detail. Tracer studies on the biosynthesis of apigenin (Fig. 1, III, R=OH) in parsley have shown that the corresponding flavanone naringenin (II, R=OH) is a far better precursor for apigenin than the corresponding 3-hydroxytlavanone dihydrokaempferol (1). Furthermore, the enzymatic oxidation of naringenin to apigenin has already been used during identification of naringenin, which had been formed enzymatically from pcoumaroyl-CoA and malonyl-CoA (5). In the present paper, we describe some properties of a cell-free extract from very young primary leaves of parsley plants which catalyses the oxidation of naringenin and 7,4’-dihydroxyflavanone (II, R=H) to the corresponding flavones. In the case of 7,4’-dihydroxyflavanone, it ‘This work was schungsgemeinschatt
supported (SFB 46).
by
Deutsche
could also be shown that the flavanone and not the isomeric chalcone (I, R=H) is the substrate of the reaction. MATERIALS
Substrates,
METHODS
Enzymes, and Reference Compounds
[2-‘ClNaringenin was obtained from 4,2’,4’,6’tetrahydroxy - [p - “Cl - chalcone - 2’ - glucoside (2). 4,2’4’-Trihydroxy-[&“C]-chalcone was a gift from Dr. W. Barz, Munster. (-)(2 S) 7,4’dihydroxy[2-‘Vlflavanone (13) was obtained by incubation of the chalcone with chalcone-flavanone isomerase. The flavanone was purified by paper chromatography with solvent D. Apigenin and cosmosiin were purchased from C. Roth, Karlsruhe. Prunin was from our laboratory collection, and 7,4’-dihydroxyflavone was a gift from Dr. E. Wong, Palmerston, New Zealand. Polyamide and cellulose MN 300 for thin layer chromatography were obtained from Macherey and Nagel, Duren. All other biochemicals were purchased from Boehringer, Mannheim. Catalase (EC 1.11.1.6.), chymotrypsin (EC 3.4.21.1.), horseradish peroxidase (EC 1.11.1.7.1, and xanthine oxidase (EC 1.2.3.2.) were obtained from Boehringer, Mannheim, and @glucosidase (EC 3.2.1.21.) from Serva, Heidelberg. Superoxide dismutase (EC
For547
Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
AND
548
SU’R’ER,
““flO”
_ R
POULTON
AND
. “o~ao~2,.,+
0
R
I
0
of flavanone to flavone. (R-H); II, naringenin or 7,4’-dihydroxyllavone
1.15.1.1.) from the blue alga Spir&na was a gift from Dr. E. Elstner, Bochum. Chalcone-flavanone isomerase (EC 551.6.) was purified from mung bean seedlings (Phaseolus aureus Roxb.) by a modification of the method of Hahlbrock et al. (12). The 45-60% (NH&SO, precipitate was redissolved in 20 mM Tris-HCl, pH 7.6, 14 mM &mercaptoethanol, and residual (NH&SO, was rdmoved by passage of the enzyme preparation through a Sephadex G-25 column, equilibrated in the same buffer. The eluate was then applied to a column of DEAE-cellulose and elution was carried out using a linear KC1 gradient up to 0.5 M KCl. Fractions possessing the highest isomerase activity were pooled and concentrated by (NH&SO, (O-0.9) precipitation, followed by desalting on a Sephadex G-25 column in this buffer. The purified enzyme had a specific activity of 95 nmol/min/mg protein.
Chromatographic
Methods
For descending paper chromatography on Whatman 3MM prewashed with methanol, 10% acetic acid and 10 mM EDTA, the following solvent systems were used: A, 30% acetic acid; B, 15% acetic acid; C, chloroform/acetic acid/water (50:45:5, by vol); D, formic acid/2 N HWwater (50:20:30, by vol); E, butanoll2 N HCl/acetic acid/water (6:1:1:2, by vol). Thin-layer chromatography was performed on polyamide and cellulose (15:6, by weight) with the solvent system: F, methanol/water/methylethylketone/acetylacetone (50:45:25:5, by vol).
and Cell Cultures
Parsley plants (Petroselinum hortense Hoff.) were grown from seeds (3). The cultivation of cell suspension cultures of parsley was carried out as described previously (4).
Cell-Free Extract Buds of very young primary leaves were ground for 5 min in a chilled mortar with quartz sand and 0.2 M Tris-HCl (pH 7.O)‘containing 14.2 rnM mercap*In vanone used.
“ofloH R
II
FIG. 1. Oxidation 4,2’,4’-trihydroxychalcone III, aplgenin (R-OH)
Plant Material
GRISEBACH
the later experiments with 7,4’-dihydroxyllaas substrate, the same buffer of pH 7.6 was
0
III I, 4,2’,4’,6’-tetrahydroxychalcone (R-OH) or 7,4’-dihydroxyflavanone (R-H).
(R-OH) (R-H);
or
toethanol. The ratio of fresh bud weightiuffer/ quartz sand was 1:1:0.5 by weight. The homogenate was centrifuged for 2 min at 3OOOg, and the supematant fluid was used as such, or after dilution with the same volume of the extraction buffer, for the incubations. The crude extract contained about 5 mg protein/ml and could be stored at -20°C for 24 h without considerable loss of activity.
Standard Incubation The incubation mixture consisted of 10 nmol of 12J’Clnaringenin (2 mWmmo1) dissolved in 10 ~1 of ethyleneglycol monomethyl ether, 215 nmol of FeSOl (1 rnM) dissolved in water, and crude extract in a total volume of 215 ~1. The reaction was started by addition of enzyme and incubated for 30 min at 30°C. At the end of this period, the incubation mixture was transferred into a reaction vessel containing 10 pg of apigenin and 10 pg of cosmosiin in 10 ~1 of ethyleneglycol monomethyl ether. The mixture was then immediately applied as a 5-cm long band to the chromatogram paper and developed with solvent system A for lo-11 h by letting the solvent drip off the paper. The ultraviolet absorbing zones (350 nm) of apigenin and cosmosiin were cut out and counted in a toluene scintillation fluid (1 liter toluene, 5 g PPO) with a Beckman LS 233 scintillation spectrometer (counting efficiency approx 55-60%). The sum of the radioactivity in apigenin and cosmosiin was taken as measure for enzyme activity.
Incubation
With Trihydroxychalcone Dihydroxyflavanone
or
Incubation with 10 nmol of 4,2’,4’-trihydroxyl/3-W-chalcone (2 mCi/mmol) or with c-)(2 S)-7,4’dihydroxy-[2-‘*Cl-flavanone was carried out as described above with the following modifications. Incubation time was loo-120 min. At the end of this period, 10 rl(10 Fg) of 7,4’-dihydroxyllavone in ethyleneglycol monomethyl ether were added to the reaction mixture. The paper chromatogram was developed with solvent system D. The flavone (R, = 0.49) was located by its bright blue fluorescence when the chromatogram was viewed in uv light. Using this solvent system the R, values for trihydroxychalcone and 7,4’-dihydroxyflavanone were 0.22 and 0.68, respectively.
ENZYMATIC
Dependence
of the Reaction
OXIDATION
on Oxygen
Incubations were carried out in Warburg vessels. The central well contained the absorption solution in which a folded filter paper was placed to enlarge the surface for better gas absorption. The absorption solution for the experiment in argon contained 300 ~1 of 30% KOH and 100 ~1 of 15% pyrogallol and the solution for the experiment in air contained 300 ~1 of 30% KOH and 100 ~1 of water. The crude extract was poured into the side arm and naringenin and the FeSOa solution were placed in the vessel. Before the reaction was started, the solutions were preincubated in the respective atmospheres for 10 min in the presence of the absorption solution.
Ultraviolet-Illumination A uv-lamp at a distance used.
of Crud-e Extmct
type TL-900 from Camag, of 0.5 cm from the quartz
West Berlin, cuvettes was
RESULTS
Zdentification ofProducts from Incubation of Naringenin with Cell-Free Extract When [2-‘4C]naringenin was incubated with a cell-free extract from buds of primary leaves of 21-day-old parsley plants, three radioactive products were detected on a paper chromatogram with solvent sys-
I
i
I
OF
549
FLAVANONE
tern A, which corresponded in their Rfvalues to apigenin, apigenin-i’-0-glucoside (cosmosiin) and naringenin-7-0-glucoside (prunin) (Fig. 2a). The identity of the radioactive zone with R,0.28 with apigenin was further established by cochromatography with authentic apigenin on paper with solvent systems B and C and on polyamide plates with solvent system F (Table I). When the radioactive zone with Rf 0.45 was eluted with water and incubated with @-glucosidase and the resulting mixture chromatographed on a polyamide plate with solvent system F, the total radioactivity appeared in the zone of apigenin. When 2 mM UDP-glucose was present in the incubation, radioactivity was located predominantly in the zones of cosmosiin and prunin (Fig. 2b). In incubations with strongly diluted crude extract or in incubations which contained mainly Sephadex G25 filtrate (see below) and only a small amount of crude extract, apigenin was the only detectable radioactive product. Dependence Enzyme
of Enzyme Activity of Leaves activity
on the Age
for the oxidation
of nar-
i
FIG. 2a. Radioscan of a paper chromatogram (solvent system A) from an incubation of [2-‘Qiaringenin with a cell-free extract from buds of primary leaves of parsley. Bars indicate the position of reference compounds. Api, apigenin; Cos, cosmosiin; Nar, naringenin; Pru, prunin. FIG. 2b. Same as 2a but with addition of 0.2 mM UDP-glucose to the incubation.
TABLE COMPARISON
OF
R,
VALUES
OF THE
MAIN RADIOACTIVE WITH REFERENCE
I PRODUCT FROM SUBSTANCES
Product Apigenin Cosmosiin Naringenin Prunin
0.28 0.28 0.45 0.61 0.83
OF [2-“C]NARINGENIN
Thin-layer
Paper chromatography A
INCUBATION
chromatography on polyamide
B
C
F
0.10
0.70 0.70 0.42
0.05 0.05 0.48
0.10 0.25
550
SLITTER.
POULTON
AND
GRISEBACH
*
ingenin to apigenin was found to a high extent only in primary buds. Primary leaves of 42-day-old plants contained less than 1% of the activity found in the buds. It was further shown that the dramatic decrease in enzyme activity could not be due to the presence of an inhibitor in the older leaves since a mixture of extracts from buds and older leaves showed an enzyme activity corresponding to the dilution of the young bud extract with the extract of older leaves (Table II). Some Properties of the Enzyme System for the Oxidation of Naringenin When the crude extract was centrifuged for 90 min at 15O,OOOg, all enzyme activity remained in the supernatant. The pH optimum of the reaction was about 7.5. The yield of apigenin at this pH was about twice as high in Tris-HCl buffer as in phosphate buffer (Fig. 3). The formation of apigenin in the fourfold diluted crude extract was linear with time for about 40 min. After 100 min the yield of apigenin reached about 50% of the added naringenin. Apigenin formation increased with protein concentrations up to about 250 pg protein in the standard incubation and remained fairly constant at higher protein concentrations. The dependence of product yield on protein concentration was concave at large dilution of the crude extract and convex at lower dilution probably because of the presence of inhibitors and cofactors in the crude extract (see below). The influence of mercaptoethanol on enzyme activity was investigated. Aliquots of a cell-free extract, prepared by homogenizing the tissue in Tris-HCl buffer alone, were then diluted with an equal volume of TABLE DEPENDENCE
OF.ENZYME
60
Counts
extract
(~1)
per minute
in apigenin”
70
75
6.0 PH
FIG. 3. pH optimum of the oxidation nin to apigenin. Experiments were carried standard incubation. 150 ~1 of the buffers to 50 ~1 of the crude extract. (0-O) 0.2 (e-0) 0.5 M Na-phosphate buffer.
Oxygen Dependence
of the Reaction
When the standard incubation was carried out in an argon atmosphere the oxidation of naringenin was less than 1% of that which occurred in the control in air (Table IV). Dependence
of the Reaction
on Fe’+ Ions
The activity of undiluted crude extracts could be stimulated approximately twofold by the addition of 1 mM Fez+. With diluted II ACTIVITY
ON LEAF
AGE
Primary leaves (42 day&B
A plus
200
200
100/100
215.104
200
1.1310
D sum of apigenin plus its 7-0-glucoside. Experiments were carried out in the standard
of naringeout in the was added Tris-HCl;
buffer containing various concentrations of mercaptoethanol. As it can be seen from Table III, mercaptoethanol has a stimulatory and protecting effect on enzyme activity. At -20°C and in the presence of 14 mM mercaptoethanol, 77% of enzyme activity remained after 2 days and 40% after 5 days.
Buds (21 day&A Crude
65
incubation
with
[2-“Clnaringenin.
B
ENZYMATIC
OXIDATION
OF
TABLE INFLUENCE Mercaptoethanol
in
III
OF MERCAPTOETHANOL
incubation
0
0
0 140 (351b
1 3250 132
551
FLAVANONE
ON’ ENZYME
ACTIVITY
3.5
3.5
7
7
35
0 1530 (11)
1 3306 810
0
1 3670 688
2018 497
35
blM)
Fe*+ (mM) Counts per minute” Counts per minute” after 24 h at 4°C
in apigenin in apigenin
1745 (43)
1
0
5371 2384
n Mean values from two experiments. * Figures in parentheses are too low to be significant. TABLE OXYGEN
DEPENDENCE
IV
OF THE ENZYMATIC
REACTIONS
Air Incubation Counts (1 Experiments
no. per minute were
1 in apigenin carried
2
2.104
out in Warburg
vessels
[mMl
MgCl, bM1 o-Phenantroline [mMl Counts per minute in apigenin n A lower but sianiticant stimulation merit.3 were carried out in the standard
as described
and Sephadex
2
100
200
in Methods.
V
OF NARINCENIN
OXIDATION
ON Fe*+
0
1”
0
0
1
0
0
0
0
0
0 4.3.103
0 8.5.103
0.01 3.1.103
was observed incubation.
extracts or with filtrates from very short Sephadex G-10 columns, 1 mM Fez+ stimulated the reaction more than tenfold. However, o-phenanthroline, a strong chelator for Fez+ (11) at a concentration of 0.1 mM, inhibited the reaction almost completely. This inhibition could be overcome by addition of 1 mM Fe’+, but not by 1 mM Mg2+ (Table V). Fe2+ ions consistently gave an approximately tenfold higher stimulation than Fe3+ ions (data not shown). Furthermore, the stimulation of naringenin oxidation by Fez+ ions was not affected by the presence of 5 mM ascorbate. Dialysis
1
1.85.104
TABLE DEPENDENCE FeS04
Argon
Gel-Filtration
The enzymatic activity of the crude extract was found to increase during the first 30 min of dialysis, suggesting the presence of inhibitory substances within the extract. Further evidence for the partial inhi-
with
Fez+ concentration
0.1 200
0.1 8.3.103
as low as 0.02 mM.
0 1 0.1 200 Experi-
bition of naringenin oxidation with the unpurified extract was shown by the fact that a 1:l mixture of crude extract and Sephadex G-25 filtrate gave an approximately threefold higher activity than with the crude extract alone. After gel-filtration of the crude extract through a 40-ml Sephadex G-25 column or following prolonged dialysis, the filtrate could not be reactivated by 1 mM Fe’+ alone (Table VI). Reactivation was achieved only by addition of the supernatant fraction of a heat-denatured extract or by addition of the concentrated dialysate. The nature of the cofactor(s) within the crude extract is as yet unclear. Since the supernatant fraction from the heat-denatured extract could reactivate the G-25 Cltrate, the cofactor(s) must be heat-stable. Further evidence for the nonproteinaceous nature of the cofactor(s) was indicated by the fact that 40 mU of chymotrypsin did not
552
SU’ITER,
POULTON
influence its ability for reactivation. Attempts to identify the unknown cofactors by means of reconstitution experiments with G-25 filtrates have so far been unsuccessful. Various potential cofactors, added at concentrations from 0.01 to 1 mM in various combinations with or without Fe’+ ions, did not restore enzyme activity in the G-25 filtrate (Table VII). Sephadex G-25 filtrates reactivated by addition of Fe’+ TABLE REACTIVATION Crude extract (/.~ll G-25 filtrate (~1)” Heat-denatured supernatant +lY’ Dialysate (/.~l)~ Counts per minute in apigenin
200 0 0 0 7.8.103
AND
GRISEBACH
ions and a supernatant fraction of a crude heat-denatured extract served as controls in these experiments. Heat-denatured yeast extract also did not serve as a cofactor source. Since flavoproteins are in general uvsensitive, the potential participation of such proteins in the reaction was further tested by illumination of the incubation mixture in a quartz cuvette for 5 and 30 VI EXPERIMENTS 0 200 0 0 300
0 200 50 0 7.9.103
0 200 0 100 4.6.103
0 0 200 0 0
a 2 ml of the crude extract was applied to a Sephadex G-25 (30 ml) column and the protein eluted with 0.2 Tris-HCl, pH 7.5, containing 7 mM mercaptoethanol. b The crude extract was heated for 2 min at 100°C and then cleared by centrifugation. c 2.5 ml of the extract were dialysed against 50 ml bidistilled water for 12 h. The dialysate was freezedried and the residue dissolved in 1.5 ml water. TABLE SUBSTANCES
Electron FAD FMN NADP+ NAD+
WHICH WERE TESTED FOR REACTIVATION
acceptors
Electron
Phenazinemethosulfate Methyleneblue Dichlorophenolindophenol Benzylviologen Methylviologen Triphenyltetrazolium-chloride Cytochrome c Menadione
NADPH NADPH-regenerating Tetrahydropteridin Ascorbic acid Catechol Protocatechuic Caffeic acid
TABLE REACTIVATION Tris-HCl buffer (~11 Crude extract (~1) Filtrate from 10 ml G-10 column Filtrate from 40 ml G-10 column Filtrate from 40 ml G-25 column FeSO, lmM1 Counts per minute in apigenin o b c d
5.8 3.5 2.1 1.8
mg mg mg mg
protein/ml. protein/ml. protein/ml. protein/ml.
VII
IN ADDITION TO Fez+ AS POTENTIAL OF SEPHADEX G-25 FILTRATES
(/.# (~1)’ (/.#
OF DIFFERENT 100 100 0 0 0 1 1.4.104
donors
COFACTORS
Metal
ions
Mg” cu2+ Zn2+ MO”
system
acid
VIII GEL FILTRATES 0 0 200 0 0 0 1.103
BY Fe’+
0 0 200 0 0 1 1.34.104
0 0 0 200 0 0 750
0 0 0 200 0 1 2.4.103
0 0 0 0 200 1 86
ENZYMATIC Unknown
-‘: 10
;I
I 10
OXIDATION
OF
553
FLAVANONE
FlO"a"One
075
050
0 25
I 075
1 0 50
I 0 25
kt
kt
0
I 0
FIG. 4a. Radioscan of a paper chromatogram (solvent system D) from an incubation of 4,2’,4’-trihydroxy-[P-14C1-chalcone with the cell-free extract. For condition see text. Bar indicates position of 7,4’-dihydroxyflavone. FIG. 4b. Same as 4a after preincubation with chalcone-flavanone isomerase.
min with ultraviolet light (350 nm or 254 nm) at 0 and 25°C. Ultraviolet light had no influence on the enzymatic activity of the extracts. The molecular size of the unknown cofactor(s) was however estimated by gel-filtration experiments. Although complete reactivation by 1 mM Fez+ was observed with a filtrate from a lo-ml Sephadex G-10 column, only partial reactivation of the filtrate by Fe !z+ ions was possible after the extract had been passed through a 40 ml G-10 column (Table VIII). It seems very likely therefore that Fez+ ions alone are retained on the smaller G-10 column, whereas the unknown cofactors are retained on the Sephadex G-25 column in addition to Fe’+ ions. Is the Substrate for Oxidation Chalcone or the Flavanone?
the
The important question whether the chalcone or the isomeric flavanone is the substrate for the oxidation could not be solved with compounds having a phloroglucinol substitution pattern in ring A. When the incubation was carried out with 4,2’,4’,6’-tetrahydroxy-[p-‘4C]-chalcone instead of naringenin, 90% of the chalcone cyclized to the flavanone within 10 s and the yield of apigenin was the same as when naringe-
nin was the substrate. The equilibrium of the isomerization at pH 7.0 lies to more than 99% on the flavanone side. Addition of partly purified chalcone-flavanone isomerase from parsley cell cultures (F. Kreuzaler, unpublished work) to the incubation had no influence on the yield of apigenin. In view of the greater stability of 4,2’,4’trihydroxychalcone,3 this chalcone and the corresponding flavanone liquiritigenin were incubated to determine whether they could also be oxidized by the parsley system. When 4,2’,4’-trihydroxy-[P-14Cl-chalcone was incubated with the enzyme preparation with addition of Fe*+, no radioactivity was present in the zone of 7,4’-dihydroxyflavone (Fig. 4a1, although in addition to an unknown compound, some flavanone was formed in this incubation by nonenzymic cyclization of the chalcone. The isomerase from parsley does not catalyse the cyclization of trihydroxychalcone (12). However, when the trihydroxychalcone was preincubated for 45 min with chalcone-flavanone isomerase from mung bean, subsequent incubation with the parsley extract and Fez+ gave strong radioactivity in the zone 3The equilibrium none (14).
constant
is 37 in favor
of flava-
554
SUlTER,
POULTON
of 7,4’-dihydroxyflavone (Fig. 4b). Controls with isomerase alone, with boiled isomerase, or with active isomerase but boiled parsley extract showed no radioactivity in the flavone zone. Without addition of Fe*+, the radioactivity in the flavone zone decreased by about 85%. The identity of the radioactive zone with R, = 0.48 (solvent D) with 7,4’-dihydroxyflavone was further established by cochromatography with an authentic reference sample on paper with solvents A, B, and E. The above results were confirmed with purified (--X2 S) 7,4’dihydroxy-2-‘*C-flavanone but, with this substrate, it was found that addition of the chalcone-flavanone isomerase to the incubation mixture partially inhibited 7,4’dihydroxyflavone formation since by catalyzing the formation of trihydroxychalcone, it lowered the substrate concentration. Could the Oxidation of Naringenin Apigenin be due to a Peroxidase?
to
Preincubation of the incubation mixture with 50 units of catalase had no influence on the yield of apigenin, nor was the reaction influenced by the addition of cyanide up to concentrations of 2 mM. Whereas addition of 10 PM Hz02 had no influence on naringenin oxidation, 1 mM Hz02 inhibited the reaction to about 50%. Sephadex G-25 filtrates could not be reactivated by addition of 1 mM Fez+ and H,Oz. In a peroxidase-catalysed reaction free radicals could be formed. However, the radical scavengers glutathione and hydroquinone added in 1 mM concentration had no influence on apigenin formation. Furthermore, superoxide dismutase did not inhibit the oxidation of naringenin. When [2-‘*C]naringenin was incubated with horseradish peroxidase and H202 and the reaction mixture chromatographed on paper with 30% acetic acid, a new unidentified radioactive product with R, = 0.85 was detected. No radioactivity was present in the apigenin zone. The oxidation of naringenin with horseradish peroxidase and H,Oz was completely inhibited by 5 units of catalase and by 14 mM mercaptoethanol. When [2-‘*Clnaringenin was incubated with xanthine and xanthine oxidase (from cow milk) in the presence and absence of
AND
GRISEBACH
Fe’+, no reaction tected.
products
could
be de-
Experiments with Cell Suspension Cultures of Parsley as Enzyme Source It had been shown previously that the activity of enzymes involved in flavonoid biosynthesis reaches maximum activity after about 24 h after onset of illumination of the cell cultures with white light (4). When cell-free extracts of parsley cells which had been illuminated for 18-22 h were incubated with [2-‘*Clnaringenin under the optimal conditions for the enzyme from parsley buds, no oxidation to apigenin could be observed. As a check for the enzymatic acitivity of the extracts, the apigenin-7-0-glucosyl transferase (2) activity was determined in parallel experiments. High activity of this enzyme was present. Furthermore, mixtures of cell-free extracts from parsley buds and parsley cell cultures were tested for enzyme activity. Both extracts were dialysed for 20 min against the Tris buffer before use. The same enzyme activities were found when different mixtures of extracts from buds and cell cultures or from buds and Tris buffer were incubated. Therefore extracts from cell cultures have neither a stimulatory nor an inhibitory effect on the enzyme activity of buds. The heat-denatured supernatant from buds could not activate the cell cultpre extract. Addition of various potential cofactors (flavin nucleotide, methylene-blue, dichlorophenol-indophein different nol, Mo5+, Fe’+, and NADPH) combinations to the cell culture extract was also without effect. DISCUSSION
The product formed from naringenin with a cell-free extract from buds of primary leaves of parsley was unequivocally identified as apigenin. The crude extracts apparently contain some UDP-glucose to convert part of naringenin and apigenin to their i’-0-glucosides with the corresponding glucosyltransferase present in young parsley leaves (3). In diluted extracts or in extracts dialysed for a short period of time, the only enzymatic product formed was apigenin.
ENZYMATIC
Chalcone
or Flavanone
-
OXIDATION
OF
eA'-
eA'-
mAr
0
0
of flavone
(VI)
0
Y
IF
FIG. 5. Formation intermediate (V).
555
FLAVANONE
from
PT
o-hydroxydibenzoylmethane
Apigenin formation requires oxygen, Fez+ ions and an additional heat-stable nonproteinaceous cofactor of low molecular weight. The additional cofactor(s) is (are) retained on a Sephadex G-25 column but only partly on a G-10 column (Mlim -700) of the same bed volume. It can therefore be estimated that the molecular weight of this factor(s) is in the range of the exclusion limit of Sephadex G-10. Previous attempts to clarify the question whether further transformations of the chalcone-flavanone intermediate proceed from the chalcone or flavanone gave equivocal results (6, 7). It is therefore gratifying that we have now proved unequivocally that the flavanone is the substrate for the oxidation to flavone. This is also interesting in view of the fact that a flavanone (naringenin) and not a chalcone is the primary product of the condensation of p-coumaroyl-CoA and malonyl-CoA catalysed by a “flavanone synthase” from parsley cell cultures (15). It has been suggested that peroxidases may take part in the biosynthesis of some flavonoids from chalcone-flavanone (8, 9). Our results clearly show that the enzyme responsible for the oxidation of naringenin to apigenin cannot be a peroxidase. The experiments with radical scavengers, superoxide dismutase and xanthine oxidase further show that 02- or OH- species are not involved in the reaction. Because 2,4,6-trihydroxydibenzoylmethane-4-glucoside was found together with the corresponding flavone chrysin’l-glucoside in leaves of a M&s species it was assumed that o-hydroxydibenzoylmethanes could be intermediates in flavone biosynthesis (Fig. 5) (16). Higher substituted dibenzoylmethanes cyclize very rapidly nonenzymatically to flavones. Tracer experiments with 2-hydroxy-[y-“Cldibenzoylmethane and leaves of Primula farinosa excreting the unsubstituted flavone (17) gave no incorporation of radioactivity into flavone which was higher than
(IV)
via
background activity obtained with caused by nonenzymic cyclization Grisebach, unpublished results).
the
enolate
boiled leaves and (L. Schill and H.
The following arguments strongly suggest that the enzyme activity which we describe here, is responsible for the formation of flavones in plants: (a) flavanone oxidase activity is found only in very young leaves of parsley, in which other enzymes involved in the flavone glycoside pathway also reach maximum activity (3). (b) The specificity of the reaction; only the flavanone but not the chalcone is oxidized, and no other product except flavone is formed. Since all other enzymes of the flavone glycoside pathway have been detected in crude extracts of parsley cell cultures (181, and since these cultures form appreciable amounts of flavones (lo), it is surprising that all attempts to find enzyme activity for oxidation of naringenin in these extracts have so far failed. Further characterization of the flavanone oxidase was severely hampered by the lack of a good enzyme source. It took 24 h to collect sufficient primary buds for the preparation of 1 ml crude extract. It is hoped that a better source can be found, which would allow the properties of this enzyme to be studied more closely. ACKNOWLEDGMENTS One of us (J.P.) thanks the Alexander von Humboldt Foundation for the award of a Research Fellowship. We thank Miss M. Kauer and Miss A. Striiter for skilled technical assistance. REFERENCES 1. GRISEBACH, Naturforsch. 2. SUTTER,
(1972) 3.
A.,
H.
BILHUBER, 746-751. ORTMANN, R., AND
ORTMANN,
tochemistry
W.
(1967)
Z.
GRISEBACH, H. Acta 258, 78-87. K., SUTTER, A., WELLMANN, E., R., AND GRISEBACH, H. (1971) Phy10, 109-116.
Biochim.
HAHLBROCK,
AND
22b,
Biophys.
556
SU’ITER,
POULTON
4. HAHLBROCK, K., EBEL, J., ORTMANN, R., Sum TER, A., WELLMANN, E., AND GRISEBACH, H. (1971) Biochim. Biophys. Actu 244, 7-15. 5. KREUZALER, F. AND HAHLBROCK, K. (1972) FEBS Lett. 28, 69-72. 6. WONG, E. AND GRISEBACH, H. (1969)Phytochemisty 8, 1419-1426. 7. PELTER, A., BRADSHAW, J., AND WARREN, R. F. (1971) Phytochemisty 10, 835-850. 8. RATHMELL, W. G. AND BENDALL, D. S. (1972) Biochem. J. 127, 125-132. 9. WONG, E. AND WILSON, J. M. (1972)Phytochemistry 11, 875. 10. KREUZALER, F. ANDHAHLBROCK, K. (1973)Phytochemistry 12, 1149-1152. 11. IRVING, H. AND MILLER, D. H. (1962) J. Chem.
AND
GRISEBACH
Sot. 5222-5237. 12. HAHLBROCK, K., WONG, E., SCHILL, L., ANDGRISEBACH, H. (1970) Phytochemistry 9.949-958. 13. HAHLBROCK, K., ZILG, H., AND GRISEBACH, H. (1970) Eur. J. Biochem. 15, 13-18. 14. BOLAND, M. J. AND WONG, E. (1975) Eur. J. Biochem. 50, 383-389. 15. KREUZALER, F. AND HAHLBROCK, K. (1975) Eur. J. Biochem., in press. 16. WILLIAMS, A. H. (1967) Chem. Znd., 1526-1527. 17. HARBORNE, J. B. (1968) Phytochemisty 7,12151230. 18. GRISEBACH, H. AND HAHLBROCK, K. (1974) in Recent Advances in Phytochemistry (Runeckles, V. C. and Conn, E. E., eds.), Vol. 8, pp. 21-52, Academic Press, New York.
