From the Botanisches Institut der Universität,
Menzinger Strasse 67, D-80638 München, Germany, the
¶ Department of Stress Response, Institute of Plant Molecular
Biology, Centre National de la Recherche Scientifique FRE 2161, 28 rue Goethe, F-67083 Strasbourg, France, and the
Institut
für Ökologische Chemie, GSF-Forschungszentrum für
Umwelt und Gesundheit, Ingolstädter Landstrasse 1, D-85764 Neuherberg, Germany
Received for publication, July 14, 2000, and in revised form, September 26, 2000
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ABSTRACT |
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Cytochrome P-450-dependent
hydroxylases are typical enzymes for the modification of basic
flavonoid skeletons. We show in this study that CYP71D9
cDNA, previously isolated from elicitor-induced soybean
(Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this
protein bound various flavonoids with high affinity (1.6 to 52 µM) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol- and
phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A,
the biosynthetic relationship of flavonoid 6-hydroxylase to
isoflavonoid biosynthesis was investigated. Recombinant
2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used
6,7,4'-trihydroxyflavanone as substrate. For its structural
identification, the chemically labile reaction product was converted to
6,7,4'-trihydroxyisoflavone by acid treatment. The structures of the
final reaction products for both enzymes were confirmed by NMR and mass
spectrometry. Our results strongly support the conclusion that, in
soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl
migration of the flavonoid B-ring during isoflavanone formation. This
is the first identification of a flavonoid 6-hydroxylase cDNA from any plant species.
Flavonoids are a diverse group of natural products that serve
important roles in plants during growth, during development, and in
defense against microorganisms and pests (1, 2). These compounds are
synthesized from phenylpropanoid- and acetate-derived precursors
through central pathways furnishing basic
C6-C3-C6 flavonoid skeletons and,
in addition, through a variety of reactions leading to a range of
modified aglycones and subsequently to their glycosylated derivatives
within each flavonoid class. Many of the enzymes of flavonoid
biosynthesis have been extensively studied (3), and recent molecular
biological approaches have complemented biochemical methods in
elucidating the mechanism and regulation of flavonoid biosynthesis (4).
Typical enzymes belonging to the complex branch pathways for the
elaboration of flavonoid skeletons are cytochrome
P-450-dependent hydroxylases (3), such as flavonoid 3'-hydroxylase (5), flavonoid 3',5'-hydroxylase (6), isoflavone 2'-hydroxylase (7), flavanone 2-hydroxylase (8), flavone synthase II
(9, 10), and 2-hydroxyisoflavanone synthase
(2HIS)1 (11-13). Whereas
flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase are
responsible for the formation of the 3',5'-hydroxylation pattern of the
flavonoid B-ring, hydroxylation of the isoflavone B-ring at the 2'
position (isoflavone 2'-hydroxylase) is one of the key reactions
leading to pterocarpan structures. The formation of flavones and
isoflavones from flavanones is catalyzed by several evolutionarily
related P-450s, either in a single concerted reaction leading directly
to the flavone double bond (flavone synthase II) or in a two-step
process, the first being a monooxygenation of the C-ring, which yields
a 2-hydroxyflavanone (flavanone 2-hydroxylase) or a
2-hydroxyisoflavanone (2HIS) intermediate. An alternative route for the
conversion of flavanones to flavones involves, instead, a
2-oxoglutarate-dependent dioxygenase (14). The A-ring
hydroxyl group in positions 5 and/or 7 is formed during the synthesis
of the flavonoid skeleton catalyzed by chalcone synthase, a member of
plant polyketide synthases (15). Additional hydroxyl groups in the
A-ring of some flavonoid classes are found in positions 6 and 8. Enzymes involved in the hydroxylation of the A-ring at these positions
have, however, not yet been described.
Isoflavone and pterocarpan derivatives play important roles in
plant-microbe interactions as phytoalexins and nodulation factors and
as phytoestrogens. In contrast to the constitutive production of
isoflavonoids in plants, pterocarpans, such as glyceollin from soybean
(Glycine max L.), are inducible and accumulate in
pathogen-infected or elicitor-treated plant tissues (16). In an attempt
to investigate transcriptionally regulated cytochromes P-450 activated
by biotic stress in soybean, the technique of differential display of
mRNA was recently employed (17). Eight full-length cDNA clones
were subsequently isolated that represented elicitor-activated
cytochromes P-450. One of these, CYP73A11 cDNA, encoded
cinnamate 4-hydroxylase, and a second one, CYP93A1 cDNA,
coded for 3,9-dihydroxypterocarpan 6a-hydroxylase (17, 18).
We now present the functional identification of CYP71D9 whose cDNA
was also previously isolated from elicitor-induced soybean cells by
using the differential display method (17). By employing heterologous
expression the CYP71D9 cDNA was demonstrated to encode a
protein capable of catalyzing the hydroxylation of ring A of flavonoid
substrates. Combined studies with recombinant 2-hydroxyisoflavanone synthase (2HIS; CYP93C1v2) indicated that A-ring hydroxylation occurs
before the 1,2-aryl shift of the flavonoid B-ring during isoflavanone formation.
Chemicals--
Daidzein (7,4'-dihydroxyisoflavone), eriodictyol
(5,7,3' 4'-tetrahydroxyflavanone), genistein
(5,7,4'-trihydroxyisoflavone), kaempferol
(3,5,7,4'-tetrahydroxyflavone), luteolin
(5,7,3',4'-tetrahydroxyflavone), naringenin
(5,7,4'-trihydroxyflavanone), and quercetin
(3,5,7,3',4'-pentahydroxyflavone) were purchased from Roth (Karlsruhe,
Germany); factor 2 (6,7,4'-trihydroxyisoflavone) and liquiritigenin
(7,4'-dihydroxyflavanone) were from Extrasynthèse (Genay,
France). Apigenin (5,7,4'-trihydroxyflavone), biochanin A
(5,7-dihydroxy-4'-methoxyisoflavone), dihydroquercetin
(3,5,7,3',4'-pentahydroxyflavanone), and isoliquiritigenin
(2',6',4-trihydroxychalcone) were from Sigma. Dihydrobiochanin A
(5,7-dihydroxy-4'-methoxyisoflavanone), dihydrokaempferol (3,5,7,4'-tetrahydroxyflavanone), formononetin
(7-hydroxy-4'-methoxyisoflavone), 7-methoxy-4'-hydroxyflavanone,
7,4'-dimethoxyflavanone, and medicarpin (3-hydroxy-9-methoxypterocarpan) were from our laboratory collection.
Cell Culture and Extraction--
Soybean (G. max L. cv. Harosoy 63) cell suspension cultures were propagated in the dark as
described earlier (19). For elicitation experiments, 6-day-old cultures
were transferred into fresh medium 12 h prior to treatment with a
Candidate cDNA Expression in Yeast--
The
Saccharomyces cerevisiae strain W303-1B, designated W(N),
and its derivatives W(R) and WAT11 (22, 23), as well as the expression
vector pYeDP60 (24), were provided by Rhône-Poulenc Agro (Lyon,
France) and D. Pompon (Gif-sur-Yvette, France). The yeast strains had
previously been engineered to either overexpress the NADPH-cytochrome
P-450 reductase from yeast (W(R)) or the Arabidopsis
thaliana isoform ATR1 (WAT11) upon galactose induction. The yeast
strain WVS1 was engineered in the same way to overexpress the
Vicia sativa P-450 reductase VS1 (accession number Z26250); VS1 was inserted into the integrative plasmid pYeDP110 (25) after polymerase chain reaction amplification for addition of BamHI and SacI restriction sites just 5' and 3'
of the coding sequence using primers 5'-CGGGATCCATGACTTCCTCTAATTCCG
(5'-end) and 5'-CGGGAGCTCTCACCAAACATCTCTTAGG (3'-end) and then
integrated into W(N) with the GAL10-CYC1 promoter at the
locus of the endogenous reductase by homologous recombination.
The coding regions of the soybean P-450s were amplified using the
primers specified earlier (26) and inserted into pYeDP60 according to
Urban et al. (27). CYP93C1v2 cDNA (AF135484; a gift from R. A. Dixon and M. Gijzen, Samuel Roberts Noble
Foundation, Ardmore, OK) (11) was amplified with the polymerase
chain reaction primers 5'-atatatggatccATGTTGCTTGAACTTGCAC (5'-end) and
5'-tatataggtaccTAATTAAGAAAGGAGTTTAG (3'-end) to generate
BamHI and KpnI restriction sites just 5' and 3'
of the coding sequence, before insertion into pYeDP60. Polymerase chain
reaction was performed as described earlier (17), and the resulting
plasmids were confirmed for identity by restriction and sequence
analyses of the CYP-coding regions. Yeast strains W(R) (for CYP93C1v2)
or WAT11 and WVS1 (for the other soybean P-450s) were transformed, and
microsomal fractions were isolated as described earlier (17, 27). CYP
expression was induced using the high density procedure (25), and
cultures of 1-5 × 108 cells/ml were used for
microsome preparation.
Assay of Flavonoid 6-Hydroxylase and 2-Hydroxyisoflavanone
Synthase Activities--
The standard assay for flavonoid
6-hydroxylase (F6H; CYP71D9, accession number: Y10490) contained in a
total volume of 100 µl of 50 mM Tricine/KOH, pH 7.9, 0.5 mM reduced glutathione, 60 µg of microsomal protein from
yeast, and 100 µM substrate (dissolved in either
Me2SO or 2-methoxy-ethanol). After equilibration for 2 min at 18 °C, the reaction was started with the addition of 20 µl of a NADPH-regenerating system (comprising 100 µM
NADPH, 160 µM glucose 6-phosphate, and 0.04 unit of
glucose 6-phosphate dehydrogenase in the reaction mixture) and
terminated by the addition of 100 µl of 3% acetic acid in ethyl
acetate. The products were extracted once with this solvent and twice
thereafter with ethyl acetate from the reaction mixture. The organic
phase was pooled and evaporated, and the residue was dissolved in 200 µl of a mixture of 40% methanol, 60% water, 0.2% acetic acid (v/v)
and analyzed by reverse-phase high performance liquid chromatography
(RP-HPLC) (LiChrosorb RP-18, 4 × 250 mm; flow rate, 1 ml
min
The assay for 2HIS contained in a total volume of 100 µl of 50 mM Tricine/KOH, pH 8.6 (28), 0.5 mM reduced
glutathione, 60 µg of microsomal protein from yeast, and 100 µM liquiritigenin or 6,7,4'-trihydroxyflavanone. The
reaction was run for 1 h at 15 °C. Products were extracted as
described for the F6H assay and were analyzed by RP-HPLC using eluents
comprising 1% acetic acid in a 50:50 mixture of methanol and
acetonitrile (solvent A) and 1% acetic acid in H2O
(solvent B). Compounds were eluted at a flow rate of 1 ml
min
For characterization of the reaction product from the combined
catalytic action of F6H and 2HIS, the incubation with
(R,S)-liquiritigenin was carried out at a larger
scale (240 times that described above for the F6H and 2HIS standard
assays). The F6H reaction was initiated by incubation at 18 °C, pH
7.9, for 1 h. This was followed by the addition of 2HIS,
adjustment of the pH to 8.6 and a switch to 15 °C for an extra hour.
The reaction was terminated, and the products were extracted as
described above. To achieve a positive identification of the
tetrahydroxylated isoflavanone product, the ethyl acetate residue was
subjected to acid treatment by stirring in 500 µl of 10% HCl (v/v)
in methanol for 1 h at room temperature. The mixture was extracted
thrice with ethyl acetate, and, upon evaporation, the pooled organic
phase was dissolved in 300 µl of methanol and analyzed by RP-HPLC as
described above for the standard 2HIS assay. Fractions of the eluate
containing the isoflavone derivative (Rt = ~16
min) were collected, concentrated, reapplied on to the column to
ascertain purity, reduced to dryness, and subjected to UV and NMR spectroscopy.
Spectrophotometric Measurements--
Spectrophotometric
measurements of total P-450 content and evaluation of substrate binding
were performed according to Omura and Sato (29) and Schalk et
al. (30), respectively. Substrate-binding spectra were recorded
using double cuvettes. Ks,
Mass Spectrometry--
Products of the F6H-catalyzed reaction
were analyzed by mass spectrometry to determine the site of
hydroxylation of the flavonoid substrates. For gas chromatography-mass
spectrometry analysis, the samples were converted to
trimethylsilyl ether derivatives with a mixture of
bis-(trimethylsilyl)trifluoroacetamide containing 1%
trimethylchlorosilane and pyridine (1:1 v/v) for 2 h at room temperature. The electronic impact analysis of 1-µl samples was performed on a Trio 2000 Micromass Quadrupole apparatus fitted with a J
and W Scientific DB 5 MS (5% phenyl, 95% methyl) column (30 m × 0.320 mm inner diameter, 0.1 µm film) using He TPH 55 at 60 kPa as carrier gas. Initial column temperature was 120 °C, held for
2 min, and ramped to 250 °C at 15 °C min NMR Spectroscopy--
Nuclear magnetic resonance spectra of
reaction products were acquired with a Bruker DMX 500 NMR spectrometer
using a 5-mm inverse geometry probehead (90°(1H) = 9.3 µs; 90°(13C) = 9.8 µs) in
acetone-d6 (2.04, 29.8 ppm) at 303 K. A
phase-sensitive (echo-antiecho selection) and sensitivity enhanced
1H,13C heteronuclear single quantum
coherence NMR spectrum of 6,7,4'-trihydroxyisoflavone was
acquired using Bruker standard software (1J(CH) = 165 Hz; acquisition time, 203 ms; spectral width, 5040 Hz (F2), 106 F1 (13C) increments with a final resolution of 83 Hz;
13C GARP decoupling: 70 µs, gradient; pulse, 1 ms;
recovery, 450 µs). The pK values of 6,7,4'-trihydroxyisoflavone
were calculated with the ACD/Labs (Pegnitz, Germany)
pKa Data Base, Version 4.5.
Yeast Expression and Functional Screening of Soybean
CYPs--
Earlier investigations (17) identified eight cDNA clones
representing cytochromes P-450 whose expression in soybean cell cultures was activated by elicitor treatment concomitantly with the
production of glyceollins, the pterocarpanoid phytoalexins of soybean:
CYP73A11, CYP82A2, CYP82A3, CYP82A4, CYP93A1, CYP93A3, CYP71D8, and
CYP71D9. Activated expression of another P-450 (CYP71A9) isolated in
the same mRNA differential display screening was refuted when
tested by Northern blot analysis. Previous analyses of the catalytic
properties of recombinant proteins expressed in yeast disclosed the
function of two of the clones. CYP73A11 cDNA encoded a
cinnamate 4-hydroxylase (17), whereas CYP93A1 cDNA coded
for 3,9-dihydroxypterocarpan 6a-hydroxylase (18). The former cytochrome P-450 thus represented a well studied enzyme of general phenylpropanoid metabolism whose action gives rise to the hydroxyl group in position 4'
of the flavonoid B-ring. More significantly, the latter cytochrome P-450 catalyzes the stereoselective and regioselective hydroxylation of
position 6a of 3,9-dihydroxypterocarpan to give
(S)-3,6a,9-trihydroxypterocarpan (glycinol), a biosynthetic
intermediate of the glyceollins (18). These earlier studies thus
demonstrated that within the isolated group of CYP clones at least two
cDNAs were related to phenylpropanoid and isoflavonoid pathways.
In an attempt to identify other candidate P-450 cDNAs involved in
flavonoid biosynthesis, all the other coding sequences were also
expressed in yeast. Because we previously showed that the level of
P-450 expression in yeast might be strongly dependent on the
coexpressed P-450 reductase (31, 32), a yeast strain overexpressing a
P-450 reductase (accession number Z26250), isolated from the legume
V. sativa, was constructed. The expression of the different
P-450s in this strain WVS1 was compared with that in the strain WAT11,
overexpressing the A. thaliana reductase ATR1 (Table
I). No expression of CYP82A4 was obtained
in either strain. For all other CYPs, except CYP71D8, twice as much
expression was obtained in WAT11 compared with WVS1. The ratio was
reversed for CYP71D8, which appeared to be more stable in the presence of the reductase from V. sativa. Microsomal fractions
isolated from the most favorable yeast strains were then systematically screened with a variety of flavonoid compounds for specific binding into the CYPs active sites. Type I binding spectra, indicative of a
displacement of solvent in the vicinity of heme (33, 34), were recorded
upon addition of naringenin to CYP71D9 and CYP82A2, dihydrokaempferol
to CYP71D8 and CYP71D9, and eriodictyol to CYP71A9, CYP71D8, CYP71D9,
and CYP82A2. Largest amplitude spectra were obtained upon binding of
eriodictyol to CYP71D8 and CYP71D9 (Table II). As an example, spectra for CYP71D9
are shown in Fig. 1. No interaction of
formononetin, genistein, or daidzein with any of the CYPs was
detected.
The preliminary screening thus indicated that some of the soybean CYPs
were binding flavonoids in their active site. For some of them, in
particular CYP71D8 and CYP71D9, displacement of solvent was effective
enough so that a positioning suitable for an oxidative attack was
likely to be achieved. To test such a possibility, recombinant yeast
microsomes were incubated with NADPH and liquiritigenin, eriodictyol,
naringenin, or dihydrokaempferol. Formation of polar metabolites was
observed with all four flavonoids but only upon incubation with CYP71D9.
Characterization of the Flavonoid 6-Hydroxylase--
To further
characterize the metabolite formed by CYP71D9, microsomes of
recombinant WAT11 yeast were incubated with liquiritigenin and
NADPH, and the ethyl acetate extract of the reaction mixture was
analyzed by RP-HPLC (Fig. 2). Following
RP-HPLC, the reaction product was identified by three criteria:
retention time during HPLC, mass spectrometry, and NMR
spectroscopy.
As shown in Fig. 2, the product P formed from liquiritigenin (S) had a
smaller retention time (6 min) than the substrate (10 min), required
NADPH for its formation, and was not formed when yeast cells were
transformed with the empty vector. Similar results were recorded with
naringenin as a substrate. The higher polarity of the products when
compared with the substrates was fully supported by mass spectrometry
of their trimethylsilyl derivatives. The metabolites of liquiritigenin
and naringenin exhibited molecular ion peaks at
m/z 488.2 and 576.2, respectively. The
retro-Diels-Alder fragment peaks were found at
m/z 296.1 (A-ring) and m/z
192.1 (B-ring) for the product formed from liquiritigenin, and at
m/z 384.2 (A-ring) and at
m/z 192.1 (B-ring) for that formed from naringenin. These results indicated that recombinant CYP71D9 protein catalyzed the monooxygenation of ring A of both flavanones (Fig. 3). Fragments hydroxylated on ring A were
also observed upon gas chromatography-mass spectrometry analysis
of the metabolites of eriodictyol and dihydrokaempferol.
To elucidate the position of hydroxylation of the A-ring,
1H NMR spectra were recorded in
acetone-d6 of the product formed from
liquiritigenin (Table III). The spectra
clearly showed that the proton signal of H-6 was absent in the
reaction product, whereas H-5 and H-8 formed singlets. All other
signals were very similar to those observed for liquiritigenin. Taken
together, the chemical characterization identified
6,7,4'-trihydroxyflavanone as the product formed from liquiritigenin in
the reaction catalyzed by the recombinant CYP71D9 protein (Fig. 3). We
conclude that the enzyme encoded by CYP71D9 is a F6H.
Catalytic Properties of Flavonoid 6-Hydroxylase--
The binding
constants (Ks) for the interaction of different
flavonoid compounds with CYP71D9 were determined with microsomes of
recombinant WAT11 (Table IV). The results
indicated that CYP71D9 exhibited highest affinity (1.6 µM) for the flavanone eriodictyol. Other flavanones,
naringenin and liquiritigenin, as well as the dihydroflavonol
dihydrokaempferol, were bound to CYP71D9 with affinities ranging from 9 to 52 µM. Methylation of the 7-hydroxyl position of the
A-ring decreased the affinity by more than 1 order of magnitude. The
highest maximal amplitudes of
Product formation with recombinant CYP71D9 in WAT11 was proportional to
time for about 10 min at a temperature of 20 °C or below. At 30 or
25 °C, activity was higher, but the enzyme was highly unstable. The
optimal pH for the reaction was 7.9. A significant increase in activity
and stability of the enzyme or amount of product of the reaction was
achieved by inclusion of reduced glutathione at 0.5 mM in
the reaction mixture. When assayed under optimal conditions, the
apparent Km value of recombinant F6H for
liquiritigenin was found to be about 7 µM. The
Kcat at 18 °C was around 50 min Biosynthetic Relationship of 6,7-Dihydroxyflavonoid Precursors to
Isoflavonoids--
Soybean has been known to contain the isoflavones
daidzein, genistein, and glycitein (7,4'-dihydroxy-6-methoxyisoflavone) and the respective 7-O-glucosides (35, 36) besides
constituents related to other flavonoid classes (37). Having identified
F6H (CYP71D9) cDNA in soybean, we attempted to analyze
its putative role in isoflavonoid biosynthesis. From the results shown
in Table V, it appeared rather unlikely that F6H might be involved in hydroxylation reactions after formation of the isoflavonoid skeleton (Fig. 3). An alternative route could be that flavanones containing a
hydroxyl group at position 6 of the A-ring might be substrates for
isoflavonoid biosynthesis. The question to be answered was whether 2HIS
(CYP93C1v2) might be capable of utilizing 6,7-dihydroxyflavanones as
substrates. Initial attempts to analyze these reactions in cell-free
extracts from soybean by incubating [14C]liquiritigenin
with a microsomal fraction from cell cultures failed, possibly because
several competing reactions were taking place.
The biosynthetic relationship of F6H and 2HIS was then studied by using
recombinant protein expressed from CYP71D9 and
CYP93C1v2 cDNAs in the yeast strains WAT11 and W(R). As
had been already shown by others (11-13, 28, 38), it was found that
2HIS readily utilized liquiritigenin to build the isoflavonoid skeleton
(Fig. 4). Under our assay conditions,
2,7,4'-trihydroxyisoflavanone (P1) eluting at 9.3 min from the HPLC
column (Fig. 4B), but not daidzein (P2) eluting at 18 min,
was the predominant product with liquiritigenin (S) as substrate. When
6,7,4'-trihydroxyflavanone (S) was incubated with the microsomal
fraction containing recombinant 2HIS two new products with retention
times of 5.4 (P1) and 15.1 min (P2), respectively, were formed (Fig.
4C). The retention time during RP-HPLC indicated that the
main product P1 was more polar than the substrate, similar to the
situation with liquiritigenin as substrate (Fig. 4, B and
C).
For product characterization, the suspected
2,6,7,4'-tetrahydroxyisoflavanone (Fig. 3) was produced from
(R,S)-liquiritigenin on a larger scale by the
combined catalytic action of recombinant F6H and 2HIS. Upon separation
of the ethyl acetate extract of the reaction mixture by HPLC (Fig.
5), it could be seen that liquiritigenin was almost completely converted. As shown in the HPLC chromatogram of
the reaction products, the amount of the fraction at
Rt of 5.4 min, containing the putative
2,6,7,4'-tetrahydroxyisoflavanone (P2) and the fraction at
Rt 14 min (6, 7, 4'-trihydroxyflavanone, P1) each accounted
for ~50%. Products in P2 appeared to be rather unstable and were
sensitive to both alkaline and acid treatments. When the ethyl acetate
extract was treated with HCl, the relative amount of the product(s) P2
greatly decreased, and the product P3 eluting at
Rt ~16 min was formed (Fig. 5). The UV
spectrum of the latter compound (
A major product of the sequential catalytic actions of recombinant F6H
and 2HIS thus most likely represented
2,6,7,4'-tetrahydroxyisoflavanone, which upon acid treatment could be
dehydrated to yield the corresponding isoflavone. The experiments
outlined in Figs. 4 and 5 therefore clearly demonstrated that
recombinant 2HIS efficiently utilized as substrate a flavanone carrying
a hydroxyl group in position 6 of the A-ring.
Natural products with hydroxyl substitution at position 6 of the
A-ring are reported for several flavonoid classes including the
isoflavonoids (37). Hydroxylation of the A-ring at this position
requires an enzyme-catalyzed reaction subsequent to chalcone skeleton
formation in the reaction mediated by chalcone synthase. A flavonoid
6-hydroxylase, of necessity, should occur in soybean because the
6,7,4'-trihydroxylated substitution pattern, as found in glycitein and
afrormosin (7-hydroxy-6, 4'-dimethoxyisoflavone), has been reported for
isoflavonoid constituents of this plant (35, 36, 41). In the current
study, we identified the catalytic function of the CYP71D9 protein,
whose full-length cDNA had been cloned earlier from elicited
soybean cell cultures (17), to be a flavonoid 6-hydroxylase. To our
knowledge, this represents the first demonstration of a cytochrome
P-450-dependent monooxygenase from plants capable of
mediating A-ring hydroxylation of flavonoid substrates. Product
characterization by mass spectrometry and NMR spectroscopy conclusively
showed that the enzyme catalyzed a regiospecific monooxygenation at
position 6 of the substrates. Whereas other CYP proteins using
flavonoids as substrates have been assigned to the families CYP75
(flavonoid 3',5'-hydroxylase, CYP75A1 and 75A3; flavonoid
3'-hydroxylase, CYP75B2) (5, 6), CYP81 (isoflavone 2'-hydroxylase,
CYP81E1) (7), and CYP93 (3,9-dihydroxypterocarpan 6a-hydroxylase,
CYP93A1; flavanone 2-hydroxylase, CYP93B1; flavone synthase II,
CYP93B2; 2HIS, CYP93C1) (8-13, 18), F6H represents the first known
member of enzymes in the CYP71 family being capable of accepting
flavonoids as substrates. The four other members of the CYP71D
subfamily whose enzyme functions have been identified so far are
CYP71D12 from Madagascar periwinkle (Catharanthus roseus) (42) and CYP71D13, CYP71D15, and CYP71D18 isolated from different species of Mentha (43). Upon functional expression in
heterologous hosts, CYP71D12 protein was demonstrated to be tabersonine
16-hydroxylase, an enzyme involved in indole alkaloid biosynthesis,
whereas the monooxygenases from mint were shown to catalyze the
regiospecific hydroxylations in position 6 or 3 of ( In the current study, in a quest for clues to the physiological
functions of the orphan soybean CYPs (17, 18), we screened for
potential substrates using spectrophotometric detection of ligand
binding. Although this method uses relatively large amounts of
recombinant material, it is much faster than assays of metabolism and
provided information about the type of compounds (e.g.
flavonoids or isoflavonoids) that are able to bind to each active site.
In the case of CYP71D9, affinity and positioning of the ligands could be predicted from saturation curves and correlated well with data from
metabolism. However, some of the other P-450s, equally induced upon
elicitation, were able to bind flavonoids in their active site but did
not metabolize them. For example CYP71D8 was observed to bind
eriodictyol with an efficiency comparable with that of CYP71D9 but
showed no sign of metabolism of the ligand. This raises the interesting
possibility that flavonoids may act as regulators of the activity of
P-450 enzymes involved in other metabolic pathways. Numerous examples
of inhibition of mammalian or insect P-450s by dietary flavonoids have
been reported with impact on the metabolism of drugs (44), the
activation of procarcinogens (45), or the biosynthesis of steroid
hormones (46, 47). In the case of plants, the binding of nonsubstrate
flavonoids to P-450 enzymes could have an impact on specific
physiological functions and result in a regulatory cross-talk between
independent pathways. It is significant that CYP71D8 and CYP71D9 do not
share more than 47% identity, thus arguing for quite divergent
functions of the two enzymes. P-450s from the subfamily CYP71D isolated
so far have been shown to be involved in diverse pathways, ranging from
indole alkaloid to monoterpenoid biosynthesis. Our report in this paper adds a third biosynthetic pathway to the list. A phylogenetic analysis
of the CYP71D sub-family (Fig. 6)
indicates that this functional diversity is related to a relatively
large evolutionary distance between its members, which are likely to
participate in still further biosynthetic routes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucan elicitor fraction (20) from Phytophthora sojae
(200 µg/ml glucose equivalents) for 3-30 h. Cells were harvested by
filtration, frozen in liquid nitrogen, and stored at
80 °C.
Microsomal fractions were isolated by a modified version of the
protocol described by Diesperger et al. (21). Frozen cells
were homogenized in a mortar with a pestle and suspended in 0.2 M Tris-HCl, pH 7.5, 15% sucrose, 30 mM
MgCl2, 1 mM phenylmethylsulfonyl fluoride, and
1 mM dithiothreitol, in the presence of Dowex 1 × 2. After filtration through a nylon mesh and centrifugation at 12,000 × g for 20 min, the microsomal fraction was collected from
the supernatant by centrifugation for 30 min at 50,000 × g, resuspended in 0.1 M
KH2PO4/K2HPO4, pH 7.4, containing 30% glycerol, frozen in liquid nitrogen, and stored at
80 °C.
1; linear gradient from 35 to 65%
methanol in 16 min; eluent 1). Compounds were detected at 290 nm and,
when (R,S)-liquiritigenin (7,4'-dihydroxyflavanone) was used as substrate, the retention times
were 10 min for the reaction product and 15 min for liquiritigenin. Amounts of products were calculated using molar extinction coefficients (
290 nm, MeOH = 6500 M
1
cm
1 for liquiritigenin;
= 15500 M
1 cm
1 for naringenin;
= 16800 M
1 cm
1 for eriodictyol)
or relative to the conversion of liquiritigenin (all other substrates
tested). The pH optimum was determined as described for the standard
assay using 50 mM Tricine/KOH buffer, pH 7-9.
1, in a linear gradient of solvent A from 25 to 70%
(eluent 2), within 18 min. Eluates were monitored at 290 nm.
Amax, and the corresponding S.D. values were
calculated from the
A390-420 nm for eight
to ten ligand concentrations using the nonlinear regression program
DNRPEASY. Flavonoids were dissolved in Me2SO.
1 and then
from 250 °C to 280 °C at 2 °C min
1.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Comparison of expression of the soybean CYPs in yeast strains
overexpressing V. sativa (WVS1) or A. thaliana ATR1 (WAT11) P450
reductase
Screening for flavonoid binding to soybean CYPs in recombinant yeast
microsomes
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Fig. 1.
Carbon monoxide and substrate-binding spectra
recorded with recombinant CYP71D9 in WAT11 yeast microsomes. The
microsomal fraction was prepared from yeast cells grown for 16 h
in the presence of galactose, resuspended, and diluted to 1 mg
ml 1 protein in 0.1 M Tris-HCl, pH 7.5, containing 30% glycerol and 1 mM EDTA. A,
difference spectrum of CO-treated reduced microsomes versus
reduced microsomes. B, spectra recorded upon addition of 200 µM of each of the indicated flavonoid compounds to the
sample cuvette containing reduced microsomes, an equal volume of
solvent being added to the reference reduced microsomes. Base lines
were recorded before addition of CO or substrate.
DHkaempferol, dihydrokaempferol.
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Fig. 2.
HPLC analysis of
(R,S)-liquiritigenin conversion in
the F6H assay. A, incubation of liquiritigenin and
NADPH with a microsomal fraction from yeast cells (strain WAT11)
expressing CYP71D9. B, assay as in A but omitting
NADPH. C, incubation with a microsomal fraction from control
yeast transformed with the vector pYeDP60 without insert. Ethyl acetate
extracts of reaction mixtures were separated on a LiChrosorb RP-18
column using eluent 1. S, substrate; P,
product.
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Fig. 3.
Scheme illustrating the biosynthesis of
isoflavones carrying a hydroxyl group at position 6 of the A-ring.
F6H mediates the introduction of the 6-hydroxyl group of the A-ring at
the flavanone level. Flavanones with or without a hydroxyl group at
position 6 are converted by the action of 2HIS to give tri- or
tetrahydroxyisoflavanones. A dehydratase catalyzes the
dehydration of the latter intermediates resulting in the formation of
the respective di- or trihydroxyisoflavones.
1H and 13C NMR spectra of liquiritigenin,
6,7,4'-trihydroxyflavanone, and 6,7,4'-trihydroxyisoflavone measured in
acetone-d6
A390-420 nm,
at saturating ligand concentration, were obtained for eriodictyol and
7-methoxy-4'-hydroxyflavanone followed by liquiritigenin. The high
light absorption of flavones above 350 nm prevented determination of
the binding constants for these compounds.
Binding constants and maximal binding amplitudes measured upon binding
of different flavonoids to recombinant CYP71D9
A390-420 nm at saturating substrate
concentrations shows how efficient is the displacement of solvent
interacting with heme resulting from the binding of each flavonoid.
High values usually indicate favourable positioning for oxidative
attack. Kinetic data were fitted using the nonlinear regression program
DNRPEASY derived by Duggleby (53) from DNRP53.
1. Complete conversion of racemic liquiritigenin was
obtained, indicating that the enzyme did not discriminate the
2S and 2R configurations of the flavanone. A
variety of compounds were tested as possible substrates for the
recombinant F6H at a substrate concentration of 100 µM
under standard assay conditions. Product analysis was performed by
RP-HPLC. As reported in Table V,
recombinant F6H catalyzed the hydroxylation of different classes of
flavonoids. Most efficient hydroxylation was observed with flavanones
(liquiritigenin, eriodictyol, and naringenin), and the dihydroflavonol,
dihydrokaempferol. Except for 7-methoxy-4'-hydroxyflavanone, which
occupied a large volume of the active site but was not properly
positioned for oxygenation, efficiency of metabolism correlated well
with the amplitudes of the type I binding spectra (Table IV). Less
efficient hydroxylation was found with flavones (apigenin and
luteolin), and little hydroxylation was seen when the flavonol,
kaempferol, or the dihydroisoflavone, dihydrobiochanin A, was used as
substrate. Virtually no hydroxylation was achieved with the isoflavones
(biochanin A, daidzein, or genistein), the pterocarpan, medicarpin, the
chalcone, isoliquiritigenin, or with 4-coumarate and
4-hydroxybenzoate.
Relative rates of conversion of different flavonoid substrates in the
reaction catalyzed by F6H
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Fig. 4.
HPLC analysis of liquiritigenin and
6,7,4'-trihydroxyflavanone conversion in the 2HIS assay.
A, reference compounds. R1,
6,7,4'-trihydroxyflavanone; R2, 6,7,4'-trihydroxyisoflavone;
R3, liquiritigenin (7,4'-dihydroxyflavanone); R4,
daidzein (7,4'-dihydroxyisoflavone). B, incubation of
(R,S)-liquiritigenin and NADPH with a microsomal
fraction from yeast W(R) expressing CYP93C1v2. C, assay as
in B using 6,7,4'-trihydroxyflavanone as substrate. Ethyl
acetate extracts of reaction mixtures were separated by RP-HPLC using
eluent 2. No products were formed in B or C in
the absence of NADPH or with microsomes from the yeast strain
transformed with empty vector. R, reference compound;
S, substrate; P, product.
max = 212, 230, 257, and 323 nm in methanol) was identical to that of authentic
6,7,4'-trihydroxyisoflavone and consistent with values reported earlier
(39). Furthermore, the NMR spectrum of the compound proved that it was
6,7,4'-trihydroxyisoflavone (Fig. 3 and Table III) also known as factor
2 (39). Owing to strong electronic interactions within the
6,7,4'-trihydroxyisoflavone system, the pK values of the individual
phenolic protons cover a wide range (pK (6), 11.96 ± 0.15; pK
(7), 7.39 ± 0.40; and pK(4'), 9.76 ± 0.15). Traces of acids
might have accumulated during purification and strongly affected the
proton chemical shift of individual positions in arbitrary directions,
and deviations up to 0.3 ppm were observed without altering the
splitting by J coupling. The 13C chemical shifts of all
cross-peaks within a heteronuclear single quantum coherence NMR
spectrum measured (40) before high vacuum treatment perfectly agreed
with those of the reference standard compound (Table III). After high
vacuum treatment (5 h, 10
3 mbar), also all of the proton
chemical shifts agreed within 0.02 ppm with those of the reference
compound.
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Fig. 5.
HPLC analysis of products formed from
(R,S)-liquiritigenin by the combined
catalytic action of recombinant F6H and 2HIS. Liquiritigenin and
NADPH were first incubated with a microsomal fraction from yeast WAT11
expressing CYP71D9 before addition of microsomes from W(R) expressing
CYP93C1v2. Reaction mixtures were extracted with ethyl acetate, and the
extracted compounds were analyzed by RP-HPLC using eluent 2 either
directly (solid line) or after treatment with 10% HCl in
methanol (dashed line). P, product.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)-limonene
leading, respectively, to the synthesis of carvone or menthol, which
are responsible for the characteristic flavours of spearmint and peppermint.
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Fig. 6.
Phylogenetic relationships in the CYP71D
sub-family of P-450 enzymes. Soybean P-450s are indicated on a
gray background, and enzymes of known functions are shown in
relief. CYP71D6 and CYP71D7 are P-450s from Solanum
chacoense (U48434 and U48435), CYP71D8, CYP71D9, and CYP71D10 are
from G. max (Y10493, Y10490 and AF022459), CYP71D11 is from
Lotus japonicus (AF000403), CYP71D12 is the tabersonine
16-hydroxylase from C. roseus (AJ238612), CYP71D13 and
CYP71D15 are ( )-limonene-3-hydroxylases from Mentha
piperita (AF124816 and AF124817), CYP71D18 is
(
)-limonene-6-hydroxylase from Mentha spicata (AF124815),
and CYP76D16 is from Nicotiana tabacum (AF166821). The
scale bar represents amino acid substitutions per
site.
Combined data obtained in the current study from binding and metabolism provide valuable information on the substrate specificity of F6H. The recombinant hydroxylase appears capable of converting a variety of flavonoid substrates containing a resorcinol- and phloroglucinol-based A-ring but with a marked preference for flavanones over flavones (Table V). The hydroxylation status of rings A and B may have a critical influence on the anchoring and orientation of the compounds in the active site (Tables IV and V). Double hydroxylation of the B-ring increases both the affinity for F6H and the efficiency of metabolism. Double hydroxylation of the A-ring also increases affinity but decreases metabolism. Binding data suggest that a hydroxyl at C5 increases the constraints on the bound substrate and the distance from the 6-position to the ferryl-oxo species that carries on the oxidative attack. Affinity, as judged by Ks, is drastically decreased, and metabolism is completely abolished upon methylation of the 7-hydroxyl group on ring A. The 7-hydroxyl group is thus essential both for anchoring the substrate and maintaining the 6-position at the proper distance to the active center. Furthermore, both the oxidation status and the substitution pattern of ring C also appear to have an impact on the binding and rate of conversion of flavonoids by F6H (Tables IV and V). In all, a number of flavanones and the dihydroflavonol, dihydrokaempferol, are hydroxylated at high efficiency, and flavones are converted to a lesser extent, whereas the flavonol, kaempferol, and (dihydro)isoflavones are barely converted. It is thus likely that the in vivo function of F6H is to introduce a 6-hydroxyl group at the flavanone level. This assumption is strongly supported by our finding that 2HIS accepts 6-hydroxyflavanones, e.g. 6,7,4'-trihydroxyflavanone, as substrates. Hydroxyl groups at position 6 of the A-ring are also known for other flavonoid classes including flavones, flavonols, and anthocyanins (37). It will be interesting to characterize the biosynthetic relationship of F6H to the flavonoid branch pathways giving rise to these compounds.
F6H cDNA was isolated initially from elicitor-treated soybean cell
cultures (17). Subsequently it was shown that elicitor treatment caused
an increase in the level of F6H mRNA exhibiting a time course
similar to that observed for several other elicitor-responsive mRNAs encoding enzymes of flavonoid biosynthesis pathways (17). It
remains to be analyzed whether among the inducibly formed flavonoid compounds 6,7-dihydroxylated derivatives might occur. This substitution pattern is represented in 6,7,4'-trihydroxyisoflavone (factor 2), a
putative precursor of the constitutively formed isoflavonoid glycitein
(35, 36). The formation of polyhydroxylated isoflavones, including
6,7,4'-trihydroxyisoflavone, from the soybean isoflavones daidzein and
glycitein has been observed during tempe (fermented soybean) production
and has been attributed to the metabolic capacity of bacteria including
Micrococcus and Arthrobacter species by performing hydroxylation and demethylation reactions at ring A (39,
48). In the present study, 6,7,4'-trihydroxyisoflavone was obtained
from enzymatically formed 2,6,7,4'-tetrahydoxyisoflavanone by acid
treatment. In vivo, this dehydration is catalyzed by a dehydratase (49). Polyhydroxylated isoflavones have been known to
exhibit anti-oxidant, anti-inflammatory, anti-allergic, and anti-carcinogenic activities and thus have an impact on mammalian biology (50). For the antioxidant activity of trihydroxyisoflavone, the
6,7-ortho-dihydroxy group seems to be essential (51, 52). In addition
to their impact on mammalian biology, it would be interesting to learn
more about the possible roles of these compounds in the interactions of
plants with their environment.
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ACKNOWLEDGEMENTS |
---|
The kind provision of yeast strain W(R) by Rhône-Poulenc Agro (Lyon, France), of strain WAT11 by Dr. D. Pompon (Gif-sur-Yvette, France), of CYP93C1v2 cDNA by Drs. R. A. Dixon and M. Gijzen (Ardmore, OK), of the V. sativa P-450 reductase cDNA VS1 by Drs. I. Benveniste and F. Durst (Department of Enzymology, CNRS-IBMP, Strasbourg, France) and the kind gift of [14C]liquiritigenin by Dr. K. Stich (Vienna, Austria) are gratefully acknowledged. The advice of Drs. A. Mithöfer and E. G. Cosio is gratefully acknowledged.
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FOOTNOTES |
---|
* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 369 and by funds from the Fonds der Chemischen Industrie (to J. E.), the Spanish Ministerio de Agricultura, Pesca y Alimentacion (to F. C.-H.), and the Alexander von Humboldt Foundation (to A. O. L.-D.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
** To whom correspondence should be addressed. E-mail: j.ebel@ botanik.biologie.uni-muenchen.de.
Published, JBC Papers in Press, Moo6277200, DOI 10.1074/jbc.M006277200
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ABBREVIATIONS |
---|
The abbreviations used are: 2HIS, 2-hydroxyisoflavanone synthase; HPLC, high performance liquid chromatography; RP, reverse-phase; F6H, flavonoid 6-hydroxylase; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine.
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