Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)-catechin to cyanidin and a novel procyanidin

FEBS Letters 580 (2006) 1642–1648

Anthocyanidin synthase from Gerbera hybrida catalyzes the conversionof (+)-catechin to cyanidin and a novel procyanidin

Frank Wellmanna,1, Markus Griesserb, Wilfried Schwabb, Stefan Martensa,Wolfgang Eisenreichc, Ulrich Materna,*, Richard Lukacina,2

a Institut fur Pharmazeutische Biologie, Philipps-Universitat Marburg, Deutschhausstrasse 17 A, D-35037 Marburg, Germanyb Fachgebiet Biomolekulare Lebensmitteltechnologie, Technische Universitat Munchen, Lise-Meitner-Str. 34, D-85354 Freising, Germanyc Lehrstuhl fur Organische Chemie und Biochemie, Technische Universitat Munchen, Lichtenberg-Str. 4, D-85747 Garching, Germany

Received 12 January 2006; revised 7 February 2006; accepted 8 February 2006

Available online 17 February 2006

Edited by Ulf-Ingo Flugge

Abstract Anthocyanidins were proposed to derive from (+)-naringenin via (2R,3R)-dihydroflavonol(s) and (2R,3S,4S)-leuco-cyanidin(s) which are eventually oxidized by anthocyanidinsynthase (ANS). Recently, the role of ANS has been put intoquestion, because the recombinant enzyme from Arabidopsisexhibited primarily flavonol synthase (FLS) activity with negligi-ble ANS activity. This and other studies led to the proposal thatANS as well as FLS may select for dihydroflavonoid substratescarrying a ‘‘b-face’’ C-3 hydroxyl group and initially form the 3-geminal diol by ‘‘a-face’’ hydroxylation. Assays with recombi-nant ANS from Gerbera hybrida fully supported the proposaland were extended to catechin and epicatechin isomers as poten-tial substrates to delineate the enzyme specificity. Gerbera ANSconverted (+)-catechin to two major and one minor product,whereas ent(�)-catechin (2S,3R-trans-catechin), (�)-epicate-chin, ent(+)-epicatechin (2S,3S-cis-epicatechin) and (�)-gallo-catechin were not accepted. The Km value for (+)-catechin wasdetermined at 175 lM, and the products were identified byLC–MSn and NMR as the 4,4-dimer of oxidized (+)-catechin(93%), cyanidin (7%) and quercetin (trace). When these incuba-tions were repeated in the presence of UDP-glucose:flavonoid 3-O-glucosyltransferase from Fragaria · ananassa (FaGT1), theproduct ratio shifted to cyanidin 3-O-glucoside (60%), cyanidin(14%) and dimeric oxidized (+)-catechin (26%) at an overallequivalent rate of conversion. The data appear to identify (+)-catechin as another substrate of ANS in vivo and shed new lighton the mechanism of its catalysis. Moreover, the enzymaticdimerization of catechin monomers is reported for the first timesuggesting a role for ANS beyond the oxidation of leucocyani-dins.� 2006 Federation of European Biochemical Societies. Publishedby Elsevier B.V. All rights reserved.

Keywords: Flavonoid biosynthesis; Oligomericproanthocyanidins; (+)-Catechin; 2-Oxoglutarate-dependentdioxygenase; Anthocyanidin synthase

Abbreviations: FaGT1, Fragaria · ananassa cv. Elsanta glucosyltrans-ferase

*Corresponding author. Fax: +49 6421 282 6678.E-mail address: [email protected] (U. Matern).

1 Present address: Werthenstein Chemie AG, CH-6105 Schachen,Switzerland.2 Present address: Chromsystems Instruments & Chemicals GmbH,Heimburg-Str. 3, D-81243 Munchen, Germany.

0014-5793/$32.00 � 2006 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2006.02.004

1. Introduction

Flavonoids comprise a class of abundant secondary metab-

olites which contribute in many ways to the growth and

subsistence of plants [1,2]. Prominent examples are the antho-

cyanin pigments and the related oligomeric proanthocyani-

dins which are under investigation for their medicinal

potential [3]. The biosynthesis of flavonoids has been studied

extensively over the last decades [4]. However, while the early

steps have been unequivocally unraveled the reactions leading

to anthocyanidins and, in particular, to proanthocyanidins

have remained under debate. All flavonoids derive from the

flavanone (2S)-naringenin, which may be oxidized to the cor-

responding flavone by the action of flavone synthase (FNS) or

hydroxylated in 3b-configuration to the (2R,3R)-dihydrofl-

avonol [5,6]. Substitution reactions may proceed at any stage.

Reduction of the dihydroflavonol by dihydroflavonol 4-reduc-

tase (DFR, Fig. 1) [7] leads to (2R,3S,4S)-leucoanthocyanidin

which was considered as the immediate precursor of anthocy-

anidin [8] initially based on supplemention experiments with

acyanic flowers of genetically defined lines of Matthiola incana

[9]. A branch pathway was proposed to convert cis-leucocy-

anidin to (2R,3S)-trans-flavan-3-ol ((+)-catechin) [10] as the

likely start unit to oligomeric proanthocyanidins (Fig. 1).

The condensation to proanthocyanidins (PA) or condensed

tannins (CT) was assumed to require flavan-3,4-diols (leuco-

cyanidins) as extension units [10,11], but more recently

(2R,3R)-cis-flavan-3-ols ((�)-epicatechins) (Fig. 1) derived

from anthocyanidins by the action of anthocyanidin reductase

(ANR) [12,13] have been suggested as the more likely precur-

sors [11–14]. Nevertheless, the oligomerization reaction has

not yet been accomplished in vitro and the precise mechanism

remains to be established [15]. Although the stereoconfigura-

tion of flavonoids at C-2 appears to be set at the level of

(2S)-naringenin and resulting necessarily in (+)-catechin and

(�)-epicatechin (Fig. 1), the diastereomers ent(�)-catechin

((2S,3R)-trans-catechin) and ent(+)-epicatechin ((2S,3S)-cis-

epicatechin) have also been reported as natural plant products

(cf. [15]). Furthermore, in some plants a bypass may exist

avoiding the 3b-hydroxylation of (2S)-flavanones and leading

to (2R,4R)-flavan-4-ols (phlobaphenes) [16] and 3-deoxyanth-

ocyanidins.

ANS belongs to the 2-oxoglutarate iron-dependent oxygen-

ases and was cloned first from Perilla frutescens [17]. Four

recombinant ANSs were used subsequently with commercial

blished by Elsevier B.V. All rights reserved.

FHT (2S)-flavanones (2R/3R)-dihydroflavonols

OOH

OH OH

OH

OH

R1

R2

OOH

OH

OH

OH

R1

R2

LAR

DFR

O+

OH

OH

OH

OH

R1

R2

OOH

OH

OH

OH

R1

R2

ANR

?

(2R,3S)-flavan-3-ols (+)-catechin

(2R,3S,4S)-leucocyanidins

?

proanthocyanidin ANS

(2R,3R)-flavan-3-ols (-)-epicatechin

anthocyanidins

FGT

anthocyanins

Fig. 1. Role of ANS in the biosynthesis of anthocyanins and proanthocyanidins. Broken arrows designate reactions which have not been confirmedexperimentally (R1, R2 = H or OH). ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; DFR, dihydroflavonol 4-reductase; FHT,flavanone 3b-hydroxylase; FGT, flavonoid 3-O-glucosyltransferase; LAR, leucoanthocyanidin reductase.

F. Wellmann et al. / FEBS Letters 580 (2006) 1642–1648 1643

leucoanthocyanidin substrates and in combination with UDP-

glucose:flavonoid 3-O-glucosyltransferase to investigate the

reaction mechanism [8]. A mechanism was postulated that pro-

ceeds from leucoanthocyanidin via 2-flaven-3,4-diol (pseudo-

base) followed by isomerization of the 2,3-double bond to the

3,4-position concomitant with a shift of the hydroxyl group

from C-4 to C-2 and removal of the C-2 hydroxyl anion under

acidic conditions to yield anthocyanidin (flavylium ion) [8].

However, the synthesis of pure leucoanthocyanidin enantiomers

is rather difficult, and the instability of flavan-3,4-diols in aque-

ous solution as well as the substrate specificity of ANS observed

in vitro cast some doubt on the role of leucoanthocyanidin as a

natural precursor of anthocyanidin [18]. In a series of studies on

recombinant ANS from Arabidopsis thaliana evidence was

presented for the initial oxidationof the substrate atC-3 [19]. Fur-

thermore, the ANS formed predominantly quercetin and cis-

and trans-dihydroquercetin (DHQ) with cyanidin being aminor

product only [19,20], and the product pattern from (2R,3S,4S)-

cis-leucocyanidin vs. that from (2R,3S,4R)-trans-leucocyanidin

implied that cis-DHQ, trans-DHQ and cyanidin resulted mostly

from the unnatural (2R,3S,4R)-trans-leucocyanidin [20]. More-

over, co-crystallization of the ANS with Fe2+, 2-oxoglutarate

and racemic trans-DHQ or enantiomerically pure (2R,3R)-

DHQ as a substrate analogue in the absence or presence of

molecular oxygen supported the stereoselective C-3 hydroxyl-

ation of the substrate and surprisingly revealed two molecules

of the substrate analogue in the active site with (2R,3R)-trans-

DHQ closest to the iron atom, whereas either enantiomer was

bound at the other location [21]. Clearly, additional data are

required to define the substrate specificity of ANS.

Recombinant ANS from Gerbera hybrida was used to deter-

mine the activity with various flavan-3-ol substrates. The selec-

tive conversion of (+)-catechin to anthocyanidin and a dimeric

flavan-3-one sheds new light on the mode of action of ANS,

because neither the mechanism proposed via isomerisation of

3-flaven-2,3-diol concomitant with the C-4/C-2 shift of the hy-

droxyl group [8] nor the lack of configurational requirement at

C-2 apply in this instance. Furthermore, the dimerization reac-

tion might be considered as a precedent for proanthocyanidin

formation.

2. Materials and methods

2.1. ChemicalsBiochemicals of analytic grade were purchased from Roth

(Karlsruhe, Germany). Reference samples of (+)-catechin, (�)-catechin,(+)-epicatechin, (�)-epicatechin, (�)-gallocatechin, cyanidin chlorideand quercetin were from Roth (Karlsruhe, Germany) or Sigma(Deisenhofen, Germany). LiChroprep RP18 (40–63 lm) was obtainedfrom Merck (Darmstadt, Germany). The solutions of flavonoids werefreshly prepared in methanol when used for enzyme incubations.

1644 F. Wellmann et al. / FEBS Letters 580 (2006) 1642–1648

2.2. Cloning and expression of ANS from Gerbera hybridaRecombinant ANS was expressed in yeast [22]. The growing of yeast

transformants and protein isolation were performed as previously de-scribed [23].

2.3. Cloning and expression of FaGT1 from Fragaria · ananassa cv.

ElsantaThe construction of the cDNA library from strawberry fruit and

the identification of the UDP-glucose:flavonoid 3-O-glucosyltransfer-ase (FaGT1) sequence has been described [24]. The open readingframe of FaGT1 (GenBank Accession number AAU09442) whichcodes for a protein of 466 amino acids was cloned into the pET-29a (+) expression vector (Novagen, Schwalbach, Germany) usingthe EcoRI and the XhoI restriction sites. Protein expression was car-ried out with the Escherichia coli strain BL21 (DE3) pLysS (Nova-gen) which was grown at 37 �C until an A600 of approximately 0.5was reached. Induction of protein expression was initiated by addingisopropyl-1-thio-b-DD-galactopyranoside (IPTG) to a final concentra-tion of 1 mM, followed by further growth at 16 �C for 8 h. Proteinpurification was carried out using the Talon� metal affinity resin(BD Biosciences Clontech, Palo Alto, USA) following the manufac-turers instructions with minor modifications. In brief, cells were har-vested by centrifugation and were disrupted with chilled mortar,pestle and glass beads (Sigma, Deisenhofen, Germany). All bufferswere modified to contain 10% glycerol, 10 mM b-mercaptoethanoland 500 mM sodium chloride. The batch/gravity-flow column purifi-cation was applied and the recombinant protein was eluted using abuffer containing 150 mM imidazole. Protein concentrations weredetermined using the Bradford microprotein assay [25]. Negative con-trols were carried out with E. coli BL21 (DE3) pLysS cells harbour-ing an empty expression vector.

2.4. Enzyme assaysActive ANS from filtered (PD10 column, Pharmacia, Freiburg, Ger-

many) crude extract from yeast cells harbouring the ANS cDNA wasincubated at 37 �C for 15 min. The standard reaction mixture (100 ll)contained 100 lM (+)-catechin, 100 lM 2-oxoglutarate, 50 lM ammo-nium iron(II) sulfate, 2.5 mM sodium ascorbate, 2 mg/ml bovine cata-lase and 50 ll protein (0.8 lg/ll) in 200 mM potassium phosphatebuffer pH 6.0. The incubations were carried out in open vials undergentle shaking and the reaction was terminated by the addition of15 ll saturated aqueous EDTA solution. After centrifugation (5 min,10000 · g) the supernatant was directly subjected to LC–MSn analysisas described below.

2.5. Liquid chromatography – mass spectrometry (LC-MSn)The system used for the analysis of the enzyme assay products was a

Bruker Daltronics esquire 3000plus ion trap mass spectrometer (BrukerDaltronics, Bremen, Germany) connected with an Agilent 1100 HPLCsystem (Agilent Technologies, Waldbronn, Germany) equipped with aquaternary pump and a variable wavelength detector. Componentswere separated on a Eurospher 100 C18 column, particle size 5 lm,10 cm · 2 mm (Grom, Rottenburg, Germany) which was held at25 �C. Solvent A was water (Merck, Darmstadt, Germany) acidifiedwith 0.05% formic acid (Roth, Karlsruhe, Germany) and solvent Bwas acetonitrile (Merck, Darmstadt, Germany). Products were sepa-rated using a linear gradient from 100% A to 100% B in 30 min witha flow rate of 0.2 ml/min. The detection wavelength was either 280or 520 nm, as indicated in the text. The electrospray ionization voltageof the capillary was set to �4000 V and the end plate to �500 V. Nitro-gen was used as dry gas at a temperature of 330 �C and a flow rate of9 l/min. The full scan mass spectra were measured in a scan range from50 to 800 m/z with a scan resolution of 13000 m/z/s. Tandem massspectrometry was carried out using helium as collision gas(3.56 · 10�6 mbar) with the collision voltage set at 1 V. All spectrawere acquired in the positive ionization mode. Data analysis was per-formed using the DataAnalysis 3.1 software (Bruker Daltronics).

2.6. Kinetic propertiesRanges of flavonoid substrate concentrations of between 16 and

100 lM were used for Km determination. The apparent Michaelis con-stant for ANS was calculated from Lineweaver–Burk plot. The proteinamounts were quantified according to Bradford [25].

2.7. Concerted reaction of ANS and FaGT1The ANS standard tests were supplemented by addition of 50 ll par-

tially purified FaGT1 (�0.2 mg protein/ml) and 10 ll of 125 mMUDP-glucose to a final volume of 200 ll and incubated at 37 �C for60 min. The flavonoids were analyzed directly by LC–MSn as describedabove.

2.8. Isolation of the dimerFor preparative purposes numerous single incubations of a total

amount of 10 mg (+)-catechin and approximately 25 mg ANS proteinwere carried out under the above mentioned conditions. Successively,the flavonoids were isolated by repeated extraction with ethyl acetate(twice 75 ll), the organic fractions pooled and finally dried under vac-uum. After resolving the residue in 2 ml of 60% aqueous methanol thedimer was separated from the reaction products by preparative RP-18column chromatography (15 cm · 3 cm). Solvent A was water (Merck,Darmstadt, Germany) acidified with 0.05% formic acid (Roth,Karlsruhe, Germany) and solvent B was acetonitrile (Merck, Darms-tadt, Germany). Products were eluted using a stepwise gradient from100 ml of 100% A to 100 ml of 50% B, whereas during each step theconcentration of B was increased by 5%. A flow rate of 2 ml/minwas applied and 20-ml fractions were collected. Fractions were ana-lyzed by LC–MSn. Fractions 21 and 22 containing the dimer werepooled, concentrated to dryness, dissolved in d6-acetone and analyzedby NMR spectroscopy.

2.9. NMR spectroscopyNMR spectra of the catechin derived dimer were recorded at 25 �C

using an AVANCE 500 spectrometer (Bruker Instruments, Karlsruhe,Germany) at transmitter frequencies of 500.1 and 125.6 MHz for 1Hand 13C, respectively. Samples were dissolved in d6-acetone (1.5 mgin 0.5 mL). Two-dimensional COSY, NOESY, HMQC, and HMBCexperiments were performed according to standard Bruker software(XWINNMR). The mixing time was 1 s in the NOESY experiment.1H and 13C NMR chemical shifts were predicted by Specinfo. The sig-nal assignments are based on proton–proton (COSY, NOESY) andproton–carbon correlation experiments (HMQC, HMBC).

3. Results and discussion

3.1. ANS assays with catechins and epicatechins

Activity assays carried out recently with recombinant ANS

from A. thaliana and employing either (2R,3S,4S)-cis-leucocy-

anidin or (2R,3S,4R)-trans-leucocyanidin as a substrate sur-

prisingly revealed quercetin or (2R,3S)-cis-dihydroquercetin

as the primary product rather than the anticipated product

cyanidin which accounted for less than 5% only [20]. These

incubations require particular experimental care, because in

aqueous solution leucocyanidins epimerize easily and cyani-

din is degraded rapidly [8]. Analogous incubations were car-

ried out subsequently with ANS from G. hybrida expressed in

yeast cells [22] and using dihydrokaempferol as a substrate in

combination with dihydroflavonol reductase from G. hybrida,

which basically confirmed the low rate of pelargonidin forma-

tion (Martens et al., unpublished). Accordingly, a more de-

tailed examination of the physiological role of ANS appears

to be necessary. In a first approach, we used the partially

purified recombinant ANS from G. hybrida for activity assays

with enantiomerically pure (+)-catechin, (�)-catechin, and

(+)-epicatechin, respectively, each over a range of concentra-

tions from 16 to 100 lM. The assay conditions were adapted

from previous work [22,26], and crude extracts from yeast

cells harbouring the empty pYES2 expression vector were

routinely used as negative controls. The incubations were

subjected directly to LC–MSn analysis, thus avoiding the

acidification of the reaction mixture prior to the separation

F. Wellmann et al. / FEBS Letters 580 (2006) 1642–1648 1645

as had been done in case of the incubations employing re-

combinant ANS from Perilla frutescens [8]. The stability of

dissolved leucocyanidins and potential products delicately de-

pends on the pH which requires particular attention. Only

(+)-catechin was accepted as substrate, yielding a red col-

oured compound. LC–MSn analysis confirmed the formation

of cyanidin from (+)-catechin due to the mass spectrum,

product ion spectrum of the pseudomolecular ion [M + H]+

of m/z 287 and the retention time of the new compound.

0

1

0

1

0

1

1.0

1.5

12 14 16 [min]

x10 6

x10 6

x10 6

A

B

C

D

m/z 575

m/z 287

m/z 449

520 nm

0

4

0

4

0

4

1.0

2.0

12 14 16 [min]

E

F

G

H

m/z 449

m/z 287

m/z 575

520 nm

x105

x105

x105

det

ecto

r re

spo

nse

det

ecto

r re

spo

nse

a b

287

557

200 250 300 350 400 450 500 550 m/z

100

557

575

200 250 300 350 400 450 500 550 m/z

100%

%

c

d

0

1

0

1

0

1

1.0

1.5

12 14 16 [min]

x10

x10

x10

A

B

C

D

m/z 575

m/z 287

m/z 449

520 nm

0

4

0

4

0

4

1.0

2.0

12 14 16 [min]

E

F

G

H

m/z 449

m/z 287

m/z 575

520 nm

x105

x105

x105

a b

0

1

0

1

0

1

1.0

1.5

12 14 16 [min]

x10

x10

x10

A

B

C

D

m/z 575

m/z 287

m/z 449

520 nm

0

4

0

4

0

4

1.0

2.0

12 14 16 [min]

E

F

G

H

m/z 449

m/z 287

m/z 575

520 nm

x105

x105

x105

a b

287

557

200 250 300 350 400 450 500 550 m/z

100

557

575

200 250 300 350 400 450 500 550 m/z

100%

% 287

557

200 250 300 350 400 450 500 550 m/z

100

287

557

200 250 300 350 400 450 500 550 m/z

100

557

575

200 250 300 350 400 450 500 550 m/z

100%

557

575

200 250 300 350 400 450 500 550 m/z

100%

%

c

d

Fig. 2. LC–MSn analyses of enzyme assays with (+)-catechin assubstrate and ANS (a) as well as of concerted enzyme assays with ANSand FaGT1 (b) and mass spectrum (c) and product ion spectrum (MS2)of m/z 575 (d) of the catechin derived dimer. Catechin derived dimer,cyanidin and cyanidin-3-glucoside were detected at ion traces m/z 575(A,E), 287 (B,F) and 449 (C,G), respectively. UV analyses at 520 nmconfirmed the formation of cyanidin and cyanidin-3-glucoside (D,H).All substances except for the catechin derived dimer were identifiedusing authentic reference material comparing retention times andmolecular masses.

Detailed mass spectral analysis revealed also quercetin

([M + H]+, m/z 303) and an additional product co-eluting

with cyanidin (Fig. 2a). This product exhibited a pseudomo-

lecular ion [M + H]+ of m/z 575 yielding a fragment of

m/z 287 in the MS2 spectrum (Fig. 2c and d). Thus, we con-

cluded a dimeric oxidized catechin as structure for the new

product. Semiquantitative analysis of the product pattern

was performed by integration of the MS signals in the respec-

tive ion traces. The catechin derived dimer (93%) with a

molecular weight of 574 g mol�1 and cyanidin (7%) were

determined as major products besides traces of quercetin

(Fig. 2a).

The product formation was essentially dependent on the

presence of ferrous ions and 2-oxoglutarate, as is typical for

this class of dioxygenases (27), and maximal activity required

the addition of ascorbate (300 lM) in accordance with previ-

ous studies [17], whereas catalase had no significant effect on

the catalytic efficiency. The substrate affinity of the Gerbera

ANS to (+)-catechin with an apparent Km value of 175 lMis within range to the substrate affinities reported for related

plant 2-oxoglutarate-dependent oxygenases.

The specifity of the Gerbera ANS for (+)-catechin is note-

worthy, because the ANSs from A. thaliana, Petunia hybrida

or G. hybrida are all capable of hydroxylating (2S)- as well

as (2R)-naringenin at C-3, beyond their catalytic activities

on (2R,3S,4S)-cis- or (2R,3S,4R)-trans-leucocyanidin and

(2R,3R)-trans-dihydroquercetin as a substrate [22,27]. In case

of the leucocyanidins, the stereoconfiguration at C-4 affected

the product selectivity leading to different ratios of quercetin

vs. dihydroquercetin [20]. This does not apply to catechins

or epicatechins. However, the 3-hydroxylation of naringenin

was reported recently to be important in biasing the sub-

strate selectivity of ANS towards the natural C-2-stereo-

chemistry (27), and the specificity of the Gerbera ANS for

(2R,3S)-trans-catechin ((+)-catechin) is thus fully compatible

with this proposal.

3.2. Incubation of (+)-catechin with ANS and FaGT1

Anthocyanidins are unlikely to accumulate in vivo, and

ANS was suggested to act in concert with UDP-glucose:flavo-

noid 3-O-glucosyltransferase, at least, as part of a membrane-

associated enzyme complex which forms the water-soluble and

stable anthocyanidin-3-O-glycosides [4,28]. The advantage of

such an interaction has also been exploited in in vitro incuba-

tions [17]. Accordingly, mixed incubations were conducted

with Gerbera ANS and flavonoid 3-O-glucosyltransferase from

Fragaria · ananassa cv. Elsanta (FaGT1) [24]. Under these

conditions the ratio of products shifted drastically towards

anthocyanin formation (14% cyanidin, 60% cyanidin 3-O-glu-

coside) at the expense of the catechin derived dimer (26%)

(Fig. 2b). Control incubations carried out with (+)-catechin

and the 3-O-glucosyltransferase only did not produce a glycos-

ylated product, which was fully compatible with the fact that

the enzyme requires C-2/C-3-oxidized flavonoid substrates

[29,30]. The results suggested that most of the cyanidin formed

during ANS incubation was conjugated immediately by the ac-

tion of FaGT1 and thus became stabilized by the glycosylation

reaction.

Nevertheless it could not be excluded that in vivo cyanidin

formation occurs via 4S-flav-2en-3,4-diol intermediate chan-

neled directly to FGT and subsequently transformed into

anthocyanin as proposed recently [20].

1646 F. Wellmann et al. / FEBS Letters 580 (2006) 1642–1648

3.3. Structure of the catechin-derived dimer

About 1.5 mg of the pure catechin-derived dimer was col-

lected from numerous incubations of (+)-catechin with recom-

binant ANS followed by separation on a preparative RP18

column. The formation of the dimer required (+)-catechin as

the substrate as well as an active ANS and was always accom-

panied by the formation of cyanidin. In contrast to cyanidin

the dimeric product was stable at room temperature. Its struc-

ture was analysed by NMR spectroscopy (Table 1).

In light of the molecular weight of the dimer, it was surpris-

ing that the 1H NMR spectrum displayed only a limited num-

ber of signals. In the downfield region of the 1H NMR

spectrum, a set of well-resolved signals were observed resem-

bling the full signal pattern of (+)-catechin. This indicates that:

(i) the structure of the dimer is closely related to the structure

of the catechin substrate; (ii) the molecule is characterized by

an inherent symmetry giving rise to isochronous signals of cor-

responding atoms in the symmetrical subunits of the molecule;

(iii) the two monomers are not connected via carbon atoms of

the aromatic rings bearing hydrogen atoms in the original cat-

echin substrate (e.g., C-8 which is the typical nucleophilic cen-

ter for condensations with catechin).

In the upfield region of the spectrum, a spin system compris-

ing hydrogen atoms at C-2–C-4 of the catechin substrate was

not observed for the dimer. On the other hand, two singlets

at 5.21 and 3.16 ppm were observed with intensities displaying

one hydrogen atom for each site per symmetrical subunit of

the molecule. A two-dimensional COSY experiment afforded

three pairs of correlated hydrogen atoms displaying the spin

systems of the aromatic rings in the catechin motif (i.e., H-

6–H-8, H-14–H-13, and H-14–H-10). Additionally, the signal

at 5.21 was correlated with the H-10 and H-14 signals and

was therefore clearly recognized as H-2. The singlet signal at

3.16 ppm gave weak correlation peaks with the H-6 and H-8

signals. Tentatively, this signal was therefore assigned as H-4.

Information about 13C NMR chemical shifts could be

gleaned from two-dimensional HMQC and HMBC experi-

ments revealing 24 pairs of connectivities between hydrogen

and carbon atoms via one bond (HMQC experiment) or via

multiple bonds (HMBC). This connectivity pattern established

the structure of the molecule as the C-4–C-4 dimer of catechin.

Table 1NMR data of the C-4–C-4 dimer derived from (+)-catechin

Position Chemical shifts (ppm) C

d1H d13C JH

Observed Predicted Observed Predicted

10 6.94(d, 1H) 6.49 114.7 117.9 2.14 6.81(dd, 1H) 6.58 119.4 123.3 8.13 6.75(d, 1H) 6.49 114.8 117.2 8.6 5.93(d, 1H) 5.71 95.2 95.3 2.8 5.81(d, 1H) 5.75 95.7 94.0 2.2 5.21(s, 1H) 5.84 82.3 95.34 3.16(s, 1H) 4.24 24.1 31.14a 94.6 99.79 130.5 128.311 144.6 144.612 145.2 142.98a 154.7 165.67 153.6 158.15 160.53 207.1

The configuration of the molecule cannot be derived unequiv-

ocally on the basis of the present data. However, the apparent

absence of a NOESY crosspeak between H-2 and H-4 could

indicate trans configuration of the respective atoms.

3.4. Mechanistic implications

Several studies have been conducted recently on flavonoid-

related 2-oxoglutarate dependent dioxygenases aiming primar-

ily at the mechanism of C-ring oxidation [6,19,22,27,31]. These

enzymes were commonly proposed to catalyze the hydroxyl-

ation of carbon-3 configurated either to the b-face (FHT/

FNS I) or to the a-face (ANS/FLS) of the flavonoid-ring plane

[27]. The initial hydroxylation suggested for the ANS reaction

is likely to occur at carbon-3, although oxidations at either C-2

or C-4 have not been fully excluded. In case of (+)-catechin,

only the a-face hydroxylation at C-3 or a concerted oxidation

at C-3/C-2 are plausible, because a hydroxylation at C-4 would

generate leucocyanidins as a source of different products [20]

(Fig. 3, I). It is thus conceivable to assume at the first stage

of the oxidation flav-3-en-3-ol and flav-2-en-3-ol with flavan-

3-one in tautomeric equilibrium, which spontaneously oxidize

further to anthocyanidin (cyanidin from (+)-catechin) in the

presence of oxygen [32] (Fig. 3, II). Alternatively, a second cy-

cle of ANS-catalyzed hydroxylation at carbon-3 might be pos-

tulated (Fig. 3, III), in analogy to the mechanistic proposal

suggested for the ANS-catalyzed formation of quercetin from

leucocyanidins [27].

The symmetric cyanidin derived dimer formed by ANS

in vitro has, to our knowledge, no precedent in the literature.

It was obviously produced by the ANS activity, most likely

from the flav-2-en-3-ol intermediate by abstraction of hydro-

gen from C-4 leaving a radical that stabilizes by spontaneous

dimerization (Fig. 3, IV). Natural procyanidins are highly di-

verse in size and composition, and the monomers are usually

lined up by C-4/C-8 or C-4/C-6 linkages [33]. So far only the

NADPH-dependent anthocyanidin (ANR) and leucoanthocy-

anidin (LAR) reductases have been characterized, which pro-

duce leucoanthocyanidin and catechin, respectively, the

precursors of procyanidins [12,33] (Fig. 1). Biomimetic models

have demonstrated a non-enzymatic condensation reaction,

suggesting the extension of a (+)-catechin start unit by

oupling constant (Hz) Correlation pattern

H COSY NOESY HMBC

2 14 2 25, 2.2 13, 10 2, 13 10, 25 14 14 142 82 6 2(w), 6(w)

10, 14 14, 10 10, 146(w), 8(w) 2

4(s), 2, 8(w)13, 21314, 102, 6(w)8(w)

OOH

OH

OH

OH

OH

I

C3-oxidation

OOH

OH

OH

OH

OH

OH

HOOH

OH

O

OH

OH

OOH

OH

OH

OH

OH

H Fe(III)

O

H

O+

OH

OH

OH

OH

OH

OOH

OH

OH

OH

OH

H H Fe(IV)

O

O+

OH

OH

OSugar

OH

OH

VII

III

(+)-catechin

flavan-3-one

flav-2-en-3-ol

FGT

FGT

C-4–C-4-catechin derived dimer

FGT

C4-hydrogen-abstraction

H2O- elimination

no enzyme

radical abreaction

OOH

OH

OH

OH

OH

H H

IVO OH

OH

OH

OH

OH

H H

Fe(IV)

O

O+

OH

OH

OH

OH

OH

H H

O Fe(II)

cyanidin cyanidin 3-glucoside

OOH

OH

O

OH

OH

O OH

OH

O

OH

OH

2

34

56

7 8 910

4a

8a

11

1213

14

V

II

C3-oxidation

VI·

Fig. 3. Oxidation of (+)-catechin catalyzed by ANS. Flav-2-en-3-ol intermediates might be further oxidized to cyanidin and catechin derived dimer,respectively, or may serve as substrate for other reactions in flavonoid or proanthocyanidin biosynthetic pathways.

F. Wellmann et al. / FEBS Letters 580 (2006) 1642–1648 1647

(2R,3S)/(2R,3R)-quinone methide derived via flav-3-en-ol from

leucoanthocyanidin. Flav-3-en-3-ols have more generally been

considered as precursors in flavonoid- and proanthocyanidin

biosynthesis [15,34] and were postulated to arise also during

the reverse NADPH/NADH-dependent reduction of anthocy-

anidins to 2,3-cis-flavan-3-ols catalysed by ANR [12]. Irrespec-

tive of chemical model reactions there is strong evidence for an

enzyme condensing catechins and leucocyanidins [35], how-

ever, the mechanism of condensation is poorly understood.

The dimerization of (+)-catechin by ANS might be considered

as a lead to proanthocyanidin condensation, in particular since

the ANS crystallized from A. thaliana has been shown recently

to accommodate two flavonoids in the active site pocket [21].

The catalytic activity documented in this report for ANS

clearly differs from the mode of action proposed recently for

the polymerisation of flavonoids in Arabidopsis seed coat testa,

which relies on a laccase-type enzyme oxidizing epicatechin to

the corresponding o-quinone [36]. This proposal followed the

assumption of polyphenol oxidases converting flavan-3-ols to

procyanidins [37].

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