(+)-Catechin to Cyanidin and a Novel Procyanidin
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FEBS Letters 580 (2006) 1642–1648 Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)-catechin to cyanidin and a novel procyanidin Frank Wellmanna,1, Markus Griesserb, Wilfried Schwabb, Stefan Martensa, Wolfgang Eisenreichc, Ulrich Materna,*, Richard Lukacˇina,2 a Institut fu¨r Pharmazeutische Biologie, Philipps-Universita¨t Marburg, Deutschhausstrasse 17 A, D-35037 Marburg, Germany b Fachgebiet Biomolekulare Lebensmitteltechnologie, Technische Universita¨tMu¨nchen, Lise-Meitner-Str. 34, D-85354 Freising, Germany c Lehrstuhl fu¨r Organische Chemie und Biochemie, Technische Universita¨tMu¨nchen, 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 Flu¨gge 1. Introduction 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 anthocyanidin Flavonoids comprise a class of abundant secondary metab- synthase (ANS). Recently, the role of ANS has been put into olites which contribute in many ways to the growth and question, because the recombinant enzyme from Arabidopsis subsistence of plants [1,2]. Prominent examples are the antho- exhibited primarily flavonol synthase (FLS) activity with negligi- cyanin pigments and the related oligomeric proanthocyani- ble ANS activity. This and other studies led to the proposal that dins which are under investigation for their medicinal ANS as well as FLS may select for dihydroflavonoid substrates potential [3]. The biosynthesis of flavonoids has been studied carrying a ‘‘b-face’’ C-3 hydroxyl group and initially form the 3- extensively over the last decades [4]. However, while the early geminal diol by ‘‘a-face’’ hydroxylation. Assays with recombi- steps have been unequivocally unraveled the reactions leading nant ANS from Gerbera hybrida fully supported the proposal to anthocyanidins and, in particular, to proanthocyanidins and were extended to catechin and epicatechin isomers as poten- tial substrates to delineate the enzyme specificity. Gerbera ANS have remained under debate. All flavonoids derive from the converted (+)-catechin to two major and one minor product, flavanone (2S)-naringenin, which may be oxidized to the cor- whereas ent(À)-catechin (2S,3R-trans-catechin), (À)-epicate- responding flavone by the action of flavone synthase (FNS) or chin, ent(+)-epicatechin (2S,3S-cis-epicatechin) and (À)-gallo- hydroxylated in 3b-configuration to the (2R,3R)-dihydrofl- catechin were not accepted. The Km value for (+)-catechin was avonol [5,6]. Substitution reactions may proceed at any stage. determined at 175 lM, and the products were identified by Reduction of the dihydroflavonol by dihydroflavonol 4-reduc- n LC–MS and NMR as the 4,4-dimer of oxidized (+)-catechin tase (DFR, Fig. 1) [7] leads to (2R,3S,4S)-leucoanthocyanidin (93%), cyanidin (7%) and quercetin (trace). When these incuba- which was considered as the immediate precursor of anthocy- tions were repeated in the presence of UDP-glucose:flavonoid 3- anidin [8] initially based on supplemention experiments with O-glucosyltransferase from Fragaria · ananassa (FaGT1), the acyanic flowers of genetically defined lines of Matthiola incana product ratio shifted to cyanidin 3-O-glucoside (60%), cyanidin (14%) and dimeric oxidized (+)-catechin (26%) at an overall [9]. A branch pathway was proposed to convert cis-leucocy- equivalent rate of conversion. The data appear to identify (+)- anidin to (2R,3S)-trans-flavan-3-ol ((+)-catechin) [10] as the catechin as another substrate of ANS in vivo and shed new light likely start unit to oligomeric proanthocyanidins (Fig. 1). on the mechanism of its catalysis. Moreover, the enzymatic The condensation to proanthocyanidins (PA) or condensed dimerization of catechin monomers is reported for the first time tannins (CT) was assumed to require flavan-3,4-diols (leuco- suggesting a role for ANS beyond the oxidation of leucocyani- cyanidins) as extension units [10,11], but more recently dins. (2R,3R)-cis-flavan-3-ols ((À)-epicatechins) (Fig. 1) derived Ó 2006 Federation of European Biochemical Societies. Published from anthocyanidins by the action of anthocyanidin reductase by Elsevier B.V. All rights reserved. (ANR) [12,13] have been suggested as the more likely precur- sors [11–14]. Nevertheless, the oligomerization reaction has Keywords: Flavonoid biosynthesis; Oligomeric proanthocyanidins; (+)-Catechin; 2-Oxoglutarate-dependent not yet been accomplished in vitro and the precise mechanism dioxygenase; Anthocyanidin synthase 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 (À)-epicatechin (Fig. 1), the diastereomers ent(À)-catechin ((2S,3R)-trans-catechin) and ent(+)-epicatechin ((2S,3S)-cis- * Corresponding author. Fax: +49 6421 282 6678. epicatechin) have also been reported as natural plant products E-mail address: matern@staff.uni-marburg.de (U. Matern). (cf. [15]). Furthermore, in some plants a bypass may exist 1 Present address: Werthenstein Chemie AG, CH-6105 Schachen, avoiding the 3b-hydroxylation of (2S)-flavanones and leading Switzerland. to (2R,4R)-flavan-4-ols (phlobaphenes) [16] and 3-deoxyanth- 2 Present address: Chromsystems Instruments & Chemicals GmbH, ocyanidins. Heimburg-Str. 3, D-81243 Munchen, Germany. ¨ ANS belongs to the 2-oxoglutarate iron-dependent oxygen- Abbreviations: FaGT1, Fragaria · ananassa cv. Elsanta glucosyltrans- ases and was cloned first from Perilla frutescens [17]. Four ferase recombinant ANSs were used subsequently with commercial 0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.02.004 F. Wellmann et al. / FEBS Letters 580 (2006) 1642–1648 1643 FHT (2S)-flavanones (2R/3R)-dihydroflavonols DFR R2 R2 OH OH HO O HO O R1 LAR R1 OH OH OH OH OH (2R,3S,4S)-leucocyanidins (2R,3S)-flavan-3-ols (+)-catechin ? ANS proanthocyanidin R2 R2 ? OH OH + HO O HO O R1 R1 ANR OH OH OH OH anthocyanidins (2R,3R)-flavan-3-ols (-)-epicatechin FGT anthocyanins Fig. 1. Role of ANS in the biosynthesis of anthocyanins and proanthocyanidins. Broken arrows designate reactions which have not been confirmed experimentally (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. leucoanthocyanidin substrates and in combination with UDP- ation of the substrate and surprisingly revealed two molecules glucose:flavonoid 3-O-glucosyltransferase to investigate the of the substrate analogue in the active site with (2R,3R)-trans- reaction mechanism [8]. A mechanism was postulated that pro- DHQ closest to the iron atom, whereas either enantiomer was ceeds from leucoanthocyanidin via 2-flaven-3,4-diol (pseudo- bound at the other location [21]. Clearly, additional data are base) followed by isomerization of the 2,3-double bond to the required to define the substrate specificity of ANS. 3,4-position concomitant with a shift of the hydroxyl group Recombinant ANS from Gerbera hybrida was used to deter- from C-4 to C-2 and removal of the C-2 hydroxyl anion under mine the activity with various flavan-3-ol substrates. The selec- acidic conditions to yield anthocyanidin (flavylium ion) [8]. tive conversion of (+)-catechin to anthocyanidin and a dimeric However, the synthesis of pure leucoanthocyanidin enantiomers flavan-3-one sheds new light on the mode of action of ANS, is rather difficult, and the instability of flavan-3,4-diols in aque- because neither the mechanism proposed via isomerisation of ous solution as well as the substrate specificity of ANS observed 3-flaven-2,3-diol concomitant with the C-4/C-2 shift of the hy- in vitro cast some doubt on the role of leucoanthocyanidin as a droxyl group [8] nor the lack of configurational requirement at natural precursor of anthocyanidin [18]. In a series of studies on C-2 apply in this instance. Furthermore, the dimerization reac- recombinant ANS from Arabidopsis thaliana evidence was tion might be considered as a precedent for proanthocyanidin presented for the initial oxidation of the substrate at C-3 [19].Fur- formation. thermore, the ANS formed predominantly quercetin and cis- and trans-dihydroquercetin (DHQ) with cyanidin being a minor 2. Materials and methods product only [19,20], and the product pattern from (2R,3S,4S)- cis-leucocyanidin vs. that from (2R,3S,4R)-trans-leucocyanidin 2.1. Chemicals implied that cis-DHQ, trans-DHQ and cyanidin resulted mostly Biochemicals of analytic grade were purchased from Roth from the unnatural (2R,3S,4R)-trans-leucocyanidin [20]. More- (Karlsruhe, Germany). Reference samples of (+)-catechin, (À)-catechin, over, co-crystallization of the ANS with Fe2+, 2-oxoglutarate (+)-epicatechin, (À)-epicatechin, (À)-gallocatechin, cyanidin chloride and quercetin were from Roth (Karlsruhe, Germany) or Sigma and racemic trans-DHQ or enantiomerically pure (2R,3R)- (Deisenhofen, Germany). LiChroprep RP18 (40–63 lm) was obtained DHQ as a substrate analogue in the absence or presence of from Merck (Darmstadt, Germany). The solutions of flavonoids were molecular oxygen supported the stereoselective C-3 hydroxyl- freshly 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 hybrida 2.7. Concerted reaction of ANS and FaGT1 Recombinant ANS was expressed in yeast [22]. The growing of yeast The ANS standard tests were supplemented by addition of 50 ll par- transformants and protein isolation were performed as previously de- tially purified FaGT1 (0.2 mg protein/ml) and 10 ll of 125 mM scribed [23]. UDP-glucose to a final volume of 200 ll and incubated at 37 °C for 60 min. The flavonoids were analyzed directly by LC–MSn as described 2.3. Cloning and expression of FaGT1 from Fragaria · ananassa cv. above. Elsanta The construction of the cDNA library from strawberry fruit and 2.8.