Báo cáo khoa học: Flavonol 3-O-glycoside hydroxycinnamoyltransferases from Scots pine (Pinus sylvestris L.) docx

Flavonol 3-O-glycoside hydroxycinnamoyltransferases from Scots pine (Pinus sylvestris L.) Florian Kaffarnik 1, *, Werner Heller 1 , Norbert Hertkorn 2 and Heinrich Sandermann Jr 1 1 Institute of Biochemical Plant Pathology, GSF-Research Center for Environment and Health, Neuherberg, Germany 2 Institute of Ecological Chemistry, GSF-Research Center for Environment and Health, Neuherberg, Germany Several plant hydroxycinnamoyltransferases (HCTs) have been described for the biosynthesis of function- ally important secondary metabolites, e.g. phytoalexins [1–3] or flower pigments [4–7]. They most commonly use CoA esters as activated donor substrates [8] and transfer the hydroxycinnamoyl moiety to a hydroxyl or amino group of acceptor substrates. Other donor substrates such as glucosyl esters are occasionally observed [9]. Acceptor substrates include anthocyanin glycosides [4–7,10], a flavonol 3-O-glycoside [11], amines [1–3,12–15], meso-tartrate, shikimate and qui- nate [16,17], fatty acids [18] or alkaloids [19] (for an overview see [20]). The biochemistry of HCTs using anthocyanin glycosides, or amines such as agmatine, tyramine or anthranilate, as acceptor substrates has been investigated in more detail [1,3,4,10,12,14,21]. Genes encoding N-HCTs acting on amines such as tyr- amine, noradrenaline and serotonin [22,23] as well as O-HCTs acting on anthocyanins [6,7,10] have recently been cloned. In Scots pine and Norway spruce needles, flavonol 3-O-glucosides acylated at positions 3¢¢ and 6¢¢ with p-coumaric and ferulic acid are the main UV-B screen- ing pigments [24,25]. We hypothesized that the final acylation that results in a dramatic absorption increase of the molecules in the UV-B range (280–315 nm) [26] Keywords hydroxycinnamoyl-CoA flavonol 3-O- glycoside hydroxycinnamoyltransferases; diacylated flavonol 3-O-glycosides; Scots pine; Pinus sylvestris Correspondence W Heller, Institute of Biochemical Plant Pathology, GSF-Research Center for Environment and Health, D-85764 Neuherberg, Germany Fax: +49 89 3187 3383 Tel: +49 89 3187 3041 E-mail: heller@gsf.de Website: http://www.gsf.de/Forschung/ Institute/biop_intro.phtml *Present address Sainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, UK (Received 24 November 2004, revised 14 January 2005, accepted 19 January 2005) doi:10.1111/j.1742-4658.2005.04574.x Flavonol 3-O-glucosides esterified with ferulic or p-coumaric acid at posi- tions 3¢¢ and 6¢¢ are the major UV-B screening pigments of the epidermal layer of Scots pine (Pinus sylvestris ) needles. The last steps in the biosyn- thesis of these compounds are catalyzed by enzymes that transfer the acyl part of hydroxycinnamic acid CoA esters to flavonol 3-O-glucosides. A newly developed enzyme assay revealed three flavonol 3-O -glucoside hydroxycinnamoyltransferases (HCTs) in Scots pine needles with specifici- ties for positions 3¢¢,4¢¢ or 6¢¢. The positions of the acyl groups were identi- fied by cochromatography with reference compounds and by NMR spectroscopy. The enzymes were characterized by molecular mass, isoelec- tric point, and also pH and temperature optima. Substrate specificities for flavonol glycosides and hydroxycinnamic acid CoA esters as well as kinetic properties of 3¢¢- and 6¢¢HCT suggested that acylation preferably occurs with glucosides and p-coumaroyl-CoA. In addition, acylation takes place in a well-defined order, beginning at position 6¢¢ followed by acylation at posi- tion 3¢¢. These results give the first detailed characterization of flavonol 3-O-glycoside HCTs involved in the protection of plant tissues against UV-B (280–315 nm) radiation. Abbreviations HCT, hydroxycinnamoyl-CoA flavonol 3-O-glucoside hydroxycinnamoyltransferase (EC 2.3.1 ); I3G, isorhamnetin 3-O-glucoside; K3G, kaempferol 3-O-glucoside; Q3G, quercetin 3-O-glucoside. FEBS Journal 272 (2005) 1415–1424 ª 2005 FEBS 1415 is mediated by hydroxycinnamoyltransferase enzymes. These steps would introduce p-coumaric and ferulic acid residues at position 3¢¢ and ⁄ or 6¢¢, respectively, of flavonol 3-O-glucosides. In the Scots pine, 3-O-glycosides of three different flavonol types, namely kaempferol, isorhamnetin and quercetin, have been detected. Interestingly, similar diacylated flavonol 3-O- glycosides are not only found in coniferous leaves but also in the leaves of broadleaf trees, such as oak spe- cies [27,29]. This suggests that these metabolites may play an important role in UV-B screening in a variety of economically important tree species. The objective of this study was to provide biochemical information on HCTs as a basis to understand the mechanisms of biosynthesis of UV-B screening pigments in the Scots pine. In this paper, HCTs acylating flavonol 3-O-glucosides are biochemically characterized in more detail for the first time. Results and Discussion Detection of HCT activities in cell extracts from Scots pine needles Cell extracts from developing needles of Scots pine trees were assayed for the presence of HCT activities. Kaempferol, quercetin and isorhamnetin 3-O-gluco- sides (K3G, Q3G and I3G, respectively; Fig. 1) as well as the monoacylated 6¢¢-p-coumaroyl K3G (tiliroside) were tested as acceptors with p-coumaroyl-CoA as the acyl donor. Incubation of crude cell extracts with I3G and p-coumaroyl-CoA, and analysis of the products with HPLC led to four different products that were recognized as acylated compounds by their UV spectra (Fig. 2A, upper panel, peaks 1 through 4, and 2B). The other peaks of Fig. 2A gave UV-spectra typical Fig. 1. Structure of flavonol 3-O-glucosides. In Scots pine needles three different acylated flavonol 3-O-glucosides are found (R: H, kaempferol; OH, quercetin; OMe, isorhamnetin). These compounds are acylated at position 3¢¢ with p-coumaric acid and at position 6¢¢ with either p-coumaric acid or ferulic acid. A B Fig. 2. HPLC analysis of enzyme products. Crude cell extracts from Scots pine needles were assayed with isorhamnetin 3-O-glucoside (A, upper panel; I3G) and 6¢¢-p-coumaroyl-kaempferol 3-O-glucoside (A, lower panel; tiliroside) as the acceptor and p-coumaroyl-CoA as the donor substrates. I3G was chosen as the nonacylated substrate with crude cell extracts because the respective product 1 with K3G comigrated with a minor nonflavonoid hydroxycinnamoyl by-product which prevented quantification of 6¢¢-p-coumaroyl-kaempferol 3-O-glucoside. S, substrate; IS, internal standard; pinosylvin methyl ether.The peaks marked 1–6 were identified as acylated products by their diode array spectra (B). Characteristics of the UV spectra of acylated compounds are the absorption maximum at 315 nm due to the hydroxycinnamic acid moieties and shoulders at 270 and 350 nm originating from the flavonol 3-O-glycoside [26]. Differences between monoacylated (1–3) and diacylated compounds (4–6) con- sist in a higher proportion of the absorbances at 315 nm and 350 nm for diacylated compared to monoacylated compounds. Spectra shown are normalized at 315 nm. Hydroxycinnamoyltransferases from Scots pine F. Kaffarnik et al. 1416 FEBS Journal 272 (2005) 1415–1424 ª 2005 FEBS for simple nonflavonoid p-coumaric acid derivatives. K3G and I3G gave similar results (data not shown) but one of the p-coumaric acid related by-products detected in the assays with I3G as substrate comigrat- ed with tiliroside, one of the reaction products of K3G (Fig. 2A). Using tiliroside as the acceptor substrate one minor and one major diacylated product were detected (Fig. 2A, lower panel, peak 5 and 6). In con- trol assays with heat-inactivated enzyme preparations, or omitting either donor or acceptor substrate, none of the expected acylated products was obtained. Diode array spectra (Fig. 2B) showed similar absorption pat- terns for compounds 1–3 with absorption ratios of approximately 1.9 between 316 and 350 nm. These spectra differed slightly from those of compounds 4–6, which showed a shoulder of markedly lower intensity at 350 nm and absorption ratios of approximately 2.9. This is in good agreement with ratios of 1.6 and 2.5 measured for tiliroside and 3¢¢,6¢¢-di-p-coumaroyl K3G, respectively (data not shown), and can be explained by the higher absorption at 315 nm of diacylated compared to monoacylated metabolites [26,28]. Com- pounds 1–3 thus appeared to be monoacylated, and compounds 4–6 diacylated, products. Furthermore, compound 4 exhibited a comparable retention time and absorption pattern to compound 6, suggesting the same acylation pattern in both compounds. The retent- ion times of compound 1 and tiliroside (Fig. 2A, ‘S’ in lower panel) were also similar, indicating the same acy- lation position for both compounds. A small shift to longer retention time of compounds 1 and 4, relative to tiliroside and compound 6, respectively, was appar- ently caused by the slightly higher lipophilicity of the isorhamnetin relative to the kaempferol derivatives, owing to the additional methoxy function of isorham- netin. The agreement of the acylation pattern of com- pound 1 with that of tiliroside was further confirmed by coinjection experiments with tiliroside and com- pound 1 derived from enzyme assays with K3G as sub- strate (data not shown). Taken together, our results show that at least three different monoacylated and two diacylated products of flavonol 3-O-glucosides were formed by HCT activities in crude cell extracts from Scots pine needles. Coinjec- tion experiments of authentic 6¢¢-p-coumaroyl K3G standard (tiliroside) allowed the identification of the respective enzyme product observed in the chromato- grams. Separation of HCT activities The formation of several products in assays of crude cell extracts raised the question of whether different enzymes were involved. The separation of enzyme activities was successfully carried out by anion exchange chromatography of protein on Q-Sepharose after ammonium sulfate precipitation. The fractions eluting from the ion exchange column were tested with both K3G and tiliroside as substrates. Three separate activities were detected with K3G (Fig. 3A; peaks I–III). Peak I represents the protein fraction not retained by the column and gave a product corres- A B Fig. 3. Separation of hydroxycinnamoyltransferase activities by anion exchange chromatography on Q-Sepharose. Protein extracted from Scots pine needles was chromatographed on Q-Sepharose after ammonium sulfate precipitation. HCT activities of collected fractions were determined with K3G (A) and tiliroside (B) as substrates. Using K3G three different HCT activities (A, I–III) were separated giving products that corresponded to compound 1 (d), compound 2 ( ) and compound 3 (.) in Fig. 2A. In contrast, only two activities were detected with tiliroside (B, IV and V) giving compounds 5 ( )and6(.), respectively, in Fig. 2A. The solid line represents protein concentration, measured as absorption at 280 nm. The dotted line shows changes in conductivity, caused by the sodium chloride gradient applied. F. Kaffarnik et al. Hydroxycinnamoyltransferases from Scots pine FEBS Journal 272 (2005) 1415–1424 ª 2005 FEBS 1417 ponding to compound 1 in Fig. 2A. The products of peaks II and III eluting upon application of an NaCl concentration gradient corresponded respectively to compounds 2 and 3 in Fig. 2A. Peaks IV and V were detected with tiliroside (Fig. 3B), and corresponded to peaks II and III in Fig. 3A, and the products were compounds 5 and 6, respectively in Fig. 2A. In the protein fraction that was not retained by the column, no activity was detectable with tiliroside as the sub- strate (Fig. 3B). This supported the above finding that compound 1 corresponds to tiliroside, which is already acylated at position 6¢¢. Thus, it was concluded that three position-specific HCTs exist in Scots pine needles, and the activity that was not bound to the anion exchange matrix can be assigned to 6¢¢HCT. Identification of products from HCT reactions The positional specificity of the two as yet unassigned HCTs was determined via spectroscopic identification of compounds 2 and 3 enzymatically prepared by incu- bations using appropriate enzyme fractions eluted from anion exchange chromatography. Incubations were performed with K3G and p-coumaroyl-CoA as sub- strates, and the products were purified by preparative HPLC and analyzed by 1D- 1 H-NMR and 2D- 1 H- 1 H- COSY-NMR spectroscopy. The product corresponding to compound 2 of Fig. 2 showed a chemical shift for H-4¢¢ of 4.79 p.p.m. com- pared to 3.23 p.p.m. of the nonacylated K3G, indica- ting that the acyl group was at position 4 of the glucose molecule. On the other hand, the product cor- responding to compound 3 of Fig. 2 showed a chem- ical shift for H-3¢¢ of 5.02 p.p.m. compared to 3.34 p.p.m. of K3G, and was therefore acylated at position 3 of the glucose molecule. The NMR data (see Experimental procedures for details) combined with the results of cochromatography thus proved the existence of three separate position-specific enzymes, i.e. 3¢¢-, 4¢¢- and 6¢¢HCT, in Scots pine needles. Both 3¢¢- and 4¢¢HCT convert nonacylated flavonol 3-O-glucosides in addition to the 6¢¢-monoacylated tiliroside, giving the respective monoacylated 3¢¢- and 4¢¢-p-coumaroyl flavonol 3-O-glucoside (Fig. 2; com- pounds 3 and 2), and diacylated 3¢¢,6¢¢- and 4¢¢,6¢¢-di-p- coumaroyl K3G (Fig. 2; compounds 6 and 5). The simultaneous presence of 3¢¢HCT and 6¢¢HCT in crude cell extracts directly gave rise to diacylated products of flavonol 3-O-glucosides, e.g. compound 4 in Fig. 2. Consistently, this is in agreement with the acylation pattern found in Scots pine, where p-coumaric and ferulic acids were identified at positions 3¢¢ and 6¢¢ of flavonol 3-O-glucosides [28]. The discovery of products acylated at position 4¢¢ was somewhat surprising, because no corresponding metabolites have been des- cribed from Scots pine so far. However, the occurrence of only low 4¢¢HCT activities in crude cell extracts of Scots pine needles indicate that flavonol 3-O-glycosides acylated at position 4¢¢ may be present as minor com- pounds that have not been recognized yet in this plant. On the other hand, metabolites with this structural fea- ture have earlier been identified from leaves of ever- green Quercus species [27,29]. General properties of the HCT enzymes We investigated the biochemical properties of the par- tially purified and separated enzyme activities after anion exchange chromatography (Fig. 3). The appar- ent molecular masses as determined with a Superose 6 column were 47 ± 2 kDa for 3¢¢HCT and 35 ± 3 kDa for 4¢¢HCT. The value for enzymatically active 6¢¢HCT was only 9 kDa under the same conditions. This surprisingly low value was attributed either to interaction of the protein with the gel matrix or to action of proteases during purification. Therefore, an ammonium sulfate fraction was prepared in the pres- ence of protease inhibitors, and chromatography was performed on a Superdex 75 column. The apparent molecular mass of 6¢¢HCT now observed was 42 ± 3 kDa, while the values of the other two activit- ies were not altered. Molecular mass data of acyl- transferases reviewed in [20] generally ranged between 40 and 70 kDa. In the case of a trimeric quinate O-hydroxycinnamoyltransferase, however, a value as low as 15 kDa for the monomer was described [30]. Partial proteolysis has been mentioned for some other acyltransferases [3]. Isoelectric points of the partially purified proteins were determined by chromatofocusing on a Mono-P column. Both 3¢¢- and 4¢¢HCT had a pI of 4.7, whereas 6¢¢HCT appeared at pI 7.9. Maximal activities were determined for both 4¢¢- and 6¢¢HCT at pH 8 and 44 °C. Half maximal values for 4¢¢HCT were obtained at pH 6.8 and 8.5, and at 36 and 50 °C. For 6¢¢HCT, half maximal values were at pH 6.5 and 9.2, and at 36 and 52 °C. Maximal activity for 3¢¢HCT was at pH 7 and 40 °C and half maximal values were at pH 6.2 and 8.0, and at 28 and 47 °C. Kinetic parameters of 3¢¢- and 6¢¢HCT Partially purified 3¢¢- and 6¢¢ HCT, the two major HCT activities in Scots pine needles, were tested for their kinetic parameters with p-coumaroyl- and feru- loyl-CoA as donor substrates and K3G, tiliroside Hydroxycinnamoyltransferases from Scots pine F. Kaffarnik et al. 1418 FEBS Journal 272 (2005) 1415–1424 ª 2005 FEBS and 3¢¢-p-coumaroyl K3G as acceptor substrates (Table 1). Using enzyme preparations after anion exchange chromatography on Q-Sepharose 3¢¢HCT showed a distinctly lower apparent K m value with p-coumaroyl-CoA than with feruloyl-CoA, whereas 6¢¢HCT has comparable apparent K m values for both CoA esters. This is consistent with the observation that the natural flavonol glycoside metabolites are substituted at position 3¢¢ with p-coumaric acid, but at position 6¢¢ with either p-coumaric acid or ferulic acid [25,28]. Regarding the flavonoid substrate 3¢¢HCT showed a lower apparent K m value for tiliroside than for K3G, indicating a higher affinity towards the monoacylated compared with the nonacylated substrate. Addition- ally, the ratio between V max and K m was clearly in favour of tiliroside as the natural substrate of 3¢¢HCT. Furthermore, the apparent K m value of 6¢¢HCT for K3G was in the same range as the one of 3¢¢HCT for tiliroside while no activity of the 6¢¢HCT with 3¢¢- p-coumaroyl K3G was detected (Table 1). These find- ings result in a sequential acylation first at position 6¢¢ with p-coumaric or ferulic acid, followed by acylation at position 3¢¢ only with p-coumaric acid as shown in Fig. 4. This model reflects the natural occurrence of the respective metabolites [28]. However, it cannot be excluded that other factors such as compartmentation or metabolic channeling may contribute to or deter- mine the specificity of the substitution pattern in vivo [31]. Substrate specificity To test the substrate specificity of 3¢¢- and 6¢¢HCT, a number of flavonol 3-O-glycosides were analysed (Table 2). Variation of the B-ring substitution pattern of the flavonol had minor but distinct influence on the transferase activities. 3¢¢HCT showed higher activity with kaempferol and isorhamnetin 3-O-glucosides with a more lipophilic B-ring compared to quercetin Table 1. Apparent Michaelis–Menten parameters of 3¢¢-and 6¢¢HCT. The apparent Michaelis–Menten parameters were deter- mined using enzyme preparations from anion exchange chromato- graphy on Q-Sepharose which fully separated the HCT activities (Fig. 3). n.d., not detectable. Enzyme Substrate K m (lM) V max (lkatÆkg )1 ) V max ⁄ K m (katÆM )1 Ækg )1 ) 3¢¢HCT p-Coumaroyl-CoA a 24 28 1.17 Feruloyl-CoA a 115 18 0.16 K3G b 47 16 0.34 Tiliroside b 16 29 1.81 6¢¢HCT p-Coumaroyl-CoA c 14 4 0.29 Feruloyl-CoA c 13 4 0.31 K3G b 22 2.2 0.1 3¢¢-p-Coumaroyl K3G b n.d. n.d. n.d. a 100 lM tiliroside as fixed substrate. b 100 lM p-coumaroyl-CoA as fixed substrate. c 100 lM K3G as fixed substrate. Fig. 4. Suggested sequential acylation of flavonol 3-O-glucosides. The K m values of 3¢¢HCT indicated a higher affinity to tiliroside (16 l M) than to K3G (47 lM). While 6¢¢HCT did not acylate 3¢¢-monocoumaroylated K3G at position 6¢¢, the K m value for K3G (22 lM) was in the same range as that of 3¢¢HCT for tiliroside. This indicates a sequential acyla- tion of flavonol 3-O-glucosides, first at posi- tion 6¢ followed by acylation at position 3¢¢. C, p-coumaroyl; F, feruloyl. F. Kaffarnik et al. Hydroxycinnamoyltransferases from Scots pine FEBS Journal 272 (2005) 1415–1424 ª 2005 FEBS 1419 3-O-glucoside. In contrast, 6¢¢HCT preferred a more polar B-ring of the substrate showing the highest activ- ity with quercetin 3-O-glucoside. Comparing different quercetin 3-O-glycosides revealed high specificity towards glucose for both enzymes (Table 2). For 3¢¢HCT the hydroxyl group at position 4¢¢ clearly influences activity. The 3-O-b-d-gal- actoside with axial configuration exhibited only 18% activity under standard assay conditions compared to the 3-O-glucoside with equatorial configuration. On the other hand, the presence or absence of the hydroxymethyl group at position 5¢¢ has no major effect indicated by comparable activities for the 3-O-b-d-galactoside and the 3-O-a- l-arabinopyrano- side. For 6¢¢HCT the positions of 3¢¢ and 4¢¢ hydroxyl groups are less important indicated by about half the activity for the 3-O-b-d-galactoside compared to the 3-O-b-d-glucoside. The 6¢¢desoxyglycoside quercetin 3-O-a-l-rhamnoside, which deviates particularly in configuration at position 5¢¢, did not serve as a sub- strate for 3¢¢HCT. On the other hand, flavonoid 6¢¢des- oxyglycosides with d-configuration are not naturally occurring in plants and were therefore not tested. Anthocyanin substrates, such as cyanidin 3-O-glu- coside, cyanidin 3,5-di-O-glucoside and cyanidin 3,2¢-di-O-glucoside were not transformed by both 3¢¢- and 6¢¢HCT under these conditions. Anthocyanin HCTs have been shown to be active under comparable conditions [4–7,10]. In conclusion, based on the flavonol 3-O-glycoside specificity of HCTs described here, these key enzymes for the biosynthesis of UV-B screening pigments in the Scots pine may represent a separate functional group of acyltransferases. Experimental procedures Reference substances and substrates Flavonol 3-O-glycosides, as well as 6¢¢-p-coumaroyl-kaempf- erol 3-O-glucoside (tiliroside) were from Extrasynthe ` se (Lyon, France). CoA esters of p-coumaric and ferulic acids were essentially synthesized according to a published method [32]. The products (0.12 mmol) were purified using a Fracto- gel EMD DEAE 650 (S) column (gel bed 12 mL) (Merck, Darmstadt, Germany) and an A ¨ KTA Explorer system (Amersham Biosciences, Freiburg, Germany). The solvents used were 0.1 m formic acid (A) and 1.5 m sodium formate (B). After application of the crude reaction product ( 0.25 mmol in 10 mL) the column was washed with 50 mL of solvent A. A gradient from 0 to 100% B in a total volume of 110 mL was then applied, followed by 320 mL solvent B. Fractions showing appropriate UV spectra (maxima at 259 and 334 nm for p-coumaroyl-CoA, 256 and 346 nm for feru- loyl-CoA) were collected, pooled and desalted on a Dowex 50 WX 8 column (Aldrich, Steinheim, Germany). Other chemicals used were of highest available purity and were pur- chased from Sigma (Steinheim, Germany). Protein determination Protein concentration was measured according to the method of Bradford [33] using bovine serum albumin (BSA) as standard. Protein extraction Analytical scale Approximately 100 mg of needle material from seedlings or pine trees, shock frozen in liquid nitrogen, was coarsely homogenized with pestle and mortar. Fifty milligrams poly(vinylpolypyrrolidone) (PVPP) and 3 mg Celite were then added, and protein was extracted by further homoge- nization with three portions of 0.5 mL extraction buffer [100 mm sodium phosphate, 10% (w ⁄ v) sucrose, 1.5% (w ⁄ v) PEG 1450, 5 mm 1,4-dithioerythritol (DTE), pH 6.8] in an ice bath [34]. After two centrifugations (20 000 g, 4 °C, 5 min each) the supernatant was desalted on a NAP-5 column (Amersham Biosciences) according to the manufacturer’s instructions. Table 2. Comparison of relative activities of different flavonol 3- O-glycosides. Relative enzyme activities were determined using enzyme preparations from anion exchange chromatography on Q-Sepharose which fully separated the HCT activities (Fig. 3). p-Coumaroyl-CoA was the donor substrate for all measurements, and substrate concentrations of 100 l M were used for all sub- strates. Flavonol substrate Substituent at position 3¢ Relative activity (%) 3¢¢HCT 6¢¢HCT Flavonol 3-O-b- D-glucopyranosides Kaempferol 3-O-Glc (astragalin) –H 100 59 Quercetin 3-O-Glc (isoquercitrin) –OH 67 100 Isorhamnetin 3-O-Glc –OMe 101 52 Quercetin 3-O-glycosides Quercetin 3-O-b- D-glucopyranoside (isoquercitrin) –OH 100 100 Quercetin 3-O-b- D-galactopyranoside (hyperoside) –OH 18 52 Quercetin 3-O-a- L-arabinopyranoside (guaijaverin) –OH 15 0 Quercetin 3-O-a- L-rhamnopyranoside (quercitrin) –OH 0 0 Hydroxycinnamoyltransferases from Scots pine F. Kaffarnik et al. 1420 FEBS Journal 272 (2005) 1415–1424 ª 2005 FEBS Preparative scale Approximately 1700 g of needle material was harvested from field-grown trees at the time of highest specific activity (June and July), immediately frozen in liquid nitrogen and ground with a pestle and mortar. After lyophilization for 48 h, the dried material was ground for 3 min at 4 °Cinan analysis mill A10 (IKA Labortechnik, Staufen, Germany) and stored at )80 °C. Cell extracts were prepared on ice from 25 to 30 g needle powder, 60 g PVPP and 4 g Celite in 100 mm sodium phosphate buffer, pH 6.8 containing 10% (w ⁄ v) sucrose, 1.5% (w ⁄ v) PEG 1450, 1 mm DTE and 1mm EDTA on ice [28]. The extraction was followed by two centrifugation steps at 30 000 g for 10 min at 4 °C. In some experiments, Complete Protease Inhibitor cocktail (one tablet per 50 mL; Roche, Mannheim, Germany) was included. Enzyme assays Crude cell extracts Enzyme assays were performed in a total volume of 212 lL with 200 lL extract at protein concentrations between 50 and 100 lgÆmL )1 and 6 lL each of hydroxycinnamoyl-CoA (3.5 mm in H 2 O) and flavonol 3-O-glucoside (3.5 mm in methanol) at final concentrations of 0.1 mm. The reaction was started by the addition of one of the substrates. After incubation at 37 °C for 60 min 1 nmol pinosylvin methyl ether (0.177 mm in methanol) was added as internal stand- ard, and the products were extracted with two portions of 200 lL ethyl acetate. The organic phases were pooled and dried under a stream of N 2 at room temperature. The resi- due was redissolved in 80 lL 50% (v ⁄ v) acetonitrile in H 2 O, and analyzed by HPLC after centrifugation at 20 000 g for 5 min. Partially purified fractions The total assay volume was 100 lL in 100 mm sodium phosphate, 5 mm DTE, pH 6.8. The final substrate concen- trations and test procedure were as described above. Protein concentration and desalting All steps were carried out at 4 °C or on ice. The crude cell extract was fractionated by ammonium sulfate pre- cipitation (25–60% saturation). After centrifugation at 30 000 g for 30 min, an upper layer was formed, contain- ing the protein and PEG 1450 [35]. The protein–PEG- phase was separated by filtration through Miracloth and dilution into buffer A [20 mm Tris ⁄ HCl buffer, pH 7.5 containing 10% (v ⁄ v) glycerol, 1 mm DTE and 1 mm EDTA]. Desalting was performed using Sephadex G-25 (Amersham Biosciences). Anion exchange chromatography A 64 mL Q-Sepharose fast flow column (Amersham Bio- sciences) was pre-equilibrated with buffer A. The concentra- ted extract (190 mL) was loaded onto the column, and after washing with two column volumes of the same buffer, the enzyme was eluted with a gradient from 0 to 0.5 m NaCl in five column volumes at a flow rate of 7.5 mLÆmin )1 . Fractions of 10 mL were collected and assayed for HCT activity and protein concentration. Gel filtration chromatography A Superose 6 HR 10 ⁄ 30 column (Amersham Biosciences) was pre-equilibrated with a buffer containing 100 mm sodium phosphate, pH 6.8, 100 mm NaCl, 10% (v ⁄ v) gly- cerol and 1 mm DTE. Fractions from the Q-Sepharose col- umn were pooled and concentrated using Centricon YM-10 filtration units (Millipore, Eschborn, Germany). Volumes of 100 lL protein solution were applied to the column and eluted at a flow rate of 250 lLÆmin )1 . Fractions of 200 lL were collected and assayed for HCT activity and protein concentration. The column was previously calibrated with the following molecular mass markers: b-amylase (200 kDa), alcohol dehydrogenase (150 kDa), BSA (66 kDa), carboan- hydrase (29 kDa) and cytochrome c (12.4 kDa). For deter- mination of the apparent molecular mass of the 6¢¢HCT after using protease inhibitors a Superdex 75 HR 10 ⁄ 30 (Amersham Biosciences) column was pre-equilibrated with a buffer containing 100 mm sodium phosphate, pH 8.0, 100 mm NaCl, 10% (v ⁄ v) glycerol, 1 mm DTE and Com- plete Protease Inhibitor cocktail (one tablet per 50 mL). A volume of 250 lL of a desalted ammonium sulfate fraction from a cell extract prepared from young needles in the pres- ence of Complete Protease Inhibitor cocktail was applied to the column, and fractions of 200 lL were collected and assayed for HCT activity. The column was calibrated using BSA (66 kDa), ovalbumin (43 kDa), chymotrypsinogen (25 kDa), myoglobin (17.6 kDa) and ribonuclease A (13.7 kDa) as standards. Chromatofocusing A Mono-P HR 5 ⁄ 20 column (Amersham Biosciences) was pre-equilibrated with 25 mm Piperazine ⁄ HCl (pH 5.2), 10% (v ⁄ v) glycerol and 1 mm DTE or 25 mm diethanolam- ine ⁄ HCl (pH 9.5), 10% (v ⁄ v) glycerol and 1 mm DTE for determination of the isoelectric point of 3¢¢- and 4¢¢HCT or 6¢¢HCT, respectively. The pH gradient was generated in the column during the passage of a solution of Polybuffer 74 (1 : 10, pH 4.0) or Polybuffer 96 (1 : 10, pH 6.0) with 10% (v ⁄ v) glycerol and 1 mm DTE. The flow rate was 0.5 mLÆmin )1 , and fractions of 0.5 or 0.8 mL were collected and assayed for HCT activity and protein concentration. F. Kaffarnik et al. Hydroxycinnamoyltransferases from Scots pine FEBS Journal 272 (2005) 1415–1424 ª 2005 FEBS 1421 Enzyme characterization The characterization of HCT activities was performed with partially purified enzyme preparations after anion exchange chromatography. All measurements were per- formed as triplicates. For determination of pH-depend- ence, enzyme preparations were buffer-exchanged with NAP-5 columns (Amersham Biosciences) in 100 mm sodium phosphate, pH 6.5–8.5 (3¢¢- and 4¢¢HCT) or 100 mm sodium phosphate, pH 6.0–8.0 and 50 mm Tris ⁄ HCl, pH 7.0–9.5 (6¢¢HCT). For determination of the kinetic parameters K m and V max the following substrate concentrations were used: p-coumaroyl- and feruloyl-CoA 10–450 lm with fixed acceptor concentrations of 100 lm, kaempferol 3-O-glucoside 10–450 lm and tiliroside 10–350 lm with fixed donor concentrations of 100 lm. Calculation of the kinetic parameters was performed by approximation of the received data to a Michaelis–Menten function with sigma plot (Jandel Scientific, San Rafael, CA, USA). NMR analysis For structure determination of enzyme products by NMR analysis, compounds were synthezised enzymatically with suitable protein fractions after anion exchange chromato- graphy, using kaempferol 3-O-glucoside and p-coumaroyl- CoA as substrates. The enzyme assay was analogous to the standard enzyme assay, but was upgraded to a volume of 2.0 mL, and an incubation time of 100 min. A total of 90 assays (180 mL) was extracted with four portions of 120 mL ethyl acetate. The organic phases were pooled and dried in vacuo. Products were purified with a preparative HPLC system, consisting of a pump 114M, a controller 420, a system organizer 340, a detector 165 (all Beckman, Mu ¨ nchen, Germany) and an integrator C-R3A Chromato- pac (Shimadzu, Duisburg, Germany). Separation was per- formed on a 250 · 8.0 mm Spherisorb ODS2 5.0 lm column (Bischoff, Leonberg, Germany) starting with 2 min 20% (v ⁄ v) acetonitrile in water, followed by a gradient up to 50% (v ⁄ v) acetonitrile within 15 min and 3 min 50% (v ⁄ v) acetonitrile at 2.8 mLÆmin )1 . Detection was performed at 314 nm. Appropriate peaks were manually collected and identified by analytical HPLC. For comparison, K3G, til- iroside and 2¢¢,6¢¢p-di-coumaroyl kaempferol 3-O-glucoside were measured as reference substances. 1 H NMR spectra were acquired with a Bruker DMX 500 NMR spectrometer (Rheinstetten, Germany) operating at 500.13 MHz proton frequency from a few mg of sample in 750 lLCD 3 CN (d 1 H ¼ 1.93 p.p.m.) usually at 303 K with 90 deg pulses [90°( 1 H) ¼ 9.3 ls], acquisition time of 3.2 s and a relaxa- tion delay of 7 s. Gradient enhanced (length, 1 ms; recov- ery, 450 ls), absolute value 2Q-COSY NMR spectra were acquired with aq ¼ 234 ms and 470 increments in F1 at a sweep width of 4370 Hz. 4¢¢-p-coumaroyl kaempferol 3-O-glucoside (analogue to compound 2 in Fig. 2) 1 H-NMR (500 MHz, CD 3 CN, 273 K, c. 150 lg): d ¼ 8.09 (2H, AA¢; H-2¢⁄6¢), d ¼ 7.64 (H, d; H-7¢¢¢), d ¼ 7.50 (2H, AA¢; H-2¢¢¢ ⁄ 6¢¢¢), d ¼ 6.94 (2H, XX¢; H-3¢⁄5¢), d ¼ 6.82 (2H, XX¢; H-3¢¢¢ ⁄ 5¢¢¢), d ¼ 6.47 (H, d; H-8), d ¼ 6.32 (H, d; H-8¢¢¢), d ¼ 6.25 (H, d; H-6), d ¼ 5.21 (H, d; H-1¢¢), d ¼ 4,79 (H, t; H-4¢¢), d ¼ 3.64 (H, t; H-3¢¢), d ¼ 3.48 (H, dd; H-2¢¢), d ¼ 3.37 (H, dddd; H-5¢¢), d ¼ 3.30 (2H, m; H-6¢¢A, H-6¢¢B). 3¢¢-p-coumaroyl kaempferol 3-O-glucoside (analogue to compound 3 in Fig. 2) 1 H-NMR (500 MHz, CD 3 CN, 303 K, c. 350 lg): d ¼ 8.08 (2H, AA¢; H-2¢⁄6¢), d ¼ 7.70 (H, d; H-7¢¢¢), d ¼ 7.53 (2H, AA¢; H-2¢¢¢ ⁄ 6¢¢¢), d ¼ 6.95 (2H, XX¢; H-3¢⁄5¢), d ¼ 6.86 (2H, XX¢; H-3¢¢¢ ⁄ 5¢¢¢), d ¼ 6.51 (H, d; H-8), d ¼ 6.39 (H, d; H-8¢¢¢), d ¼ 6.29 (H, d; H-6), d ¼ 5.17 (H, d; H-1¢¢), d ¼ 5.02 (H, t; H-3¢¢), d ¼ 3.60 (H, t; H-2¢¢), d ¼ 3.54 (H, m; H-4¢¢), d ¼ 3.48 (H, m; H-6¢¢A), d ¼ 3.43 (H, dd; H-6¢¢B), d ¼ 3.28 (H, ddd; H-5¢¢). HPLC analysis Analysis of enzyme products HPLC separation was performed according to [24] with the following modifications: a 250 · 4.6 mm Spherisorb ODS2 5.0 lm column was run for 3 min with solvent A [1.9% (v ⁄ v) formic acid, 0.1% (w ⁄ v) ammonium formate in water] followed by a gradient for 7 min to 35% solvent B [1.9% (v ⁄ v) formic acid, 0.1% (w ⁄ v) ammonium formate, 9.6% (v ⁄ v) water in acetonitrile], 7 min to 44% B, 5 min to 79% B and 1 min to 100% B, detection was performed at 314 nm. Acknowledgements We thank Susanne Stich for excellent technical assist- ance and Giovanni Romussi, Genova, for providing a sample of 2¢¢,6¢¢-di-p-coumaroyl kaempferol 3-O-glu- coside. References 1 Hohlfeld H, Schu ¨ rmann W, Scheel D & Strack D (1995) Partial purification and characterization of hydroxycinnamoyl-Coenzyme A: tyramine hydroxycin- namoyltransferase from cell suspension cultures of Sola- num tuberosum. 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Phytochemistry 45, 1415–1418. 35 Moya-Leon MA & John P (1995) Purification and bio- chemical characterization of 1-aminocyclopropane-1-car- boxylate oxidase from banana fruit. Phytochemistry 39, 15–20. Hydroxycinnamoyltransferases from Scots pine F. Kaffarnik et al. 1424 FEBS Journal 272 (2005) 1415–1424 ª 2005 FEBS . Flavonol 3-O-glycoside hydroxycinnamoyltransferases from Scots pine (Pinus sylvestris L. ) Florian Kaffarnik 1, *, Werner Heller 1 , Norbert. Inhibitor cocktail (one tablet per 50 mL). A volume of 250 lL of a desalted ammonium sulfate fraction from a cell extract prepared from young needles in the