Tài liệu Báo cáo khoa học: Octaketide-producing type III polyketide synthase from Hypericum perforatum is expressed in dark glands accumulating hypericins pdf

Octaketide-producing type III polyketide synthase from Hypericum perforatum is expressed in dark glands accumulating hypericins Katja Karppinen 1 , Juho Hokkanen 2, *, Sampo Mattila 2 , Peter Neubauer 3 and Anja Hohtola 1 1 Department of Biology, University of Oulu, Finland 2 Department of Chemistry, University of Oulu, Finland 3 Department of Process and Environmental Engineering, University of Oulu, Finland Hypericum perforatum L., St John’s wort, is a medici- nal plant that is widely utilized for the treatment of mild to moderate depression [1,2]. Hypericins, a group of red-pigmented naphthodianthrones including hypericin and pseudohypericin, as well as their intimate precursors protohypericin and proto- pseudohypericin (Fig. 1), are considered the principal agents in the range of biological activities reported for H. perforatum [3–5]. Hypericins, together with other bioactive compounds of the crude plant extract, such as hyperforins and flavonoids, have been found to con- tribute to the antidepressant activity of the plant [1–3]. Hypericin is the most potent natural photosensitizer described to date, and its photodynamic activities allow hypericin to also act as an antiviral and antitu- moral agent [3,6–8]. In H. perforatum, hypericins have been suggested to act as the plant’s defence against insects [9]. H. perforatum is characterized by the presence of dark glands in the aerial parts of the plant [10–13]. Keywords dark glands; hypericin; octaketide synthase; St. John’s wort (Hypericum perforatum L.); type III polyketide synthase Correspondence K. Karppinen, Department of Biology, University of Oulu, PO Box 3000, FIN-90014 Oulu, Finland Fax: +358 8 553 1061 Tel: +358 8 553 1544 E-mail: katja.karppinen@oulu.fi *Present address Novamass Ltd, Oulu, Finland Database Nucleotide sequence data have been submitted to the DDBJ ⁄ EMBL ⁄ GenBank databases under the accession number EU635882 (Received 26 April 2008, revised 18 June 2008, accepted 27 June 2008) doi:10.1111/j.1742-4658.2008.06576.x Hypericins are biologically active constituents of Hypericum perforatum (St John’s wort). It is likely that emodin anthrone, an anthraquinone precursor of hypericins, is biosynthesized via the polyketide pathway by type III polyketide synthase (PKS). A PKS from H. perforatum, HpPKS2, was investigated for its possible involvement in the biosynthesis of hyperic- ins. Phylogenetic tree analysis revealed that HpPKS2 groups with function- ally divergent non-chalcone-producing plant-specific type III PKSs, but it is not particularly closely related to any of the currently known type III PKSs. A recombinant HpPKS2 expressed in Escherichia coli resulted in an enzyme of  43 kDa. The purified enzyme catalysed the condensation of acetyl-CoA with two to seven malonyl-CoA to yield tri- to octaketide prod- ucts, including octaketides SEK4 and SEK4b, as well as heptaketide aloe- sone. Although HpPKS2 was found to have octaketide synthase activity, production of emodin anthrone, a supposed octaketide precursor of hypericins, was not detected. The enzyme also accepted isobutyryl-CoA, benzoyl-CoA and hexanoyl-CoA as starter substrates producing a variety of tri- to heptaketide products. In situ RNA hybridization localized the HpPKS2 transcripts in H. perforatum leaf margins, flower petals and stamens, specifically in multicellular dark glands accumulating hypericins. Based on our results, HpPKS2 may have a role in the biosynthesis of hypericins in H. perforatum but some additional factors are possibly required for the production of emodin anthrone in vivo. Abbreviations CHS, chalcone synthase; DIG, digoxigenin; IPTG, isopropyl thio-b- D-galactoside; OKS, octaketide synthase; PCS, pentaketide chromone synthase; PKS, polyketide synthase; STS, stilbene synthase. FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS 4329 Dark glands appear as black or dark red multicellular nodules that occur near the leaf margins, stems, flower petals and stamens [10–12,14]. Hypericum species with dark glands are known to produce hypericins [15]. The correlation between the concentrations of hypericins and the existence of dark glands in H. perforatum tis- sues has shown the presence of these red pigments in the glands [12,16,17]. Hypericin has also been shown to accumulate in red glands in the sepals in H. elodes [15]. It has been proposed that hypericins not only accumulate, but are also biosynthesized in the dark glands [12,18]. The localization of hypericins in the nodular structures is considered to have evolved as a mechanism for the plant to avoid the potential auto- toxicity of these compounds [19]. The biosynthesis of hypericins is currently poorly understood, but the polyketide pathway is likely to play a central role [12,20]. Plant-specific type III poly- ketide synthases (PKSs) are involved in the biosynthe- sis of a large variety of plant secondary metabolites, including chalcones, stilbenes, benzophenones, acri- dones, phloroglucinols and benzalacetone derivatives [21]. The enzymes catalyse the formation of complex natural products by condensing various CoA-thioesters with malonyl-CoA in a reaction sequence that closely parallels fatty acid biosynthesis [22]. The functional diversity of simple homodimeric type III PKSs is derived from small differences in the active site that influence the substrate specificities, the number of con- densation reactions and the mechanisms of cyclization reactions [22,23]. In some cases, the reaction inter- mediates are also modified by interaction with other enzymes [23]. The type III PKS involved in the biosyn- thesis of hypericins has been suggested to condensate one molecule of acetyl-CoA with seven molecules of malonyl-CoA to form an octaketide chain that subse- quently undergoes cyclizations and decarboxylation, leading to the formation of emodin anthrone (Fig. 1), a precursor of hypericins [3,12,20]. However, there are no reports on the characterization of the type III PKS with octaketide synthase (OKS) activity which is responsible for the formation of emodin anthrone. The final stages of hypericin biosynthesis are conducted by the gene product of hyp-1, encoding for the phenolic coupling protein that catalyses the oxidative dimeriza- tion of emodin anthrone to hypericin [3,20]. To date, four different PKS family genes have been cloned from the genus Hypericum. Chalcone synthase (CHS) and benzophenone synthase have been cloned from both H. androsaemum [24] and H. perforatum [25]. In addition, in a recent study, we described the cloning of two previously uncharacterized cDNAs from H. perforatum encoding for PKSs, designated as HpPKS1 and HpPKS2 [25]. Expression of HpPKS2 was found to correlate with the concentration of hyp- ericins in H. perforatum tissues and HpPKS2 is thus a candidate gene for the biosynthesis of hypericins [25]. In this study, the role of H. perforatum HpPKS2 is investigated in more detail. We describe the functional characterization of HpPKS2 and the exact localization of HpPKS2 transcripts in H. perforatum leaves and flower buds using in situ RNA hybridization. HpPKS2 was found to be an OKS and is specifically expressed in the dark glands accumulating hypericins. Thus, the results imply that HpPKS2 may have a role in the bio- synthesis of hypericins in H. perforatum. The failure of HpPKS2 to catalyse the formation of emodin anthrone in vitro, but produce other octaketides instead, is dis- cussed in terms of a possible need for some additional factors for the production of emodin anthrone. CoAS O Acetyl-CoA + 7 × malonyl-CoA OKS O H OH O OH O Emodin OH OH OHO Oxidation OOO O OOO SEnz O SEK4 O O OH O O OH OH SEK4b O OOH O O OH OH in vitro Cyclizations, decarboxylation in vivo OH O OH OH CH 3 R OH O OH OH R = CH 3 , Hypericin R = CH 2 OH, Pseudohypericin R = CH 3 , Protohypericin R = CH 2 OH, Protopseudohypericin OH O OH CH 3 R OH O OH OH O H Oxidative dimerization 3 2 O Emodin anthrone Fig. 1. Putative reaction of OKS involved in the biosynthesis of hypericins in H. perforatum. In vivo OKS is suggested to condensate one ace- tyl-CoA with seven malonyl-CoA to form an octaketide chain that subsequently undergoes cyclizations and decarboxylation to form emodin anthrone. It is possible that in vitro OKS affords shunt products SEK4 and SEK4b in the absence of some additional, yet unidentified factors. Octaketide synthase from Hypericum perforatum K. Karppinen et al. 4330 FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS Results During amplification of the HpPKS2 coding sequence from H. perforatum, several cDNA clones of HpPKS2 that differed slightly from one another were encoun- tered. The deduced amino acid sequences of the clones shared 99–100% identity. The HpPKS2 clone that was found to be the most abundant of the different cDNA clones in H. perforatum was selected for investigation in this study. The nucleotide sequence of the clone has been deposited in GenBank under the accession num- ber EU635882. However, because the HpPKS2 clones showed such high sequence similarity and thus their expression in H. perforatum tissues could not be distin- guished from each other, the general name HpPKS2 is used in this study. Phylogenetic analysis The overall similarity of the deduced amino acid sequence of HpPKS2 with other type III PKS family proteins was investigated using a neighbor-joining tree (Fig. 2). Phylogenetic analysis showed that the members of the plant-specific type III PKSs grouped into CHSs and non-CHSs, except stilbene synthases (STSs) from Fabaceae and Gymnosperms. In these cases, the STSs were closer to CHSs of the same or related species than other non-chalcone-forming PKSs. HpPKS2 grouped with functionally divergent non-chalcone-forming plant-specific type III PKSs, including OKS and pentaketide chromone synthase (PCS) from Aloe arborescens [26,27]. However, HpPKS2 was positioned on a sub-branch of its own without any particularly closely related proteins. Expression of HpPKS2 in Escherichia coli To study the enzymatic function of HpPKS2 in more detail, the coding region of the HpPKS2 cDNA was functionally expressed in Escherichia coli strain M15 [pREP4] with pQE30 vector. When E. coli cells harbouring the recombinant plasmid were grown at Oryza sativa CHS (AB000801) Zea mays CHS (X60205) Ruta graveolens CHS (AJ297789) Gerbera hybrida CHS (Z38096) Arabidopsis thaliana CHS (AF112086) Vitis vinifera CHS (X75969) Hypericum androsaemum CHS (AF315345) Sorbus aucuparia CHS (DQ286037) Camellia sinensis CHS (D26593) Petunia hybrida CHS (X04080) Hydrangea macrophylla CHS (AB011467) Picea mariana CHS (AF227627) Pinus sylvestris CHS (X60754) Pinus sylvestris STS (S50350) Pueraria lobata CHS (D10223) Phaseolus vulgaris CHS (X06411) Pisum sativum CHS (X63333) Medicago sativa CHS (L02902) Glycine max CHS (X53958) Arachis hypogaea STS (L00952) Vitis vinifera STS (S63221) Rheum palmatum BAS (AF326911) Humulus lupulus VPS (AB015430) Hydrangea macrophylla CTAS (AB011468) Hydrangea macrophylla STCS (AF456445) Ruta graveolens ACS (AJ297788) Gerbera hybrida 2-PS (Z38097) Rheum palmatum ALS (AY517486) Plumbago indica PKS (AB259100) Phalaenopsis sp. BBS (X79903) Bromheadia finlaysoniana BBS (AJ131830) Sorbus aucuparia BIS (DQ286036) Hypericum androsaemum BPS (AF352395) Wachendorfia thyrsiflora PKS1 (AY727928) Ipomoea purpurea CHS-B (U15947) Ipomoea purpurea CHS-A (U15946) Aloe arborescens PCS (AY823626) Aloe arborescens OKS (AY567707) Hypericum perforatum HpPKS2 (EU635882) Aspergillus oryzae csyB (AB206759) Aspergillus oryzae csyA (AB206758) Fusarium graminearum FG08378.1 (XM_388554) Magnaporthe grisea MG04643.4 (XM_362198) Streptomyces griseus RppA (AB018074) 100 100 100 100 100 100 100 99 94 90 57 54 51 87 100 98 100 100 0,1 Fabaceae Gymnosperms divergent PKSs CHSs plants fungi bacteria Oryza sativa CHS (AB000801) Zea mays CHS (X60205) Ruta graveolens CHS (AJ297789) Gerbera hybrida CHS (Z38096) Arabidopsis thaliana CHS (AF112086) Vitis vinifera CHS (X75969) Hypericum androsaemum CHS (AF315345) Sorbus aucuparia CHS (DQ286037) Camellia sinensis CHS (D26593) Petunia hybrida CHS (X04080) Hydrangea macrophylla CHS (AB011467) Picea mariana CHS (AF227627) Pinus sylvestris CHS (X60754) Pinus sylvestris STS (S50350) Pueraria lobata CHS (D10223) Phaseolus vulgaris CHS (X06411) Pisum sativum CHS (X63333) Medicago sativa CHS (L02902) Glycine max CHS (X53958) Arachis hypogaea STS (L00952) Vitis vinifera STS (S63221) Rheum palmatum BAS (AF326911) Humulus lupulus VPS (AB015430) Hydrangea macrophylla CTAS (AB011468) Hydrangea macrophylla STCS (AF456445) Ruta graveolens ACS (AJ297788) Gerbera hybrida 2-PS (Z38097) Rheum palmatum ALS (AY517486) Plumbago indica PKS (AB259100) Phalaenopsis sp. BBS (X79903) Bromheadia finlaysoniana BBS (AJ131830) Sorbus aucuparia BIS (DQ286036) Hypericum androsaemum BPS (AF352395) Wachendorfia thyrsiflora PKS1 (AY727928) Ipomoea purpurea CHS-B (U15947) Ipomoea purpurea CHS-A (U15946) Aloe arborescens PCS (AY823626) Aloe arborescens OKS (AY567707) Hypericum perforatum HpPKS2 (EU635882) Aspergillus oryzae csyB (AB206759) Aspergillus oryzae csyA (AB206758) Fusarium graminearum FG08378.1 (XM_388554) Magnaporthe grisea MG04643.4 (XM_362198) Streptomyces griseus RppA (AB018074) 100 100 100 100 100 100 100 99 94 90 57 54 51 87 100 98 100 100 0,1 Fabaceae Gymnosperms CHSs plants fungi bacteria Fabaceae Gymnosperms FabaceaeFabaceae GymnospermsGymnosperms CHSsCHSs Functionally CHSs plants fungi bacteria plants fungi bacteria Fig. 2. Phylogenetic analysis of type III PKS enzymes. The tree was constructed using the neighbor-joining algorithm. The numbers at the forks are bootstrap values that indicate the per cent values for obtaining this particular branching in 1000 replicates; only values > 50% are shown. The indicated scale represents 0.1 amino acid substitu- tions per site. The GenBank accession num- bers are followed by the names of the species. ACS, acridone synthase; ALS, aloe- sone synthase; BAS, benzalacetone syn- thase; BBS, bibenzyl synthase; BIS, biphenyl synthase; BPS, benzophenone syn- thase; CHS, chalcone synthase; CTAS, 4-coumaroyltriacetic acid synthase; OKS, octaketide synthase; PCS, pentaketide chromone synthase; 2-PS, 2-pyrone synthase; STCS, stilbene carboxylate synthase; STS, stilbene synthase; VPS, valerophenone synthase. K. Karppinen et al. Octaketide synthase from Hypericum perforatum FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS 4331 37 °C after induction with isopropyl thio-b-d-galacto- side (IPTG), all the induced HpPKS2 proteins became insoluble. Similar phenomena have been reported previously in the expression of some plant- specific type III PKSs in E. coli [28,29], and in many cases, a low temperature has been used to obtain recombinant PKS in a soluble form [28,30–32]. There- fore, the culture temperature was lowered to 16 °C after induction with IPTG. Under these culture condi- tions, IPTG-induced E. coli cells produced the soluble HpPKS2 protein, as shown on SDS ⁄ PAGE gel (Fig. 3). Because the recombinant HpPKS2 protein contained an additional hexahistidine tag at the N-terminus, it enabled us to obtain the enzyme with high purity after purification with Ni-NTA agarose. After purification, commonly  2.5 mg of pure recombinant HpPKS2 was obtained from 1 g of E. coli cell pellet. The purified enzyme gave a band with a molecular mass of  43 kDa on SDS ⁄ PAGE gel (Fig. 3). Enzyme activity of recombinant HpPKS2 The enzymatic activity of the purified recombinant HpPKS2 was tested for suggested emodin anthrone- forming activity by using acetyl-CoA as a starter sub- strate. Some of the products (Fig. 4), determined by UPLC ⁄ ESIMS, were simple a-pyrones with a linear keto side chain (A1, A2, A3) showing loss of CO 2 from the parent ion ([M-H-44] ) ) in the negative ioni- zation mode (Fig. 5A). Other characteristic fragments at m ⁄ z 125 corresponding to [C 6 H 5 O 3 ] ) (pyrone moi- ety) and m ⁄ z 167 corresponding to [C 8 H 7 O 4 ] ) were also detected for some a-pyrones, depending on the chain length of the particular compound. Octaketides SEK4 (A4) and SEK4b (A7), as well as heptaketide aloesone (A9), were also found from incubations. The proposed fragmentation patterns of SEK4 and SEK4b in the negative ionization mode are presented in Fig. 5B,C, respectively. A heptaketide aloesone was identified based on its UV spectrum and the structure was confirmed by its fragmentation in the positive ionization mode (Fig. 5D). HpPKS2 also produced pentaketide chromone A8 (2,7-dihydroxy-5-methyl- chromone) and heptaketide chromone A10 [1-(5,7- dihydroxy-4-oxo-4H-chromen-2-yl)pentane-2,4-dione]. The structure A8 was identified based on its UV spectrum, exact mass and retention behaviour [26]. Structure A10 was identified based on its exact mass and fragment ion at m ⁄ z 189 (loss of acyl side chain) in ESI ) conditions. Also, heptaketide phenylpyrone A6 [6-(2,4-dihydroxy-6-(2-oxopropyl)phenyl)-4-hydro- xy-2H-pyran-2-one] showing loss of CO 2 from the parent ion ([M-H-44] ) ), but no other fragments under ESI ) conditions, was identified. Although HpPKS2 showed the expected OKS activity, emodin anthrone, a supposed octaketide precursor of hypericins, was not detected. Recombinant HpPKS2 was also examined for its ability to use other CoA-thioesters as starter sub- strates. It was found that HpPKS2 accepted all tested starter units (isobutyryl-CoA, benzoyl-CoA and hexa- noyl-CoA) to produce a variety of tri- to heptaketide products (Fig. 4), most of which were identified as a-pyrones with a linear keto side chain (B1, B2, B3, B4, C1, C2, C3, C5, C7, D1, D2, D4, D6). In addition, chromones B8 [1-(5,7-dihydroxy-4-oxo-4H- chromen-2-yl)-5-methylhexane-2,4-dione] and C6 [5,7- dihydroxy-2-(2-oxo-2-phenylethyl)-4H-chromen-4-one], phloroglucinols B6 [6-methyl-1-(2,4,6-trihydroxyphe- nyl)heptane-1,3,5-trione] and D5 [1-(2,4,6-trihydroxy- phenyl)decane-1,3,5-trione], as well as phenylpyrones B5 [6-(2,4-dihydroxy-6-(3-methyl-2-oxobutyl)phenyl) -4-hydroxy-2H-pyran-2-one], C4 [6-(3,5-dihydroxy biphenyl-2-yl)-4-hydroxy-2H-pyran-2-one] and D3 [6-(2,4-dihydroxy-6-pentylphenyl)-4-hydroxy-2H-pyran- 2-one] were detected. Identification of the compounds was made based on their similar fragmentation, UV characteristics and retention behaviour compared with the corresponding products obtained using acetyl- CoA as a starter substrate. None of the above-men- tioned products was found from negative control reactions that contained heat-denatured enzyme with corresponding substrates. 1234 M kDa 116.0 66.2 45.0 35.0 25.0 18.4 14.4 Fig. 3. SDS ⁄ PAGE analysis of recombinant HpPKS2 expressed in E. coli. (1) Total proteins from E. coli without induction, (2) total pro- teins from E. coli induced with IPTG, (3) soluble proteins, (4) puri- fied recombinant HpPKS2 protein, (M) protein molecular mass marker, with sizes (kDa) indicated at the right. Octaketide synthase from Hypericum perforatum K. Karppinen et al. 4332 FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS Heptaketides Hexaketides Pentaketides Substrate Tetraketides Triketides OO OH OOOO C2 RT UPLC = 1.50 min m/z 355 [M-H] - λ max 274 nm OO OH OHOH RT UPLC = 1.89 min m/z 295 [M-H] - λ max 307 nm C4 OO OH O O C7 RT UPLC = 2.63 min m/z 271 [M-H] - λ max 316 nm O OOH OH O C6 RT UPLC = 2.54 min m/z 295 [M-H] - λ max 242, 285, 340 nm OO OH OOO C1 RT UPLC = 1.25 min m/z 313 [M-H] - λ max 264 nm CoAS O Benzoyl-CoA OO OH O C3 RT UPLC = 1.77 min m/z 229 [M-H] - λ max 246, 284 nm OO OH RT UPLC = 1.93 min m/z 187 [M-H] - λ max 219, 233, 318 nm C5 OO OH OHOH D3 RT UPLC = 2.42 min m/z 289 [M-H] - λ max 298 nm O O O OH O D6 RT UPLC = 3.16 min m/z 265 [M-H] - λ max 281 nm O OH O OOO D1 RT UPLC = 1.79 min m/z 307 [M-H] - λ max 261 nm OHOH OH OO O D5 RT UPLC = 3.13 min m/z 307 [M-H] - λ max 288 nm CoAS O Hexanoyl-CoA O OH O O D2 RT UPLC = 2.39 min m/z 223 [M-H] - λ max 284 nm OO OH D4 RT UPLC = 2.54 min m/z 181 [M-H] - λ max 227, 284 nm OO OH OHOH O B5 RT UPL C = 1.77 min m/z 303 [M-H] - λ max 238, 286 nm OHOH OH O OO RT UPL C = 2.18 min m/z 279 [M-H] - λ max 238, 287 nm B6 OO OH O OOO B2 RT UPL C = 1.12 min m/z 321 [M-H] - λ max 264 nm OO OH O OO B1 RT UPL C = 0.96 min m/z 279 [M-H] - λ max 262 nm OOH OH O O O B8 RT UPL C = 2.89 min m/z 303 [M-H] - λ max 230, 281, 324, 337, 404 nm CoAS O Isobutyryl-CoA OO OH O B3 RT UPL C = 1.28 min m/z 195 [M-H] - λ max 284 nm OO OH RT UPL C = 1.43 min m/z 153 [M-H] - λ max 225, 284 nm B4 Octaketides O OOH O O OH OH A7 RT UPL C = 1.31 min m/z 317 [M-H] - λ max 230, 280 nm O OOH O O OH OH A4 RT UPL C = 0.84 min m/z 317 [M-H] - λ max 284 nm OO OH OH OH O A6 RT UPL C = 1.17 min m/z 275 [M-H] - λ max 282 nm CoAS O Acetyl-CoA OO OH OOO RT UPL C = 0.45 min m/z 251 [M-H] - λ max 270 nm A1 OO OH O A2 RT UPL C = 0.52 min m/z 167 [M-H] - λ max 285 nm O O OHOH A8 RT UPL C = 1.37 min m/z 191 [M-H] - λ max 309 nm RT UPL C = 0.62 min m/z 125 [M-H] - λ max 283 nm A3 OO OH O O OH O A9 RT UPL C = 1.45 min m/z 231 [M-H] - λ max 243, 251, 292 nm O O OH OH OO A10 RT UPL C = 1.54 min m/z 275 [M-H] - λ max 235, 276 nm Heptaketides Hexaketides Pentaketides Substrate Tetraketides Triketides OO OH OOOO C2 RT UPLC = 1.50 min m/z 355 [M-H] - λ max 274 nm OO OH OHOH RT UPLC = 1.89 min m/z 295 [M-H] - λ max 307 nm C4 OO OH O O C7 RT UPLC = 2.63 min m/z 271 [M-H] - λ max 316 nm O OOH OH O C6 RT UPLC = 2.54 min m/z 295 [M-H] - λ max 242, 285, 340 nm OO OH OOO C1 RT UPLC = 1.25 min m/z 313 [M-H] - λ max 264 nm CoAS O Benzoyl-CoA OO OH O C3 RT UPLC = 1.77 min m/z 229 [M-H] - λ max 246, 284 nm OO OH RT UPLC = 1.93 min m/z 187 [M-H] - λ max 219, 233, 318 nm C5 OO OH OHOH D3 RT UPLC = 2.42 min m/z 289 [M-H] - λ max 298 nm O O O OH O D6 RT UPLC = 3.16 min m/z 265 [M-H] - λ max 281 nm O OH O OOO D1 RT UPLC = 1.79 min m/z 307 [M-H] - λ max 261 nm OHOH OH OO O D5 RT UPLC = 3.13 min m/z 307 [M-H] - λ max 288 nm CoAS O Hexanoyl-CoA O OH O O D2 RT UPLC = 2.39 min m/z 223 [M-H] - λ max 284 nm OO OH D4 RT UPLC = 2.54 min m/z 181 [M-H] - λ max 227, 284 nm OO OH OHOH O B5 RT UPL C = 1.77 min m/z 303 [M-H] - λ max 238, 286 nm OHOH OH O OO RT UPL C = 2.18 min m/z 279 [M-H] - λ max 238, 287 nm B6 OO OH O OOO B2 RT UPL C = 1.12 min m/z 321 [M-H] - λ max 264 nm OO OH O OO B1 RT UPL C = 0.96 min m/z 279 [M-H] - λ max 262 nm OOH OH O O O B8 RT UPL C = 2.89 min m/z 303 [M-H] - λ max 230, 281, 324, 337, 404 nm CoAS O Isobutyryl-CoA OO OH O B3 RT UPL C = 1.28 min m/z 195 [M-H] - λ max 284 nm OO OH RT UPL C = 1.43 min m/z 153 [M-H] - λ max 225, 284 nm B4 Octaketides O OOH O O OH OH A7 RT UPL C = 1.31 min m/z 317 [M-H] - λ max 230, 280 nm O OOH O O OH OH A4 RT UPL C = 0.84 min m/z 317 [M-H] - λ max 284 nm OO OH OH OH O A6 RT UPL C = 1.17 min m/z 275 [M-H] - λ max 282 nm CoAS O Acetyl-CoA OO OH OOO RT UPL C = 0.45 min m/z 251 [M-H] - λ max 270 nm A1 OO OH O A2 RT UPL C = 0.52 min m/z 167 [M-H] - λ max 285 nm O O OHOH A8 RT UPL C = 1.37 min m/z 191 [M-H] - λ max 309 nm RT UPL C = 0.62 min m/z 125 [M-H] - λ max 283 nm A3 OO OH O O OH O A9 RT UPL C = 1.45 min m/z 231 [M-H] - λ max 243, 251, 292 nm O O OH OH OO A10 RT UPL C = 1.54 min m/z 275 [M-H] - λ max 235, 276 nm Heptaketides Hexaketides Pentaketides Substrate Tetraketides Triketides Heptaketides Hexaketides Pentaketides Substrate Tetraketides Triketides OO OH OOOO C2 RT UPLC = 1.50 min m/z 355 [M-H] - λ max 274 nm OO OH OHOH RT UPLC = 1.89 min m/z 295 [M-H] - λ max 307 nm C4 OO OH O O C7 RT UPLC = 2.63 min m/z 271 [M-H] - λ max 316 nm O OOH OH O C6 RT UPLC = 2.54 min m/z 295 [M-H] - λ max 242, 285, 340 nm OO OH OOO C1 RT UPLC = 1.25 min m/z 313 [M-H] - λ max 264 nm CoAS O Benzoyl-CoA OO OH O C3 RT UPLC = 1.77 min m/z 229 [M-H] - λ max 246, 284 nm OO OH RT UPLC = 1.93 min m/z 187 [M-H] - λ max 219, 233, 318 nm C5 OO OH OHOH D3 RT UPLC = 2.42 min m/z 289 [M-H] - λ max 298 nm O O O OH O D6 RT UPLC = 3.16 min m/z 265 [M-H] - λ max 281 nm O OH O OOO D1 RT UPLC = 1.79 min m/z 307 [M-H] - λ max 261 nm OHOH OH OO O D5 RT UPLC = 3.13 min m/z 307 [M-H] - λ max 288 nm CoAS O Hexanoyl-CoA O OH O O D2 RT UPLC = 2.39 min m/z 223 [M-H] - λ max 284 nm OO OH D4 RT UPLC = 2.54 min m/z 181 [M-H] - λ max 227, 284 nm OO OH OHOH O B5 RT UPL C = 1.77 min m/z 303 [M-H] - λ max 238, 286 nm OHOH OH O OO RT UPL C = 2.18 min m/z 279 [M-H] - λ max 238, 287 nm B6 OO OH O OOO B2 RT UPL C = 1.12 min m/z 321 [M-H] - λ max 264 nm OO OH O OO B1 RT UPL C = 0.96 min m/z 279 [M-H] - λ max 262 nm OOH OH O O O B8 RT UPL C = 2.89 min m/z 303 [M-H] - λ max 230, 281, 324, 337, 404 nm CoAS O Isobutyryl-CoA OO OH O B3 RT UPL C = 1.28 min m/z 195 [M-H] - λ max 284 nm OO OH RT UPL C = 1.43 min m/z 153 [M-H] - λ max 225, 284 nm B4 Octaketides O OOH O O OH OH A7 RT UPL C = 1.31 min m/z 317 [M-H] - λ max 230, 280 nm O OOH O O OH OH A4 RT UPL C = 0.84 min m/z 317 [M-H] - λ max 284 nm OO OH OH OH O A6 RT UPL C = 1.17 min m/z 275 [M-H] - λ max 282 nm CoAS O Acetyl-CoA OO OH OOO RT UPL C = 0.45 min m/z 251 [M-H] - λ max 270 nm A1 OO OH O A2 RT UPL C = 0.52 min m/z 167 [M-H] - λ max 285 nm O O OHOH A8 RT UPL C = 1.37 min m/z 191 [M-H] - λ max 309 nm RT UPL C = 0.62 min m/z 125 [M-H] - λ max 283 nm A3 OO OH O O OH O A9 RT UPL C = 1.45 min m/z 231 [M-H] - λ max 243, 251, 292 nm O O OH OH OO A10 RT UPL C = 1.54 min m/z 275 [M-H] - λ max 235, 276 nm OO OH OOOO C2 RT UPLC = 1.50 min m/z 355 [M-H] - λ max 274 nm OO OH OHOH RT UPLC = 1.89 min m/z 295 [M-H] - λ max 307 nm C4 OO OH O O C7 RT UPLC = 2.63 min m/z 271 [M-H] - λ max 316 nm O OOH OH O C6 RT UPLC = 2.54 min m/z 295 [M-H] - λ max 242, 285, 340 nm OO OH OOO C1 RT UPLC = 1.25 min m/z 313 [M-H] - λ max 264 nm CoAS O Benzoyl-CoA OO OH O C3 RT UPLC = 1.77 min m/z 229 [M-H] - λ max 246, 284 nm OO OH RT UPLC = 1.93 min m/z 187 [M-H] - λ max 219, 233, 318 nm C5 OO OH OOOO C2 RT UPLC = 1.50 min m/z 355 [M-H] - λ max 274 nm OO OH OOOO C2 RT UPLC = 1.50 min m/z 355 [M-H] – λ max 274 nm OO OH OHOH RT UPLC = 1.89 min m/z 295 [M-H] - λ max 307 nm C4 OO OH OHOH RT UPLC = 1.89 min m/z 295 [M-H] – λ max 307 nm C4 OO OH O O C7 RT UPLC = 2.63 min m/z 271 [M-H] - λ max 316 nm OO OH O O C7 RT UPLC = 2.63 min m/z 271 [M-H] – λ max 316 nm O OOH OH O C6 RT UPLC = 2.54 min m/z 295 [M-H] - λ max 242, 285, 340 nm O OOH OH O C6 RT UPLC = 2.54 min m/z 295 [M-H] – λ max 242, 285, 340 nm OO OH OOO C1 RT UPLC = 1.25 min m/z 313 [M-H] - λ max 264 nm OO OH OOO C1 RT UPLC = 1.25 min m/z 313 [M-H] – λ max 264 nm CoAS O Benzoyl-CoA CoAS O Benzoyl-CoA OO OH O C3 RT UPLC = 1.77 min m/z 229 [M-H] - λ max 246, 284 nm OO OH O C3 RT UPLC = 1.77 min m/z 229 [M-H] – λ max 246, 284 nm OO OH RT UPLC = 1.93 min m/z 187 [M-H] - λ max 219, 233, 318 nm C5 OO OH RT UPLC = 1.93 min m/z 187 [M-H] – λ max 219, 233, 318 nm C5 OO OH OHOH D3 RT UPLC = 2.42 min m/z 289 [M-H] - λ max 298 nm O O O OH O D6 RT UPLC = 3.16 min m/z 265 [M-H] - λ max 281 nm O OH O OOO D1 RT UPLC = 1.79 min m/z 307 [M-H] - λ max 261 nm OHOH OH OO O D5 RT UPLC = 3.13 min m/z 307 [M-H] - λ max 288 nm CoAS O Hexanoyl-CoA O OH O O D2 RT UPLC = 2.39 min m/z 223 [M-H] - λ max 284 nm OO OH D4 RT UPLC = 2.54 min m/z 181 [M-H] - λ max 227, 284 nm OO OH OHOH D3 RT UPLC = 2.42 min m/z 289 [M-H] - λ max 298 nm OO OH OHOH D3 RT UPLC = 2.42 min m/z 289 [M-H] – λ max 298 nm O O O OH O D6 RT UPLC = 3.16 min m/z 265 [M-H] - λ max 281 nm O O O OH O D6 RT UPLC = 3.16 min m/z 265 [M-H] – λ max 281 nm O OH O OOO D1 RT UPLC = 1.79 min m/z 307 [M-H] - λ max 261 nm O OH O OOO D1 RT UPLC = 1.79 min m/z 307 [M-H] – λ max 261 nm OHOH OH OO O D5 RT UPLC = 3.13 min m/z 307 [M-H] - λ max 288 nm OHOH OH OO O D5 RT UPLC = 3.13 min m/z 307 [M-H] – λ max 288 nm CoAS O Hexanoyl-CoA CoAS O Hexanoyl-CoA O OH O O D2 RT UPLC = 2.39 min m/z 223 [M-H] - λ max 284 nm O OH O O D2 RT UPLC = 2.39 min m/z 223 [M-H] – λ max 284 nm OO OH D4 RT UPLC = 2.54 min m/z 181 [M-H] - λ max 227, 284 nm OO OH D4 RT UPLC = 2.54 min m/z 181 [M-H] – λ max 227, 284 nm OO OH OHOH O B5 RT UPL C = 1.77 min m/z 303 [M-H] - λ max 238, 286 nm OHOH OH O OO RT UPL C = 2.18 min m/z 279 [M-H] - λ max 238, 287 nm B6 OO OH O OOO B2 RT UPL C = 1.12 min m/z 321 [M-H] - λ max 264 nm OO OH O OO B1 RT UPL C = 0.96 min m/z 279 [M-H] - λ max 262 nm OOH OH O O O B8 RT UPL C = 2.89 min m/z 303 [M-H] - λ max 230, 281, 324, 337, 404 nm CoAS O Isobutyryl-CoA OO OH O B3 RT UPL C = 1.28 min m/z 195 [M-H] - λ max 284 nm OO OH RT UPL C = 1.43 min m/z 153 [M-H] - λ max 225, 284 nm B4 OO OH OHOH O B5 RT UPL C = 1.77 min m/z 303 [M-H] - λ max 238, 286 nm OO OH OHOH O B5 RT UPL C = 1.77 min m/z 303 [M-H] – λ max 238, 286 nm OHOH OH O OO RT UPL C = 2.18 min m/z 279 [M-H] - λ max 238, 287 nm B6 OHOH OH O OO RT UPL C = 2.18 min m/z 279 [M-H] – λ max 238, 287 nm B6 OO OH O OOO B2 RT UPL C = 1.12 min m/z 321 [M-H] - λ max 264 nm OO OH O OOO B2 RT UPL C = 1.12 min m/z 321 [M-H] – λ max 264 nm OO OH O OO B1 RT UPL C = 0.96 min m/z 279 [M-H] - λ max 262 nm OO OH O OO B1 RT UPL C = 0.96 min m/z 279 [M-H] – λ max 262 nm OOH OH O O O B8 RT UPL C = 2.89 min m/z 303 [M-H] - λ max 230, 281, 324, 337, 404 nm OOH OH O O O B8 RT UPL C = 2.89 min m/z 303 [M-H] – λ max 230, 281, 324, 337, 404 nm CoAS O Isobutyryl-CoA CoAS O Isobutyryl-CoA OO OH O B3 RT UPL C = 1.28 min m/z 195 [M-H] - λ max 284 nm OO OH O B3 RT UPL C = 1.28 min m/z 195 [M-H] – λ max 284 nm OO OH RT UPL C = 1.43 min m/z 153 [M-H] - λ max 225, 284 nm B4 OO OH RT UPL C = 1.43 min m/z 153 [M-H] – λ max 225, 284 nm B4 Octaketides O OOH O O OH OH A7 RT UPL C = 1.31 min m/z 317 [M-H] - λ max 230, 280 nm O OOH O O OH OH A4 RT UPL C = 0.84 min m/z 317 [M-H] - λ max 284 nm OO OH OH OH O A6 RT UPL C = 1.17 min m/z 275 [M-H] - λ max 282 nm CoAS O Acetyl-CoA OO OH OOO RT UPL C = 0.45 min m/z 251 [M-H] - λ max 270 nm A1 OO OH O A2 RT UPL C = 0.52 min m/z 167 [M-H] - λ max 285 nm O O OHOH A8 RT UPL C = 1.37 min m/z 191 [M-H] - λ max 309 nm RT UPL C = 0.62 min m/z 125 [M-H] - λ max 283 nm A3 OO OH O O OH O A9 RT UPL C = 1.45 min m/z 231 [M-H] - λ max 243, 251, 292 nm O O OH OH OO A10 RT UPL C = 1.54 min m/z 275 [M-H] - λ max 235, 276 nm Octaketides O OOH O O OH OH A7 RT UPL C = 1.31 min m/z 317 [M-H] - λ max 230, 280 nm O OOH O O OH OH A4 RT UPL C = 0.84 min m/z 317 [M-H] - λ max 284 nm Octaketides O OOH O O OH OH A7 RT UPL C = 1.31 min m/z 317 [M-H] - λ max 230, 280 nm O OOH O O OH OH A7 RT UPL C = 1.31 min m/z 317 [M-H] – λ max 230, 280 nm O OOH O O OH OH A4 RT UPL C = 0.84 min m/z 317 [M-H] - λ max 284 nm O OOH O O OH OH A4 RT UPL C = 0.84 min m/z 317 [M-H] – λ max 284 nm OO OH OH OH O A6 RT UPL C = 1.17 min m/z 275 [M-H] - λ max 282 nm CoAS O Acetyl-CoA OO OH OOO RT UPL C = 0.45 min m/z 251 [M-H] - λ max 270 nm A1 OO OH O A2 RT UPL C = 0.52 min m/z 167 [M-H] - λ max 285 nm O O OHOH A8 RT UPL C = 1.37 min m/z 191 [M-H] - λ max 309 nm RT UPL C = 0.62 min m/z 125 [M-H] - λ max 283 nm A3 OO OH O O OH O A9 RT UPL C = 1.45 min m/z 231 [M-H] - λ max 243, 251, 292 nm O O OH OH OO A10 RT UPL C = 1.54 min m/z 275 [M-H] - λ max 235, 276 nm OO OH OH OH O A6 RT UPL C = 1.17 min m/z 275 [M-H] - λ max 282 nm OO OH OH OH O A6 RT UPL C = 1.17 min m/z 275 [M-H] – λ max 282 nm CoAS O Acetyl-CoA CoAS O Acetyl-CoA OO OH OOO RT UPL C = 0.45 min m/z 251 [M-H] - λ max 270 nm A1 OO OH OOO RT UPL C = 0.45 min m/z 251 [M-H] – λ max 270 nm A1 OO OH O A2 RT UPL C = 0.52 min m/z 167 [M-H] - λ max 285 nm OO OH O A2 RT UPL C = 0.52 min m/z 167 [M-H] – λ max 285 nm O O OHOH A8 RT UPL C = 1.37 min m/z 191 [M-H] - λ max 309 nm O O OHOH A8 RT UPL C = 1.37 min m/z 191 [M-H] – λ max 309 nm RT UPL C = 0.62 min m/z 125 [M-H] - λ max 283 nm A3 OO OH RT UPL C = 0.62 min m/z 125 [M-H] – λ max 283 nm A3 OO OH OO OH O O OH O A9 RT UPL C = 1.45 min m/z 231 [M-H] - λ max 243, 251, 292 nm O O OH O A9 RT UPL C = 1.45 min m/z 231 [M-H] – λ max 243, 251, 292 nm O O OH OH OO A10 RT UPL C = 1.54 min m/z 275 [M-H] - λ max 235, 276 nm O O OH OH OO A10 RT UPL C = 1.54 min m/z 275 [M-H] – λ max 235, 276 nm Fig. 4. Structures of enzymatic reaction products of H. perforatum HpPKS2 with different starter substrates. The structures were deter- mined by UPLC ⁄ ESIMS. K. Karppinen et al. Octaketide synthase from Hypericum perforatum FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS 4333 Localization of HpPKS2 transcripts in H. perforatum tissues In order to obtain more insight into the role of HpPKS2 in H. perforatum, in situ RNA hybridization studies were performed. Digoxigenin (DIG)-labelled HpPKS2 RNA probes were used to hybridize fixed tissue sections of the leaves and flower buds of H. perforatum in order to localize exactly the HpPKS2 transcripts in the tissues. After hybridization of the cross-sections of the leaves with a HpPKS2 RNA anti- sense probe, a dark blue signal that indicates HpPKS2 expression was clearly observed in the leaf margins (Fig. 6A). The signal was specifically localized in the multicellular nodular structures between the lower epi- dermis and the photosynthetic parenchymal cells of the H. perforatum leaves. Under test conditions, no signifi- cant background staining was observed, and the HpPKS2 probe specificity was confirmed by the absence of signal in the negative control sections of the leaves hybridized with HpPKS2 RNA sense probe (Fig. 6B). In the hybridized sections of the flower buds, a strong dark blue signal for HpPKS2 transcripts was localized in the petals (Fig. 6C) and the stamens between anthers (Fig. 6E), also restricted to multi- cellular nodules. The nodules that showed the HpPKS2 expression in flower buds were structurally similar to those found to contain HpPKS2 transcripts in the leaf sections. No signal was observed in the corresponding areas of the negative controls of the flower bud sections hybridized with HpPKS2 RNA sense probe (Fig. 6D,F). Multicellular nodules showing HpPKS2 expression in both leaves and flower buds consisted of a core of large cells that was surrounded by one to three flat cell layers. The HpPKS2 transcripts were found to be pres- ent in the large cells and also in the some of the inner- most flat cells of the nodules. In the flower petals of H. perforatum, two types of multicellular nodules that share the same anatomical organization in the cross- sections have been reported previously [10]. Spheroidal nodules similar to those observed in the leaf margins are also present in the petal margins, whereas the nod- ules in the interior parts of the petals are elongated tubulars [10,12]. In this study, nodules in both the margins and the interior parts of the petals were found to contain HpPKS2 transcripts. Localization of hypericins in H. perforatum tissues As reported previously [25], the leaf margins and flower buds contain the highest amounts of hypericins in H. perforatum. To see exactly where the red hyperic- ins are located, unstained cross-sections of the leaves and flower buds of H. perforatum were observed under a microscope. The dark red hypericins could be easily located because they remained in the paraffin sections and did not disappear until the in situ RNA hybridiza- tion. Leaf cross-sections showed dark red material in multicellular nodules in the leaf margins (Fig. 7A). The nodules were included between the lower OO OH R OOO -CO 2 O OOH OOH O OH m/z 191 O OOH O O OH OH O O OH O O O OH -CO 2 -CH 2 O -CO 2 -CO 2 or O OOH O O OH OH OH O O OH OH OH OH OH O OOH O O OH O OOH OH OH O O OH OO OH R OOO -CO 2 OO OH R OOO m/z 125 m/z 167 -CO 2 O OOH OOH O OH O OOH O O OH OH O OOH OOH O OH m/z 317 m/z 125 O OOH O O OH OH O O OH O O O OH O O OH O O O OH m/z 231 m/z 189 -CO 2 -CH 2 O -CO 2 -CO 2 m/z 317 m/z 273 m/z 229 m/z 287 m/z 243 or O OOH O O OH OH OH O O OH OH OH OH OH O OOH O O OH O OOH OH OH O O OH A B C D Fig. 5. MS fragmentation patterns of (A) a-pyrones, (B) SEK4 and (C) SEK4b in the negative ionization mode, and (D) aloesone in the positive ionization mode. Fragment ions were identified based on their exact masses. Octaketide synthase from Hypericum perforatum K. Karppinen et al. 4334 FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS epidermis and the photosynthetic parenchymal cells of leaves, and they comprised a core of large cells surrounded by flat cell layers (Fig. 7B). Red material was present in the large cells and also in the some of the innermost flat cells of the nodules. Dark red multicellular nodules of the same structure were also observed in cross-sections of the flower buds (Fig. 7C). Smaller red nodules were present in the flower petals (Fig. 7D), whereas larger ones were found in the stamens between anthers (Fig. 7E). The red material AB CD EF Fig. 6. In situ RNA localization of HpPKS2 transcripts in leaves and flower buds of H. perforatum. Cross-section of (A) leaf, (C) petal of flower bud and (E) stamen of flower bud hybridized with DIG-labelled HpPKS2 RNA antisense probe. (B,D,F) Corresponding sections were hybridized with HpPKS2 RNA sense probe. Arrows point to multicellular nodules. Bars = 100 lm. A B C D E Fig. 7. Localization of hypericins in leaves and flower buds of H. perforatum. Unstained cross-sections of (A) leaf (B) showing red pigmented nodules in leaf margins and (C) flower bud (D) showing red pigmented nodules in petal and (E) in stamen. Small arrows point to multicellular nodules. Bars = 100 lm. K. Karppinen et al. Octaketide synthase from Hypericum perforatum FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS 4335 was present in the nodules of both the margins and the interior parts of the flower petals. Discussion Despite the fact that hypericins are pharmacologically important compounds of H. perforatum, a widely used herbal remedy for the treatment of depression [1,2], there is little information available about the biosynthe- sis of these compounds. To date, only one gene has been cloned and characterized from the biosynthetic route leading to hypericins. The enzymatic product of hyp-1 has been shown to catalyse the final stages of hypericin biosynthesis [20]. It has been proposed that type III PKS would attend to the formation of emodin anthrone, the initial key reaction step in the biosynthesis of hypericins [20], but no such activity has been reported. In this study, the role of a newly found PKS from H. perforatum, HpPKS2 [25], was investigated for its possible involvement in the biosynthesis of hypericins. Phylogenetic analysis showed that the plant-specific type III PKS family proteins grouped into CHSs and other functionally divergent PKSs (Fig. 2). The only exceptions were STSs from Fabaceae and Gymno- sperms grouping with CHSs from the same or related species. STSs have been proposed to have evolved independently from CHSs several times, which explains their presence in several clusters in the phylogenetic tree [31–33]. HpPKS2 of H. perforatum grouped with functionally divergent non-chalcone-forming plant-specific type III PKSs. The grouping of HpPKS2 with non-CHSs indicates that HpPKS2 is not involved in the biosynthesis of flavonoids in H. perforatum. The functionally divergent PKSs include, for example, OKS and PCS from A. arborescens, which accept malonyl-CoA or acetyl-CoA as a starter substrate to produce octaketides (SEK4 and SEK4b) and pentake- tide chromone (5,7-dihydroxy-2-methyl-chromone), respectively [26,27]. However, HpPKS2 was not partic- ularly closely related to any of the currently known type III PKSs, which indicates that it is a novel plant-specific type III PKS family protein. We have previously reported that the deduced amino acid sequence of HpPKS2 shares only < 52% identity with previously isolated type III PKSs [25]. HpPKS2 expressed in E. coli resulted in an enzyme of  43 kDa (Fig. 3). The size coincides with a predicted molecular mass of 43.1 kDa for HpPKS2, calculated using bioinformatics tools [25], and with that of a subunit size typical to plant-specific type III PKSs. The plant-specific type III PKSs are reported to be homodimeric proteins with a subunit size of 40–45 kDa [21,23]. Functional characterization of the purified recombi- nant HpPKS2 revealed the expected OKS activity. But instead of producing emodin anthrone, an octaketide precursor of hypericins, the enzyme catalysed the con- densation of one molecule of acetyl-CoA with seven molecules of malonyl-CoA to form unnatural octake- tides SEK4 and SEK4b (Fig. 4). SEK4 and SEK4b, the longest polyketides known to be produced by type III PKSs, have also been shown to be the products of OKS from A. arborescens [26] and shunt products of minimal type II PKS from Streptomyces coelicolor [34,35]. The A. arborescens OKS, along with HpPKS2, is the only enzyme among unmodified plant-specific type III PKSs that has been shown to have OKS activity. Because the aloe does not accumulate SEK4 and SEK4b, the aloe OKS has been suggested to be involved in the biosynthesis of anthrones and anthraquinones in the plant and SEK4 ⁄ SEK4b produced in the absence of additional tailoring enzymes in vitro [26,36]. The A. arborescens OKS preferred malonyl-CoA as a starter substrate for the production of SEK4 and SEK4b. Because SEK4 and SEK4b could not be found from incubations with starter substrates other than acetyl-CoA in this study, it is likely that HpPKS2 used only acetyl-CoA as a starter substrate for production of SEK4 and SEK4b. HpPKS2 also catalysed the formation of tri- to heptaketide products, using acetyl-CoA as a starter substrate (Fig. 4). Triketide and tetraketide pyrones are often biosynthesized in vitro by PKSs when incu- bated with acetyl-CoA as a starter substrate [37–39]. Penta- to octaketides are more rare products. Two dif- ferent pentaketide products have previously been reported to be produced by unmodified plant-specific type III PKSs. These are 5,7-dihydroxy-2-methylchro- mone produced by PCS from A. arborescens [27] and a-pyrone by PKS from Plumbago indica [39]. The pen- taketide chromone structure A8 has not previously been reported to be produced by plant-specific type III PKS. The hexaketide a-pyrone A1 produced by HpPKS2 can be classified as a derailment product of type III PKS. The structure is also the product of P. indica PKS, along with hexaketide phenylpyrone [39]. Because the pyrones are not found in P. indica tissues, it has been suggested that the PKS in vivo would be involved in the biosynthesis of naphthoqui- none plumbagin and the pyrones produced in vitro in the absence of accessory enzymes [39]. Of the three heptaketides produced by HpPKS2 using acetyl-CoA as a starter substrate, one was aloesone. Aloesone has previously been reported as a product of aloesone synthase of Rheum palmatum [31], a plant known to be rich with chromones, napthalenes and anthraquinones. Octaketide synthase from Hypericum perforatum K. Karppinen et al. 4336 FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS Aloesone was also the product of A. arborescens OKS, along with SEK4 and SEK4b, after a single amino acid mutation, i.e. replacement of glycine by alanine, as in the case of aloesone synthase in the Gly207 site [26]. HpPKS2 has serine in the corresponding site. To our knowledge, the other two heptaketides produced by HpPKS2, chromone A10 and phenylpyrone A6, have not previously been reported as products of plant-specific type III PKSs. Notably, OKS and PCS from A. arborescens, PKS from P. indica, aloesone synthase from R. palmatum and now HpPKS2 from H. perforatum all share mechanistically related reac- tions, such as accepting acetyl-CoA ⁄ malonyl-CoA as a starter substrate, performing high numbers of conden- sations and two to three cyclization reactions. Because most type III PKSs perform only one to three exten- sions and catalyse the formation of one six-membered ring, it can be assumed that the above-mentioned PKSs may be involved in the biosynthesis of structur- ally similar types of compounds in plants. The acceptance of other, larger starter substrates by HpPKS2 shows that the enzyme has a broad substrate acceptance, as reported for other type III PKSs [22–24,26,27,32,37,39,40]. HpPKS2 accepted both aro- matic and aliphatic CoA-esters as starter units. By using isobutyryl-CoA, benzoyl-CoA and hexanoyl- CoA as starter substrates, HpPKS2 produced tri- to heptaketide products, mostly pyrones (Fig. 4). In addi- tion to pyrones, some chromones and phloroglucinols were also produced. It should be noted that with star- ter substrates other than acetyl-CoA, HpPKS2 was not able to produce octaketides but only afforded shorter products supporting the view that acetyl-CoA could be the real starter substrate for HpPKS2 in vivo. To our knowledge, the compounds produced by HpPKS2 in vitro, which were mostly pyrones, have not been described as constituents of H. perforatum. Sev- eral recombinant plant-specific type III PKSs are known to biosynthesize metabolites, especially pyrones, that have not been described as being accumulated by their plants of origin [26,32,37,39,40]. The products have been found to be typical for in vitro incubations of type III PKSs with non-physiological substrates, non-optimal assay conditions and are also suggested to be produced in the absence of co-operating tailoring enzymes [23,26,30,32,39]. To date, the only character- ized example of such a co-operating interaction of plant-specific type III PKS with tailoring enzyme is the biosynthesis of 6¢-deoxychalcone [23]. Typically, type I and type II PKSs consist of many additional subunits, including ketoreductases, cyclases and aromatases, that are often needed for the production of specific cyclized polyketide products [34,35,41–43]. These additional subunits interact with PKS to stabilize the highly reac- tive polyketide chain preventing non-specific cycliza- tions. It is not currently known whether emodin anthrone biosynthesis requires additional enzymes and thus it is possible that HpPKS2 failed to produce emo- din anthrone in this study because of the absence of additional tailoring enzymes in vitro. To further s tudy the r ole of HpPKS2 in H. perforatum, in situ RNA hybridization studies to locate HpPKS2 transcripts were performed. HpPKS2 expression was found to localize specifically in multicellular nodules in the leaf margins, flower petals and stamens of H. per- foratum (Fig. 6). These types of structures present in the H. perforatum tissues have been described previ- ously by several authors, and are referred to as dark glands [10,17,18,44]. In this study, the same nodules were also found to contain dark red material (Fig. 7). The red material in the dark glands has previously been found to consist of hypericins, and their accumu- lation is shown to be restricted to only the dark glands in H. perforatum [12,16–18]. The obtained results are consistent with our previous study in which the expres- sion of HpPKS2, measured using real-time PCR, was shown to correlate with the concentrations of hyperic- ins in different H. perforatum tissues [25]. Recently, emodin, which is an oxidized derivative of emodin anthrone (Fig. 1), has also been found to accumulate at high concentrations in the dark glands of H. perfo- ratum [12]. The presence of emodin in only the dark glands in H. perforatum suggests that emodin biosyn- thesis may take place in the glands [12]. The restriction of HpPKS2 expression and the presence of both hyper- icins and emodin specifically in the same cells imply that HpPKS2 may have a role in the biosynthesis of hypericins in H. perforatum. The localization of the HpPKS2 transcripts in the dark glands that accumu- late hypericins is very similar to the expression pattern of type III PKS from Humulus lupulus. Valerophenone synthase from H. lupulus is responsible for the biosyn- thesis of the phloroglucinol skeleton of hop resin, and it has been shown to be expressed specifically in secre- tory structures called ‘lupulin glands’ accumulating the resin [45]. The HpPKS2 transcripts were found to accumulate into both the large cells and some of the innermost flat cells of the multicellular nodules. This indicates that if HpPKS2 is involved in the biosynthesis of hypericins, then at least the early phase of biosynthesis, i.e. the formation of emodin anthrone, may occur in both cell types. Kornfeld et al. [18] hypothesized that the biosynthesis of hypericins takes place in the peripheral flat cells rather than in the large interior cells of the nodules. K. Karppinen et al. Octaketide synthase from Hypericum perforatum FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS 4337 Based on these results, H. perforatum HpPKS2 is a novel plant-specific type III PKS having OKS activity. Furthermore, our findings show a strong connection between HpPKS2 expression and the accumulation of hypericins, indicating that HpPKS2 may have a role in the initial key reaction step in the biosynthesis of hypericins in H. perforatum. However, although the enzyme is capable of carrying out the expected number of condensation reactions in vitro, it fails in the cyclization of the produced octaketide chain to emodin anthrone. The formation of derailment products by HpPKS2 may mean that the biosynthesis of emodin anthrone requires some additional, as yet unidentified factors that are missing in vitro. Recently, several type III PKSs have been isolated that do not, in vitro, produce the metabolites that they are expected to cata- lyse and that are found in their plant of origin. There- fore, further studies are needed to elucidate the reasons for these failures to reveal the actual biosynthesis mechanism of many plant polyketides, including hypericins. Experimental procedures Construction of expression plasmid cDNA from H. perforatum leaves was prepared as described previously [25]. The coding region of HpPKS2 was amplified from the cDNA by PCR, using forward primer 5¢-CATATTG GGATCCATGGGTTCCCTTGAC-3¢ (the translation start codon is in bold and the BamHI site is underlined) and reverse primer 5¢-ACGCT GGTACC TTAGAGAGGCACACTTCG-3¢ (the translation stop codon is in bold and the KpnI site is underlined). The PCR was performed with DyNazymeÔ II DNA polymerase (Finnzymes, Espoo, Finland). The PCR conditions were denaturation at 94 °C for 5 min, followed by 40 cycles of amplification at 94 °C for 1 min, 60 °C for 2 min and 72 °C for 2 min, and a final extension at 72 °C for 10 min. The amplified PCR product was purified by electrophoresis on a 1% (w ⁄ v) ethidium bromide-stained agarose gel. The PCR fragment of the expected size ( 1.2 kb) was excised from the gel and further purified using MontageÒ DNA Gel Extraction Kit (Millipore, Bedford, MA, USA). The purified PCR product was digested with BamHI and KpnI (Isogen, Bioscience, Maarssen, The Netherlands) and ligated into the BamHI ⁄ KpnI site of expression vector pQE30 (Qiagen, Hilden, Germany). Thus, the recombinant enzyme contains an additional hexahistidine tag at the N-terminus. The resulting recombinant plasmid pQE30– HpPKS2 was confirmed by sequencing, using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) and an ABI 310 DNA sequencer (Model 377; Applied Biosystems). Expression of recombinant HpPKS2 The recombinant plasmid pQE30–HpPKS2 was transferred into the E. coli host strain M15 [pREP4] (Qiagen) for pro- tein expression. E. coli cells harbouring the plasmid were grown in Luria–Bertani liquid medium in the presence of ampicillin (100 lgÆmL )1 ) and kanamycin (25 lgÆmL )1 )at 30 °C until the D 600 of the culture reached 0.6. After the culture had been cooled on ice, IPTG (Roche, Basel, Swit- zerland) was added to the culture in a final concentration of 0.4 mm to induce protein expression. The culture was incubated further at 16 °C for 20 h. Enzyme purification E. coli cells were harvested by centrifugation (4000 g for 20 min) and resuspended in a lysis buffer (50 mm sodium phosphate buffer, pH 8.0, containing 500 mm NaCl, 10 mm b-mercaptoethanol, 1% Tween 20 and 20 mm imidazole). The cells were disrupted using lysozyme (1 mgÆmL )1 ) and sonication (Type UP50H; Dr Hielscher GmbH, Teltow, Germany). The lysate was diluted twofold with the same buffer and centrifuged at 17 000 g for 30 min. The super- natant was collected for purification of recombinant protein under native conditions according to the protocol of the QIAexpressionist [46], using Ni-NTA agarose. Unbound proteins were washed away with a wash buffer (50 mm sodium phosphate buffer, pH 7.0, containing 500 mm NaCl, 10 mm b-mercaptoethanol, 10% glycerol, 1% Tween 20 and 20 mm imidazole) and the recombinant protein was eluted with an elution buffer (50 mm sodium phosphate buffer, pH 7.0, containing 500 mm NaCl, 10 mm b-mercaptoethanol, 10% glycerol and 250 mm imidazole). After purification, the protein concentration was deter- mined according to Bradford [47], using BSA (Sigma, St Louis, MO, USA) as a standard. The purity of the protein was verified by SDS ⁄ PAGE, using 12% separation and 3% stacking gels. The proteins were run along with protein markers (Fermentas, Vilnius, Lithuania) at 180 V, using a Mini-Protean II electrophoresis system (Bio-Rad, Hercules, CA, USA) followed by staining with Coomassie Brilliant Blue R-250 (Merck, Darmstadt, Germany). Polyketide synthase assays Purified recombinant HpPKS2 (100 lg) was mixed with 200 lm starter substrates (acetyl-CoA, isobutyryl-CoA, ben- zoyl-CoA or hexanoyl-CoA; Sigma) and 300 l m malonyl- CoA (Sigma). An assay buffer (0.5 m potassium phosphate buffer, pH 6.8, containing 2.8 mm b-mercaptoethanol and 10 lm dithiothreitol) was then added to 500 lL. For con- trol reactions, the enzyme was heat-denatured. Incubations were carried out at 30 °C for 90 min. Reactions were stopped by adding 50 lL of 20% HCl, and the products were then extracted twice with 250 lL of ethyl acetate. Octaketide synthase from Hypericum perforatum K. Karppinen et al. 4338 FEBS Journal 275 (2008) 4329–4342 ª 2008 The Authors Journal compilation ª 2008 FEBS [...]... 25 Karppinen K & Hohtola A (2008) Molecular cloning and tissue-specific expression of two cDNAs encoding polyketide synthases from Hypericum perforatum J Plant Physiol 165, 1079–1086 26 Abe I, Oguro S, Utsumi Y, Sano Y & Noguchi H (2005) Engineered biosynthesis of plant polyketides: chain length control in an octaketide-producing plant type III polyketide synthase J Am Chem Soc 127, 12709–12716 27 Abe... conditions were maintained throughout the procedure Phylogenetic tree construction In total, 44 amino acid sequences of type III PKS family proteins including H perforatum HpPKS2 were aligned using the clustal w program The protruding ends of the sequences were truncated and eventually 396 amino acids, including gaps, were aligned A phylogenetic tree was constructed using the neighbor-joining method, with... 4341 Octaketide synthase from Hypericum perforatum K Karppinen et al producing novel multifunctional type III polyketide synthase from Huperzia serrata FEBS J 274, 1073–1082 41 Hertweck C, Xiang L, Kalaitzis JA, Cheng Q, Palzer M & Moore BS (2004) Context-dependent behavior of the enterocin iterative polyketide synthase: a new model for ketoreduction Chem Biol 11, 461–468 42 Kalaitzis JA & Moore BS... vector (Promega, Madison, WI, USA) DIG-labelled sense and antisense probes were prepared from the linearized plasmid by in vitro transcription with SP6 or T7 RNA polymerase, using DIG RNA Labelling Kit according to the manufacturer’s instructions (Roche) In situ RNA hybridization analysis Before hybridization, rehydrated tissue sections were treated with proteinase K (1 lgÆmL)1 in 100 mm Tris ⁄ HCl and 50... 18.2 MW) prior to injection into UPLC The UPLC eluents were 0.1% acetic acid (BDH Laboratory Supplies, Poole, UK) in UP grade water (A) and acetonitrile (B) (HPLC grade; Merck) The initial gradient condition was 90% A and 10% B, changing linearly to 60% B in 4 min followed by 1 min of isocratic elution and 2 min of equilibration with initial conditions, giving a total analysis time of 7 min The eluent... 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Plant–environment interactions: accumulation of hypericin in dark glands of Hypericum perforatum Ann Bot 98, 793–804 ˇ ˇ ´ ´ ´ ´ 13 Kosˇ uth J, Katkovcinova Z, Olexova P & Cellarova E (2007) Expression of the hyp-1 gene in early stages of development of Hypericum perforatum L Plant Cell Rep 26, 211–217 ˇ ´ ´ 14 Repcak M & Martonfi P (1997) The localization of secondary substances in Hypericum perforatum flower... production of hypericins in two selected Hypericum perforatum shoot cultures is related to differences in black gland structure Plant Physiol Biochem 45, 24–32 19 Walker L, Sirvent T, Gibson D & Vance N (2001) Regional differences in hypericin and pseudohypericin concentrations and five morphological traits among Hypericum perforatum plants in the northwestern United States Can J Bot 79, 1248–1255 20 Bais HP,... of hypericin in St John’s wort (Hypericum perforatum L.) J Biol Chem 278, 32413–32422 21 Schroder J (1997) A family of plant-specific polyketide ¨ synthases: facts and predictions Trends Plant Sci 2, 373–378 22 Austin MB & Noel JP (2003) The chalcone synthase superfamily of type III polyketide synthases Nat Prod Rep 20, 79–110 23 Schroder J (2000) The family of chalcone synthase related proteins: functional... calculated and measured masses were < 3 mDa) in both ESI+ and ESI) conditions Octaketide synthase from Hypericum perforatum Tissue fixation and embedding H perforatum leaves and flower buds were collected from the botanical garden of the University of Oulu, Finland The samples excised from the plants were fixed in 4% (w ⁄ v) paraformaldehyde and 0.25% (v ⁄ v) glutaraldehyde in 0.1 m sodium phosphate buffer (pH . Octaketide-producing type III polyketide synthase from Hypericum perforatum is expressed in dark glands accumulating hypericins Katja Karppinen 1 ,. specifically in multicellular dark glands accumulating hypericins. Based on our results, HpPKS2 may have a role in the biosynthesis of hypericins in H. perforatum