Mechanistic Studies on Three 2-Oxoglutarate-dependent Oxygenases of Flavonoid Biosynthesis
2004; Elsevier BV; Volume: 279; Issue: 2 Linguagem: Inglês
10.1074/jbc.m309228200
ISSN1083-351X
AutoresJonathan J. Turnbull, Jun‐ichiro Nakajima, Richard W.D. Welford, Mami Yamazaki, Kazuki Saito, Christopher J. Schofield,
Tópico(s)Plant biochemistry and biosynthesis
ResumoAnthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3β-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid “C ring”; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 atmospheres. In the 18O2/16OH2 atmosphere close to complete incorporation of a single 18O label was observed in the dihydroflavonol products (e.g. (2R,3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate. Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3β-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid “C ring”; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 atmospheres. In the 18O2/16OH2 atmosphere close to complete incorporation of a single 18O label was observed in the dihydroflavonol products (e.g. (2R,3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate. The flavonoids are a large class of plant secondary metabolites. They contain a 15-carbon phenylpropanoid core, which is extensively modified by rearrangement, alkylation, oxidation, and glycosylation (1.Bohm B.A. Introduction to Flavonoids. Harwood Academic Publishers, Reading, UK1998Google Scholar). In plants the flavonoids fulfill a diverse array of roles including pigmentation and protection against UV photodamage and can act as signaling molecules (1.Bohm B.A. Introduction to Flavonoids. Harwood Academic Publishers, Reading, UK1998Google Scholar, 2.Harborne J.B. Williams C.A. Phytochemistry. 2000; 55: 481-504Crossref PubMed Scopus (3258) Google Scholar). They have been reported to possess a variety of biomedicinal properties including antimalarial, antioxidant, and antitumor activities. Furthermore flavonoids have been shown to modulate the hypoxic response in endothelial cells, an effect that might be mediated by their ability to chelate iron or directly inhibit hydroxylase enzymes involved in the response (3.Wilson W.J. Poellinger L. Biochem. Biophys. Res. Commun. 2002; 293: 446-450Crossref PubMed Scopus (60) Google Scholar, 4.Welford R.W.D. Schlemminger I. McNeill L.A. Hewitson K.S. Schofield C.J. J. Biol. Chem. 2003; 278: 10157-10161Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Due to their historic importance in genetic studies and their biomedicinal properties, flavonoid biosynthesis is of interest to both biologists and chemists (1.Bohm B.A. Introduction to Flavonoids. Harwood Academic Publishers, Reading, UK1998Google Scholar, 5.Robinson R. Nature. 1936; 137: 172-173Crossref Scopus (10) Google Scholar, 6.Scott-Moncrieff R. J. Genet. 1936; 32: 111-170Crossref Scopus (39) Google Scholar). The flavonoid biosynthetic pathway has been extensively studied in plants by a combination of genetic and labeling studies (7.Heller W. Forkmann G. Harborne J.B. The Flavonoids: Advances in Research Since 1986. Chapman & Hall, London1993: 499-535Crossref Google Scholar, 8.Forkmann G. Heller W. Comprehensive Natural Products Chemistry. Elsevier Science, Amsterdam1999: 713-748Crossref Google Scholar, 9.Springob K. Nakajima J. Yamazaki M. Saito K. Nat. Prod. Rep. 2003; 20: 288-303Crossref PubMed Scopus (309) Google Scholar). Advances in genetic techniques and recombinant expression technologies have enabled proposals regarding the substrate and product selectivities of individual enzymes to be studied in vitro. The steps leading to the flavonoid core have now been established (see Fig. 1). Formation of chalcone 1 is catalyzed by a type III polyketide synthase known as chalcone synthase via condensation of para-coumaryl-CoA 2 and three malonyl-CoA thioesters 3 (10.Jez J.M. Noel J.P. J. Biol. Chem. 2000; 275: 39640-39646Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11.Ferrer J.L. Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 1999; 6: 775-784Crossref PubMed Scopus (578) Google Scholar). Chalcone isomerase then catalyzes the intramolecular cyclization of chalcone 1 to give (2S)-naringenin 4, a precursor of many flavonoids 1Flavonoid classes are named according to the oxidation state of the “C” ring as flavones, flavanones, flavonols, etc. Each may have different hydroxylation patterns on the “A”/“B” rings. The enzymes of the biosynthetic pathway accept a variety of A/B ring hydroxylation patterns. Where a class name is used, an example of a compound belonging to that class follows. For clarity in the figures the A and B rings are not always drawn. Use of Cahn-Ingold-Prelog nomenclature for the assignment of the absolute stereochemistry at the C-3 position of the C ring can be confusing (especially for prochiral C-3-gem-diols) due to the “priority changes” in different classes of flavonoid. Hence in the text, the faces of the flavonoid C ring are differentiated by α or β. When the A ring is drawn on the left with C-4 oxygen bond pointing down the page, the face above the plane of the paper is defined as β, while the face beneath the plane of the paper is α. including the anthocyanin (e.g. cyanin 5) subfamily (12.Jez J.M. Bowman M.E. Dixon R.A. Noel J.P. Nat. Struct. Biol. 2000; 7: 786-791Crossref PubMed Scopus (279) Google Scholar). Oxidation reactions involving both heme and non-heme iron-dependent enzymes play a central role in flavonoid biosynthesis (7.Heller W. Forkmann G. Harborne J.B. The Flavonoids: Advances in Research Since 1986. 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In anthocyanin (e.g. 5) biosynthesis the steps involving oxidation of (2S)-flavanones (e.g. (2S)-naringenin 4) and (2R,3S,4S)-cis-leucoanthocyanidins (e.g. (2R,3S,4S)-cis-leucocyanidin (LCD) 2The abbreviations used are: LCD, leucocyanidin; 2OG, 2-oxoglutarate; FHT, flavanone 3β-hydroxylase; ANS, anthocyanidin synthase; FNS, flavone synthase; FLS, flavonol synthase; DHQ, dihydroquercetin; DHK, dihydrokaempferol; HPLC, high pressure liquid chromatography; LCMS, liquid chromatography-mass spectrometry; MES, 2-(N-morpholino)ethanesulfonic acid; MOPSO, 3-(N-morpholino)-2-hydroxy-propanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. 6) are catalyzed by flavanone 3β-hydroxylase (FHT) (15.Forkmann G. Stotz G. Planta. 1984; 161: 261-265Crossref PubMed Scopus (24) Google Scholar, 16.Britsch L. Grisebach H. Eur. J. Biochem. 1986; 156: 569-577Crossref PubMed Scopus (138) Google Scholar, 17.Britsch L. Arch. Biochem. 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The available evidence suggests that catalysis by 2OG oxygenases proceeds via bidentate binding of 2OG 7 to the active site iron (42.Schofield C.J. Zhang Z.-H. Curr. Opin. Struct. Biol. 1999; 9: 722-731Crossref PubMed Scopus (343) Google Scholar, 43.Ryle M.J. Hausinger R.P. Curr. Opin. Chem. Biol. 2002; 6: 193-201Crossref PubMed Scopus (204) Google Scholar). Substrate binding is thought to enable dioxygen to displace a ligating water from the catalytic iron center (44.Zhang Z. Ren J. Stammers D.K. Baldwin J.E. Harlos K. Schofield C.J. Nat. Struct. Biol. 2000; 7: 127-133Crossref PubMed Scopus (254) Google Scholar, 45.Zhou J. Kelly W.L. Bachmann B.O. Gunsior M. Townsend C.A. Solomon E.I. J. Am. Chem. Soc. 2001; 123: 7388-7398Crossref PubMed Scopus (146) Google Scholar). Oxidative decarboxylation of 2OG 7 then occurs, producing succinate 10, CO2, and a ferryl [Fe(IV)=O ↔ Fe(III)–O·] 11 intermediate, which can subsequently affect hydroxylation or desaturation of the substrate (Fig. 2) (46.Price J.C. Barr E.W. Tirupati B. Bollinger J.M. Krebs C. Biochemistry. 2003; 42: 7497-7508Crossref PubMed Scopus (564) Google Scholar, 47.Rohde J.U. In J.H. Lim M.H. Brennessel W.W. Bukowski M.R. Stubna A. Munck E. Nam W. Que L. Science. 2003; 299: 1037-1039Crossref PubMed Scopus (772) Google Scholar). Recent crystallographic work has demonstrated that ANS contains the double-stranded β-helix common to other 2OG oxygenases and identified the residues involved in substrate binding (38.Aravind, L., and Koonin, E. V. (2001) Genome Biol. http://genomebiology.com/2001/2/3/RESEARCH/0007Google Scholar, 48.Wilmouth R.C. Turnbull J.J. Welford R.W.D. Clifton I.J. Prescott A.G. Schofield C.J. Structure. 2002; 10: 93-103Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar). ANS and FLS are closely related (50–60% sequence similarity at the polypeptide level) but display a lower level of similarity to FHT and FNS I ( 85%) with only a trace amount (∼2%) of the anticipated two-electron oxidation product, cyanidin 12, being observed (Fig. 3B) (50.Turnbull J.J. Nagle M.J. Seibel J.F. Welford R.W.D. Grant G. Schofield C.J. Bioorg. Med. Chem. Lett. 2003; 13: 3853-3857Crossref PubMed Scopus (35) Google Scholar). Incubations of (2R,3S,4R)-trans-LCD 13 with ANS gave cis-DHQ 14, trans-DHQ 8/15, quercetin 9, and cyanidin 12 (Fig. 3C) (21.Turnbull, J. J., Sobey, W. J., Aplin, R. T., Hassan, A., Firmin, J. L., Schofield, C. J., and Prescott, A. G. (2000) Chem. Commun. 2473-2474Google Scholar, 49.Welford, R. W. D., Turnbull, J. J., Claridge, T. D. W., Prescott, A. G., and Schofield, C. J. (2001) Chem. Commun. 1828-1829Google Scholar, 50.Turnbull J.J. Nagle M.J. Seibel J.F. Welford R.W.D. Grant G. Schofield C.J. Bioorg. Med. Chem. Lett. 2003; 13: 3853-3857Crossref PubMed Scopus (35) Google Scholar). ANS also catalyzes the natural reaction of FLS, i.e. conversion of (2R,3R)-trans-DHQ 8 to quercetin 9 (Fig. 3A) (21.Turnbull, J. J., Sobey, W. J., Aplin, R. T., Hassan, A., Firmin, J. L., Schofield, C. J., and Prescott, A. G. (2000) Chem. Commun. 2473-2474Google Scholar). Both ANS and FLS have also been found to react with the unnatural substrate naringenin 4 (Fig. 3, D and E) (26.Martens S. Forkmann G. Britsch L. Wellmann F. Matern U. Lukacin R. FEBS Lett. 2003; 544: 93-98Crossref PubMed Scopus (92) Google Scholar, 28.Prescott A. Stamford N.P.J. Wheeler G. Firmin J.L. Phytochemistry. 2002; 60: 589-593Crossref PubMed Scopus (99) Google Scholar, 29.Lukacin R. Wellmann F. Britsch L. Martens S. Matern U. Phytochemistry. 2003; 62: 287-292Crossref PubMed Scopus (92) Google Scholar, 49.Welford, R. W. D., Turnbull, J. J., Claridge, T. D. W., Prescott, A. G., and Schofield, C. J. (2001) Chem. Commun. 1828-1829Google Scholar). These and other results led to the proposal that ANS mediates oxidation of the natural (2R,3S,4S)-cis-LCD 6 substrate via initial oxidation at C-3 followed by loss of water from the 3,3-gem-diol 16 to give a C-2,C-3 enol (a (4S)-flav-2-en-3,4-diol 17). In vitro, this may be (partially) retained in the active site to undergo a further round of oxidation to give quercetin 9. It has been proposed that in vivo the (4S)-flav-2-en-3,4-diol 17 may be directly channeled to flavonoid glycosyltransferase, which is responsible for cyanin 5 formation (22.Nakajima J. Tanaka Y. Yamazaki M. Saito K. J. Biol. Chem. 2001; 276: 25797-25803Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 50.Turnbull J.J. Nagle M.J. Seibel J.F. Welford R.W.D. Grant G. Schofield C.J. Bioorg. Med. Chem. Lett. 2003; 13: 3853-3857Crossref PubMed Scopus (35) Google Scholar) (Fig. 1). Here we report work that enhances mechanistic understanding of the non-heme dioxygenases of flavonoid biosynthesis by carrying out assays of ANS, FHT, and FLS in 18O-labeled dioxygen and water with a variety of substrates. Materials—Chemicals were purchased from Sigma and were of at least analytical or molecular biology grade except for the following: organic solvents (Rathburn and Riedel-de-Haen); gases (BOC Gases); 18O2 (Isotec, Inc.); kanamycin sulfate (Amersham Biosciences); isopropyl-β,d-thiogalactoside, and dithiothreitol (Melford Laboratories Ltd.); molecular weight markers (BDH Ltd.); Bacto-tryptone, yeast extract and Bacto-agar (Difco and Oxoid Ltd.); agarose and acrylamide/bisacrylamide stock (Anachem); competent cells (Stratagene); commercial leucocyanidin and leucopelargonidin (Industrial Research Ltd. and DihydroChem Technologies); (2S)-naringin and (2R)-naringin (a gift from Dr. J. L. Firmin, John Innes Centre, Norwich, UK and purchased from Extrasynthese); apigenin, anthocyanidin, dihydrokaempferol, eriodictyol, kaempferol, luteolin, and pelargonidin (Sequioa Biochemicals Ltd.); hyamine hydroxide (ICN Radiochemicals); and OptiPhase “Safe” (Wallac Scintillation Products). Cell Growth, Lysis, and Protein Purification—A. thaliana ANS was prepared and purified as reported previously (51.Turnbull J.J. Prescott A.G. Schofield C.J. Wilmouth R.C. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 425-427Crossref PubMed Scopus (20) Google Scholar). For FLS the pET-3a A. thaliana FLS plasmid was transformed into Escherichia coli BL21(DE3) Gold cells, and an overnight starter culture was used as a 1% inoculum in 600 ml of 2TY medium in 2-liter unbaffled flasks containing ampicillin (100 μg ml-1). After inoculation, growth was continued at 37 °C and 250 rpm until induction with 0.2 mm isopropyl-β,d-thiogalactoside when the A600 reached 0.6–0.8. Growth continued at 37 °C for 3.5 h before harvesting the cells and storage at -80 °C until further use. A clear lysate containing the recombinant A. thaliana FLS was prepared in the same manner as for A. thaliana ANS. The soluble portion of the cell lysate was applied to a Q-Sepharose FF column pre-equilibrated at 50 mm Tricine, pH 7.3 before elution using a linear gradient of NaCl from 0 to 0.5 m NaCl over ∼5 column volumes. FLS eluted at ∼230 mm NaCl, and the fractions containing FLS at ∼45–55% purity (by SDS-PAGE analysis) were pooled and further purified. The FLS protein was concentrated and buffer-exchanged into 50 mm MES, pH 6.15, 10% (w/v) glycerol before loading onto the reactive green C-19 column equilibrated using the same buffer. The protein was eluted with a linear gradient of NaCl from 0 to 2 m NaCl. Fractions of ∼90–95% purity were pooled, concentrated, buffer-exchanged, and then stored at -80 °C. Petunia hybrida ANS was prepared as reported previously (22.Nakajima J. Tanaka Y. Yamazaki M. Saito K. J. Biol. Chem. 2001; 276: 25797-25803Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). P. hybrida FHT and FLS were prepared from corresponding cDNAs amplified by PCR as for P. hybrida ANS. 1-[14C]2-Oxoglutarate Assays—Standard assay conditions were based on those reported previously (52.Sabourin P.J. Bieber L.L. J. Biol. Chem. 1982; 257: 7460-7467Abstract Full Text PDF PubMed Google Scholar) and comprised a total assay volume of 100 μl containing the following components: 100 mm MOPSO, pH 6.2, 20 mm ascorbate, 160 μm 2OG (5% 1-[14C]), 40 μm FeSO4·7H2O, 0.1 mg ml-1 bovine serum albumin, 10 μl ml-1 catalase, 0.1 mg ml-1 enzyme, 800 μm substrate (MeOH). After initiation of the enzymatic reaction a tube containing 200 μl of hyamine hydroxide was added, and the vial was sealed. The assays were incubated at 30 °C for 10 min before work-up (4.Welford R.W.D. Schlemminger I. McNeill L.A. Hewitson K.S. Schofield C.J. J. Biol. Chem. 2003; 278: 10157-10161Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). 18O Labeling Experiments—For experiments in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 environments, reagent solutions were initially mixed under an argon atmosphere (<0.4–0.8 ppm O2) in a Belle Technology® glove box to a total volume of 500, 70, and 70 μl, respectively. The FeSO4·7H2O, ascorbate, 2OG, substrates (except the unstable leucoanthocyanidins), 5-ml tubes, solid reagents, and rubber septa were ported into a glove box 24 h before use. The leucoanthocyanidin substrates (dissolved in Milli-Q H2O) and purified ANS and FLS were ported into the glove box immediately prior to use. H218O used for assays in 16O2/H218O and 18O2/H218O environments was 95% 18O-labeled. The MOPSO buffer used was prepared by addition of 1 m MOPSO, pH 6.2 to the 95% 18O-labeled H2O. The assay reagents were all dissolved in either Milli-Q H2O or MeOH. This lead to a ∼80% 18O-labeled aqueous environment. The reagents were mixed in the following order to give final concentrations of 50 mm MOPSO, pH 6.2, 40 mm ascorbate, 20 mm 2OG, 40 μm FeSO4·7H2O, 0.2 mg ml-1 enzyme, 800 μm substrate (MeOH). HPLC/LCMS Assays—HPLC/LCMS was carried out using a Synergi C-18 MAX column (250 mm × 4.6 mm; bead size, 4 μm, Phenomenex). Analyses utilized a photodiode array detector and a Micromass ZMD mass spectrometer (electrospray ionization). All HPLC grade buffers were filtered through 0.2-μm filters and sparged with helium(g) at 100 ml min-1 for 20 min before use. Buffers were acidified using formic acid, 2% (v/v) for HPLC and 0.05% (v/v) for LCMS buffers. Prior to injection 1% (v/v) formic acid was added to samples, and they were centrifuged at 13,000 rpm for 10 min. After injection samples were eluted isocratically for 10 min in 10% MeOH, and then a gradient was run from 10 to 80% MeOH over 25 min followed by 10 min of eluting isocratically with 80% MeOH. Substrate Synthesis—(2R,3S,4R)-trans-LCD 13 and (2R,3S,4S)-cis-LCD 6 were synthesized and purified as reported previously (50.Turnbull J.J. Nagle M.J. Seibel J.F. Welford R.W.D. Grant G. Schofield C.J. Bioorg. Med. Chem. Lett. 2003; 13: 3853-3857Crossref PubMed Scopus (35) Google Scholar, 53.Stafford H.A. Lester H.H. Porter L.J. Phytochemistry. 1985; 24: 333-338Crossref Scopus (31) Google Scholar). (2R)- and (2S)-naringenin 4 were produced by naringinase-mediated cleavage of the glycosidic bond of (2R)- and (2S)-naringin, respectively (30 °C, pH 4.0, 100 mm NaH2PO4 buffer for 30 min). (2R)- and (2S)naringenin were extracted from the reaction mixtures by solid phase extraction. Strata C18E solid phase extraction columns (3-ml, 500-mg sorbent bed) from Phenomenex were mounted onto an IST Vacmaster manifold, and solvent was washed through the columns in vacuo. All columns were primed with MeOH (4 ml) and equilibrated with H2O (4 ml) before the sample was loaded. The column was washed sequentially with 4-ml aliquots of 20 and 40% MeOH (v/v) before elution of the purified (2R)- and (2S)-naringenin with 6 ml of 100% MeOH (v/v). The MeOH was removed in vacuo, and the water was removed by lyophilization. A racemic mixture of cis/trans-DHQ was produced by C-2 epimerization of commercial DHQ as reported previously (54.Kiehlmann E. Li E.P.M. J. Nat. Prod. 1995; 58: 450-455Crossref Scopus (44) Google Scholar). cis-DHQ was purified from the cis/trans-DHQ mixture as follows. Racemic cis/trans-DHQ was dissolved in MeOH (10 mg, 1 ml) and loaded onto a Phenomenex Luna C-18 250-mm × 10-mm (bead size, 4 μm) column pre-equilibrated with 50 mm NaH2PO4, pH 6.8, 20% MeOH (v/v) and run at 3.6 ml min-1. The column was run isocratically for 15 min in 50 mm NaH2PO4, pH 6.8, 20% MeOH (v/v) before a gradient was run to 50 mm NaH2PO4, pH 6.8, 50% MeOH (v/v) over 25 min followed by 15 min of isocratic elution. The peaks corresponding to the racemic DHQ diastereomers were collected, diluted to <10% MeOH by addition of Milli-Q H2O, and freeze-dried. The purified DHQ (trans- and cis-) diastereomers were dissolved in the minimum volume of 50 mm NaH2PO4, pH 6.8, 20% MeOH (v/v) and then loaded onto Strata C18E solid phase extraction columns pre-equilibrated as for the purification of the naringinase reaction mixture above. The columns were washed with 10 ml of Milli-Q H2O before elution of DHQ in 6 ml of 100% MeOH (v/v). The presence of the DHQ isomers was confirmed by UV analysis of the eluent before evaporation of the MeOH at 30 °C in vacuo and further drying on a high vacuum line. NMR analysis in MeOH-[2H]4 confirmed the identity of the purified racemic cis-DHQ containing <10% trans-DHQ. Selected 1H NMR data (54.Kiehlmann E. Li E.P.M. J. Nat. Prod. 1995; 58: 450-455Crossref Scopus (44) Google Scholar): trans-DHQ δH (500 MHz): 4.6 (d, 1H, J 11.5 Hz, H-3), 5.0 (d, 1H, J 11.5 Hz, H-2), 5.8–6.0 (2ca d, 1H, J 2.0 Hz, H-6, H-8), 6.7–7.0 (m, 3H, H-2′, H-3′, H-4′); cis-DHQ δH (500 MHz): 4.2 (d, 1H, J 3.0 Hz, H-3), 5.3 (d, 1H, J 3.0 Hz, H-2), 5.8–6.0 (2ca d, 1H, J 2.0 Hz, H-6, H-8), 6.7–7.0 (m, 3H, H-2′, H-3′, H-4′). The Effect of Different Flavonoid Substrates on the Rate of ANS- and FLS-catalyzed Oxidative Decarboxylation of 2OG— Prior to the labeling studies we assessed the activities of ANS and FLS (both A. thaliana) in the presence of a variety of natural and unnatural substrates by measuring release of 14CO2 from 1-[14C]2OG 7 (Fig. 2 and Tables I and II). While it is known that prime substrate oxidation is not always fully coupled to that of 2OG 7 (4.Welford R.W.D. Schlemminger I. McNeill L.A. Hewitson
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