From the School of Pharmaceutical Sciences and the 21st Century COE Program, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan
Received for publication, March 31, 2003 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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Recent crystallographic and protein engineering studies on alfalfa (Medicago sativa) CHS, a homodimeric 42-kDa protein, revealed the active site machinery of the chalcone-forming reaction that proceeds through starter molecule loading at Cys-164, malonyl-CoA decarboxylation, and polyketide chain elongation, followed by cyclization and aromatization of the enzyme-bound tetraketide intermediate (Fig. 2) (1117). The catalytic center of CHS is composed of four amino acid residues: the catalytic triad of Cys-164, His-303, and Asn-336 and the "gatekeeper" Phe-215, absolutely conserved in all of the known type III PKSs. Interestingly, BAS is the only exception that lacks the active site Phe-215; the conserved residues 214LF are uniquely replaced by IL (numbering in M. sativa CHS) (Fig. 3) (5). In CHS, Phe-215, located at the junction between the active site cavity and the CoA binding tunnel, has been proposed to facilitate decarboxylation of malonyl-CoA and help orient substrates and intermediates during the sequential condensation reactions (Fig. 2) (12, 18). We proposed a hypothesis that the absence of Phe-215 in BAS may be the reason why the polyketide chain elongation is terminated at the diketide stage and followed by a second decarboxylation reaction to generate benzalacetone. In order to test the hypothesis, a mutant R. palmatum BAS in which the residues 214IL were substituted by LF was constructed. Furthermore, to investigate the structure-function relationship of the type III PKS enzymes, we also carried out functional analysis of a series of Phe-215 mutants of CHS from Scutellaria baicalensis (19).
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EXPERIMENTAL PROCEDURES |
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Site-directed MutagenesisR. palmatum BAS mutant (I214L/L215F) and S. baicalensis CHS mutants (L214I/F215L, F215W, F215Y, F215S, F215A, F215H, and F215C) were constructed using the QuikChange site-directed mutagenesis kit (Stratagene) and a pair of primers as follows (mutated codons are underlined): BAS I214L/L215F (5'-CATCTGGACTCCATGATAGGCCAAGCATTATTTGGCGATGGG-3' and 5'-AGCCCCATCGCCAAATAATGCTTGGCCTATCATGGAGTCCA-3'), CHS L214I/F215L (5'-TTGACAGCCTGGTTGGGCAGGCGATTTTAGGCGATGGCG-3' and 5'-CGCCATCGCCTAAAATCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215W (5'-TTGACAGCCTGGTTGGGCAGGCGCTGTGGGGCGATGGCG-3' and 5'-CGCCATCGCCCCACAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215Y (5'-TTGACAGCCTGGTTGGGCAGGCGCTGTATGGCGATGGCG-3' and 5'-CGCCATCGCCATACAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215S (5'-TTGACAGCCTGGTTGGGCAGGCGCTGTCAGGCGATGGCG-3' and 5'-CGCCATCGCCTGACAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215A (5'-TTGACAGCCTGGTTGGGCAGGCGCTGGCAGGCGATGGCG-3' and 5'-CGCCATCGCCTGCCAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215H (5'-TTGACAGCCTGGTTGGGCAGGCGCTGCATGGCGATGGCG-3' and 5'-CGCCATCGCCATGCAGCGCCTGCCCAACCAGGCTGTCAA-3'), CHS F215C (5'-TTGACAGCCTGGTTGGGCAGGCGCTGTGTGGCGATGGCG-3' and 5'-CGCCATCGCCACACAGCGCCTGCCCAACCAGGCTGTCAA-3').
Enzyme Expression and PurificationAfter confirmation of the sequence, the plasmid was transformed into E. coli BL21(DE3)pLysS. The cells harboring the plasmid were cultured to an A600 of 0.6 in LB medium containing 100 µg/ml ampicillin at 30 °C. Then 0.4 mM isopropyl-1-thio--D-galactopyranoside was added to induce protein expression, and the culture was incubated further at 30 °C for 16 h. The E. coli cells were harvested by centrifugation and resuspended in 50 mM potassium phosphate buffer, pH 8.0, containing 0.1 M NaCl. Cell lysis was carried out by the freeze-thaw method and centrifuged at 15,000 g for 60 min. The supernatant was passed through a column of Pro-BondTM resin (Invitrogen), which contained Ni2+ as an affinity ligand. After washing with 50 mM potassium phosphate buffer, pH 7.9, containing 0.5 M NaCl and 40 mM imidazole, the recombinant BAS was finally eluted with 15 mM potassium phosphate buffer, pH 7.5, containing 10% glycerol and 500 mM imidazole. Finally, the enzyme preparation was desalted by Bio-Gel P6DG desalting gel. Protein concentration was determined by the Bradford method (Protein Assay; Bio-Rad) with bovine serum albumin as a standard.
Enzyme ReactionThe standard reaction mixture contained 27 µM 4-coumaroyl-CoA, 54 µM malonyl-CoA, and 20 µg of the purified enzyme in a final volume of 500 µl of 100 mM potassium phosphate buffer, pH 8.0, containing 1 mM EDTA. Incubations were carried out at 30 °C for 20 min, and stopped by the addition of 50 µl of 20% HCl. By the acid treatment, naringenin chalcone is thus converted to racemic naringenin through a nonstereospecific ring-C closure. Furthermore, early released triketide and tetraketide intermediate are also converted to lactone derailment products, BNY and CTAL. The products were then extracted with 600 µl of ethyl acetate and concentrated by N2 flow. The residue was dissolved in an aliquot of MeOH containing 0.1% trifluoroacetic acid and separated by reverse-phase HPLC (JASCO 880) on a TSK-gel ODS-80Ts column (4.6 x 150 mm; TOSOH) with a flow rate of 0.8 ml/min. Gradient elution was performed with H2O and MeOH, both containing 0.1% trifluoroacetic acid: 05 min, 30% MeOH; 517 min, linear gradient from 30 to 60% MeOH; 1725 min, 60% MeOH; 2527 min, linear gradient from 60 to 70% MeOH. Elutions were monitored by a multichannel UV detector (MULTI 340, JASCO) at 290, 330, and 360 nm; UV spectra (198400 nm) were recorded every 0.4 s.
On-line LC-ESIMS spectra were measured with a Hewlett-Packard HPLC 1100 series (Wilmington, DE) coupled to a Finnigan MAT LCQ ion trap mass spectrometer (San Jose, CA) fitted with an electrospray ionization source. HPLC separations were carried out under the same conditions as described above. The electrospray ionization capillary temperature and capillary voltage were 275 °C and 3.0 V, respectively. The tube lens offset was set at 20.0 V. All spectra were obtained in both negative and positive mode over a mass range of m/z 120350, at a range of one scan every 2 s. The collision gas was helium, and the relative collision energy scale was set at 30.0% (1.5 eV). Enzyme reactions with nonphysiological substrates and analysis of their product were carried out as described before (19, 21, 22).
Enzyme KineticsSteady state kinetic parameters were determined by using [2-14C]malonyl-CoA (1.8 mCi/mmol) as a substrate. The experiments were carried out in triplicate using four concentrations of 4-coumaroyl-CoA (11.0, 5.5, 2.8, and 1.4 µM) in the assay mixture, containing 28 µM malonyl-CoA, 4.4 µg of purified enzyme, 1 mM EDTA, in a final volume of 500 µl of 100 mM potassium phosphate buffer. Incubations were carried out at 30 °C for 20 min. The reaction products were extracted and separated by HPLC, and the radioactivity in each fraction was determined by a liquid scintillation counter (Aloka LSC-3100). The kinetic parameters were calculated for formation of major product of the enzyme reactions at optimum pH, except for those of BAS I214L/L215F mutant, which was measured for formation of naringenin chalcone. Lineweaver-Burk plots of data were employed to derive the apparent Km and kcat values (average of triplicates ± S.D.) using EnzFitter software (BIOSOFT).
Three-dimensional Homology ModelingThe model of R. palmatum BAS was produced by the SWISS-MODEL package (available on the World Wide Web at expasy.ch/spdbv/) provided by the Swiss-PDB-Viewer program (23). A standard homology modeling procedure was applied based on the sequence homology of residues 5384 of R. palmatum BAS and the x-ray crystal structures of CHS superfamily enzymes (6368% identity) including M. sativa CHS (1BI5A.pdb, 1BQ6A.pdb, 1CGKA.pdb, 1CGZA.pdb, 1CHWA.pdb, 1CHWB.pdb, 1CMLA.pdb), M. sativa CHS C164A mutant (1D6FA.pdb), M. sativa CHS N336A mutant (1D6HA.pdb), M. sativa CHS H303Q mutant (1D6IA.pdb, 1D6IB.pdb), M. sativa CHS G256A mutant (1I86A.pdb), M. sativa CHS G256V mutant (1I88A.pdb, 1I88B.pdb), M. sativa CHS G256L mutant (1I89A.pdb, 1I89B.pdb), M. sativa CHS G256F mutant (1I8BA.pdb, 1I8BB.pdb), M. sativa CHS F215F mutant (1JWXA.pdb), and G. hybrida 2-PS (1EE0A.pdb, 1EE0B.pdb, 1QLVA.pdb, and 1QLVB.pdb). The corresponding Ramachandran plot was also created with Swiss PDB-Viewer software to confirm that the majority of residues grouped in the energetically allowed regions. Calculation of cavity volumes (Connolly's surface volumes) was then performed with the CASTP program (available on the World Wide Web at cast.engr.uic.edu/cast/) (24). The cavity volume of BAS was estimated to be 881 Å3, whereas that of M. sativa CHS (1CGKA.pdb) and G. hybrida 2-PS (1EE0A.pdb) were 1019 and 298 Å3, respectively.
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RESULTS |
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In the absence of a crystal structure of BAS, we constructed a homology model based on the sequence identity of residues 5384 of R. palmatum BAS with those of M. sativa CHS (11) and G. hybrida 2-PS (25), whose x-ray crystal structures were recently reported by Noel and co-workers. In the Ramachandran plot calculated for the model, most amino acid residues group in the energetically allowed regions with only few exceptions, primarily glycine residues that can adopt /
angles in all four quadrants. The homology model predicted that R. palmatum BAS has the same overall fold as M. sativa CHS (Fig. 4), with cavity volume estimated to be 881 Å3, which is almost as large as that of M. sativa CHS (1019 Å3) but much larger than that of G. hybrida 2-PS (298 Å3) (25). This suggested that the active site cavity of BAS is easily large enough to accommodate the tetraketide chalcone product.
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Enzyme Activity of BAS I214L/L215F MutantIn order to test the hypothesis that the substitution of the conserved 214LF residues by IL in R. palmatum BAS would account for the interruption of the chain extension at the diketide stage, a mutant enzyme in which the residues 214IL were replaced by LF was constructed. The BAS I214L/L215F mutant was heterologously overexpressed in E. coli as a recombinant protein with a His tag at the C terminus as in the case of wild-type BAS, and purified to homogeneity by a nickel-chelate column. As we had expected, BAS I214L/L215F mutant exhibited chalcone-forming activity and performed sequential condensations with three acetate units from malonyl-CoA to produce a tetraketide naringenin chalcone (6), although the activity was rather weak, and a triketide pyrone, BNY (4) was obtained as a major product (Fig. 5B). It was thus confirmed that the residues 214IL are indeed involved in the formation of a diketide benzalacetone and that the polyketide chain elongation was interrupted because of the absence of the active site Phe-215 in BAS.
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Interestingly, the chalcone-forming activity of the mutant enzyme was dependent on pH, which was maximum at pH 6.5, whereas at pH 8.0, most of the reactions were terminated at the triketide stage, leading to formation of BNY as a major product along with a trace amount of benzalacetone and CTAL (Fig. 6B). Enzyme kinetics analysis revealed that BAS I214L/L215F mutant showed Km = 33.5 µM and kcat = 0.169 min1 for 4-coumaroyl-CoA and Km = 46.6 µM and kcat = 0.181 min1 for malonyl-CoA (chalcone-forming activity at pH 6.5) (Table I), whereas wild-type BAS showed Km = 10.0 µM and kcat = 1.79 min1 for 4-coumaroyl-CoA and Km = 23.3 µM and kcat = 1.78 min1 for malonyl-CoA (benzalacetone-forming activity at pH 8.0). The mutant enzyme thus exhibited 36-fold decreases in kcat/Km for 4-coumaroyl-CoA and 20-fold decreases in kcat/Km for malonyl-CoA compared with wild-type BAS.
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The observed pH dependence of the activity of the mutant enzyme prompted us to reinvestigate the pH profile of the enzyme activity of wild-type BAS. Consistent with our previous report (5), it was reconfirmed that the formation of benzalacetone was maximum within a range of pH 8.08.5; however, interestingly, there was a dramatic change in the product profile under acidic pH. Instead of a diketide benzalacetone, formation of a triketide pyrone was dominant, and BNY was obtained almost as a single product at pH 6.0 (Fig. 6A). This indicated that both wild-type and I214L/L215F mutant BAS performed increased numbers of condensation reactions under acidic conditions.
Enzyme Activity of CHS L214I/F215L MutantIn order to further investigate the structure-function relationship between BAS and CHS enzyme, the conserved active site residues 214LF of S. baicalensis CHS were in turn substituted by IL as in the case of BAS. As a result, the chalcone-forming activity of CHS L214I/F215L mutant was significantly reduced, and BNY was obtained as a major product (Fig. 5D), indicating that most of the chain elongation reactions were terminated at the triketide stage. The BNY forming activity was maximum at pH 7.0, whereas production of tetraketides, chalcone and CTAL, was prominent under acidic conditions within a range of pH 6.06.5 (Fig. 6D). In contrast, wild-type S. baicalensis CHS shows maximum chalcone-forming activity at pH 8.0 (Fig. 6C). Interestingly, both wild-type and L214I/F215L mutant CHS did not produce benzalacetone derived from the diketide intermediate, which was confirmed by liquid chromatography-mass spectrometry analysis.
CHS L214I/F215L mutant showed Km = 23.5 µM and kcat = 0.056 min1 for 4-coumaroyl-CoA and showed Km = 125 µM and kcat = 0.054 min1 for malonyl-CoA (BNY-forming activity at pH 7.0) (Table I), whereas wild-type S. baicalensis CHS showed Km = 5.8 µM and kcat = 0.824 min1 for 4-coumaroyl-CoA and showed Km = 25.5 µM and kcat = 0.862 min1 for malonyl-CoA (chalcone-forming activity at pH 8.0). The mutant enzyme thus exhibited 60-fold decreases in kcat/Km for 4-coumaroyl-CoA and 77-fold decreases in kcat/Km for malonyl-CoA.
Substrate Specificities of BAS I214L/L215F and CHS L214I/F215L MutantIn previous studies, we have demonstrated that S. baicalensis CHS has remarkably broad substrate specificities toward starter and extension substrate of the polyketide formation reaction (19, 21, 22, 26, 27). Thus, instead of 4-coumaroyl-CoA, the enzyme accepted a variety of starter molecules, both aromatic and aliphatic CoA esters of different chain length including benzoyl-CoA (7) and hexanoyl-CoA (8), and efficiently converted to a series of chemically and structurally different unnatural polyketides (Fig. 7C). In contrast, R. palmatum BAS showed rather narrow substrate specificities and did not accept aliphatic CoA esters such as hexanoyl-CoA and acetyl-CoA, whereas benzoyl-CoA was converted to a triketide pyrone (9) (Fig. 7A) (5).
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To further investigate the effect of the mutations, the enzyme activities of BAS I214L/L215F and CHS L214I/F215L mutant were tested with the less bulky starter analogs. Benzoyl-CoA (7) as a substrate, BAS I214L/L215F, afforded a triketide and a tetraketide pyrone (9, 10), whereas hexanoyl-CoA, now accepted as a substrate, was converted to a triketide pyrone (12) (Fig. 7B). The mutation thus resulted in increased number of condensations compared with wild-type BAS. On the other hand, the CHS L214I/F215L mutant, which also afforded BNY as a major product, exhibited a similar product profile, which resulted from the decreased number of chain elongations compared with wild-type CHS (Fig. 7D). Interestingly, in either case, formation of a benzalacetone-type diketide was not detected in the assay mixture.
Enzyme Activity of CHS Phe-215 Point MutantsIn addition to CHS L214I/F215L mutant, six more Phe-215 mutants of S. baicalensis CHS (F215W, F215Y, F215S, F215A, F215H, and F215C) were constructed and tested for the enzyme activities. Previous studies by Noel and co-workers (12) showed that M. sativa CHS F215W, F215Y, and F215S mutations resulted in formation of increased levels of truncated products and that the F215S mutant preferentially accepted nonphysiological N-methylanthraniloyl-CoA as a starter to produce an unnatural tetraketide pyrone (18).
In S. baicalensis CHS, the point mutations significantly reduced chalcone-forming activities and altered functional behavior of the enzyme to various degrees (Fig. 8). Each of the Phe-215 mutants exhibited 9209-fold decreases in kcat/Km for 4-coumaroyl-CoA and 72815-fold decreases in kcat/Km for malonyl-CoA compared with wild-type CHS (Table I). Consistent with the previous report, most of the S. baicalensis CHS mutants afforded increased levels of BNY derived from a triketide intermediate as in the case of the above mentioned L215I/F215L mutant (Fig. 8). Notably, F215H, F215S, and F215H mutant still retained good catalytic activities and yielded BNY as a major product. On the other hand, it was remarkable that the F215Y mutation, only a small modification of the active site by an additional hydroxyl group, resulted in almost complete loss of the enzyme activity. In contrast, M. sativa CHS F215Y mutant still retained chalcone-forming activity, suggesting subtle species difference of the active site structure between S. baicalensis and M. sativa (12). Most interestingly, none of the mutants produced benzalacetone, which indicated that additional subtle structural differences exist between the active site of BAS and CHS besides the residues 214LF.
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DISCUSSION |
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The CHS-based homology model of R. palmatum BAS predicted that BAS has the same overall fold as M. sativa CHS with cavity volume (881 Å3) almost as large as that of M. sativa CHS (1019 Å3) but much larger than that of G. hybrida 2-PS (298 Å3). This suggested that the active site of BAS itself is easily large enough to perform the sequential condensations and to accommodate the tetraketide chalcone product. It has been reported that the shape and volume of the active site cavity greatly influence the substrate specificity and control the final length of the polyketide (25).
In M. sativa CHS, the conformationally flexible "gatekeeper" Phe-215, located at the active site entrance, is thought to facilitate decarboxylation of malonyl-CoA and help orient substrates and intermediates during the sequential condensation reactions, thus controlling the number of the elongation steps (12, 18). Indeed, our site-directed mutagenesis of S. baicalensis CHS (L214I/F215L, F215W, F215Y, F215S, F215A, F215H, and F215C) significantly reduced chalcone-forming activities and generally afforded increased levels of BNY derived from a triketide intermediate (Fig. 8), which was agreed well with the earlier report on M. sativa CHS mutants (F215S, F215Y, and F215W) by Noel and co-workers (12).
Interestingly, none of the S. baicalensis CHS Phe-215 mutants produced benzalacetone, indicating that there are additional subtle structural differences between the active site of BAS and CHS besides the residues 214LF. Further modification of the active site was thus required for functional conversion of CHS to BAS. Point mutations such as G149D, V210I, T264S, and S332K (numbering in M. sativa CHS) may be good candidates for the termination of the elongation reaction and decarboxylation of the corresponding diketide intermediate, since these residues that are well conserved in plant CHS superfamily enzymes and located at or close to the active site are uniquely absent in R. palmatum BAS (Fig. 3). Furthermore, the T197C mutation would be also interesting because of the reasons described below (2).
The primary driving force for decarboxylation reaction is thought to be the formation of carbondioxide (an entropic effect) and a stabilized carbanion. Decarboxylation is thus facilitated by initial protonation of the -carbonyl group in
-keto acid and by stabilization of the resulting anion, as proposed in the malonyl-CoA decarboxylation (Fig. 2). Alternatively, enzymatic decarboxylation may also involve a Schiff base formation between the
-carbonyl and active site basic residue, which permits facile protonation of the imine (29). However, there appear to be no appropriate basic residues uniquely present in the active site of BAS for the decarboxylation of a diketide intermediate to generate benzalacetone. On the other hand, Austin and Noel (2) proposed a possibility that BAS utilizes a second active site cysteine for the decarboxylation reaction based on their observation that Thr-197 of CHS sterically altered in a number of divergent type III PKSs is replaced by a reactive cysteine in BAS. As mentioned above, T197C mutation would be thus very interesting; however, in the absence of the crystal structure of BAS, the precise geometry of the active site of the enzyme still remains to be unknown.
Alternatively, the decarboxylation of a diketide intermediate may be also catalyzed by His-303 and Asn-336, the same active site residues involved in the malonyl-CoA decarboxylation by forming an "oxyanion hole" that accommodates the negatively charged transition state (Fig. 2D). In the absence of the aromatic Phe-215, polarizing the -carbonyl group of 4-coumaroyl acetic acid (or its equivalent) derived from a diketide intermediate would similarly facilitate a second decarboxylation reaction, thus leading to formation of benzalacetone. This hypothesis may be supported by our observation that S. baicalensis CHS (as well as A. hypogaea STS) also catalyzed an unusual decarboxylation reaction of a diketide intermediate to generate a benzalacetone-type compound as a major product when incubated with 4-coumaroyl-CoA and a nonphysiological extender methylmalonyl-CoA (26). Interestingly, 4-coumaroylacetic acid, the diketide without decarboxylation process, has never been detected in the assay mixture.
The pH dependence of the polyketide chain elongations was remarkable. Except for wild-type CHS that showed maximum chalcone-forming activity at pH 8.0, most of the BAS and CHS Phe-215 mutants efficiently catalyzed an increased number of chain extensions under acidic conditions. Most notably, wild-type R. palmatum BAS exhibited a dramatic change from benzalacetone to BNY production at acidic pH. The pH change thus modulated between formation of a decarboxylated diketide product and a triketide pyrone, but interestingly products derived from decarboxylation of a triketide intermediate have never been detected. Presumably, the pH change significantly affected charge distribution in the enzyme-substrate and enzyme-intermediate complex, including the most important charged interactions of the catalytic triad of Cys-164, His-303, and Asn-336, thus greatly influenced the chain elongation reactions. The pH dependence of malonyl-CoA decarboxylation reaction of wild-type M. sativa CHS has been reported to be optimum under acidic condition (13); however, rate-limiting step of the polyketide formation reaction in each enzyme remains to be elucidated.
In summary, we have demonstrated mechanistic consequences of substitution of the crucial active site residue involved in the polyketide chain elongation reaction and provided a structural basis for understanding the structure-function relationship between BAS and CHS enzyme. Further comparative analyses of functionally divergent type III PKS enzymes promise to reveal intimate structural details of the enzyme-catalyzed processes and suggest strategies for manipulating substrate and product specificities of the polyketide formation reactions.
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FOOTNOTES |
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To whom correspondence should be addressed: University of Shizuoka, School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka 422-8526, Japan. Tel./Fax: 81-54-264-5662; E-mail: abei{at}ys7.u-shizuoka-ken.ac.jp.
1 The abbreviations used are: CHS, chalcone synthase (EC 2.3.1.74
[EC]
); BAS, benzalacetone synthase (EC 2.3.1.-); PKS, polyketide synthase; STS, stilbene synthase; 2-PS, 2-pyrone synthase; CoA, coenzyme A; BNY, bisnoryangonin; CTAL, 4-coumaroyltriacetic acid lactone; HPLC, high pressure liquid chromatography.
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REFERENCES |
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