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First published online March 18, 2005; 10.1104/pp.104.054502 Plant Physiology 137:1375-1388 (2005) © 2005 American Society of Plant Biologists Partial Reconstruction of Flavonoid and Isoflavonoid Biosynthesis in Yeast Using Soybean Type I and Type II Chalcone Isomerases1,[w]Donald Danforth Plant Science Center, St. Louis, Missouri 63132
Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. These compounds play important roles in many essential physiological processes. In addition, flavonoids and isoflavonoids have direct but complex effects on human health, ranging from reducing cholesterol levels and preventing certain cancers to improving women's health. In this study, we cloned and functionally characterized five soybean (Glycine max) chalcone isomerases (CHIs), key enzymes in the phenylpropanoid pathway that produces flavonoids and isoflavonoids. Gene expression and kinetics analysis suggest that the soybean type I CHI, which uses naringenin chalcone as substrate, is coordinately regulated with other flavonoid-specific genes, while the type II CHIs, which use a variety of chalcone substrates, are coordinately regulated with an isoflavonoid-specific gene and specifically activated by nodulation signals. Furthermore, we found that some of the newly identified soybean CHIs do not require the 4'-hydroxy moiety on the substrate for high enzyme activity. We then engineered yeast (Saccharomyces cerevisiae) to produce flavonoid and isoflavonoid compounds. When one of the type II CHIs was coexpressed with an isoflavone synthase, the enzyme catalyzing the first committed step of isoflavonoid biosynthesis, various chalcone substrates added to the culture media were converted to an assortment of isoflavanones and isoflavones. We also reconstructed the flavonoid pathway by coexpressing CHI with either flavanone 3  -hydroxylase or flavone synthase II. The in vivo reconstruction of the flavonoid and isoflavonoid pathways in yeast provides a unique platform to study enzyme interactions and metabolic flux.
Flavonoids and isoflavonoids are major plant secondary metabolites that mediate diverse biological functions and exert significant ecological impacts. Approximately 20% of the carbon fixed by photosynthesis is believed to be channeled into the phenylpropanoid pathway, generating the majority of the phenolic compounds found in nature, including flavonoids and isoflavonoids (Weisshaar and Jenkins, 1998  ). These compounds play important roles in many essential physiological processes. For example, lignins are major cell wall components, flavonols are UV protectants, and anthocyanins are the primary attractants for pollinators in many species (Dixon and Steele, 1999). In addition, flavonoids and isoflavonoids have direct but complex effects on human health. For example, resveratrol found in red wine has been shown to double the life span of yeast (Saccharomyces cerevisiae; Howitz et al., 2003), the flavonols and tannins found in green tea can drastically reduce the likelihood of stroke and heart disease, and the isoflavones produced by soybean (Glycine max) can prevent many hormone-dependent cancers and improve women's health (Beecher, 2003). Despite being one of the most studied plant secondary-metabolic pathways, surprising new functions and characteristics of these compounds are still being discovered.
The general phenylpropanoid pathway is conserved in all plant species. In this core pathway, the amino acid Phe is converted into p-coumaroyl-CoA in three enzymatic steps. In most species, chalcone synthase (CHS; EC 2.3.1.74) then condenses p-coumaroyl-CoA with 3 molecules of malonyl-CoA to form 4, 2', 4', 6'-tetrahydroxychalcone (naringenin chalcone). As shown in Figure 1, naringenin chalcone can be further metabolized to (2S)-5, 7, 4'-trihydroxyflavanone (naringenin) by chalcone isomerase (CHI; EC 5.5.1.6) to form the primary C15 flavonoid skeleton. Recently, Shimada et al. (2003)
During plant-microbe interactions, many of the genes encoding the pathway enzymes are coordinately regulated at the transcriptional level, and a number of the transcription factors that bind to the conserved cis-elements of flavonoid pathway genes have been identified (Ni et al., 1996 ; Weisshaar and Jenkins, 1998). In addition, several enzymes of the phenylpropanoid pathway were shown to form protein complexes that efficiently direct metabolic flux toward the production of a particular compound (Dixon and Steele, 1999; Ovadi and Srere, 2000; Achnine et al., 2004). For example, Arabidopsis (Arabidopsis thaliana) CHS, CHI, and dihydroflavonol 4-reductase may interact during flavonoid biosynthesis (Winkel-Shirley, 1999). Understanding the partitioning of metabolites between the flavonoid and isoflavonoid pathways is essential to uncover and utilize the mechanisms of pathway regulation. For instance, to engineer the production of economically important isoflavones in nonlegume plants, the regulation of naringenin accumulation is critical, because heterologously expressed IFS must directly compete with endogenous enzymes for this substrate (Yu et al., 2000; Liu et al., 2002; Yu and McGonigle, 2005). We undertook this study to establish a system for the in vivo functional analysis of metabolic enzymes. Initially, we cloned and characterized the soybean CHIs responsible for naringenin and liquiritigenin production. We found that the type II CHIs were coordinately regulated developmentally and during plant-microbe interactions with other isoflavonoid biosynthetic enzymes, whereas the type I CHI was coordinately regulated with other flavonoid pathway enzymes. When we expressed the CHIs in yeast and added chalcones to the culture media, the chalcones were taken up by the yeast and rapidly converted to flavanones. Moreover, when the CHIs were coexpressed with IFS in yeast, chalcone substrates were converted to flavanone and then to isoflavones as a result of the coordinate activity of both enzymes. Coexpression of CHI with F3H or FNSII resulted in the production of dihydroflavonols or flavones, respectively, from chalcones. Novel compounds were also produced through the addition of a number of chalcones not common to most plants. This in vivo assay system thus provides a unique platform for the functional analysis of interactions between metabolic enzymes, particularly for the membrane-bound Cyt P450s.
Identification of CHI Expressed Sequence Tags in Soybean
Seven different CHI tentative unique contigs were identified in soybean using the Plant Genome Database (Dong et al., 2004
The deduced amino acid sequences of CHI1A, CHI1B2, CHI2, CHI3, and CHI4A were aligned to MsaCHI1 using the ClustalW algorithm (Fig. 2). Of the contigs identified, CHI1A was most homologous to MsaCHI1 with 79.3% identity. MsaCHI1 was 62.8% identical to CHI1B2, 46.9% identical to CHI2, 27.4% identical to CHI3, and 19.3% identical to CHI4. Twenty residues were absolutely conserved between MsaCHI1 and the soybean CHIs (Fig. 2). Surprisingly, none of these residues were active site residues. Residues lining the active site of the MsaCHI1 crystal structure (Jez et al., 2000
CHI3 is predicted to be between 55 and 72 residues longer at the N terminus than the other CHIs identified in this study (Fig. 2). Four Web-based programs, ChloroP, iPSORT, Predotar, and TargetP, were used to ascertain whether the elongated N terminus of CHI3 encodes a signal peptide that might control subcellular targeting. The nuclear-encoded, plastid-targeted soybean ribulose 1,5-bisphosphate carboxylase small subunit was included for comparison. All four programs predicted both CHI3 and ribulose 1,5-bisphosphate carboxylase small subunit to be targeted to the chloroplast (see Supplemental Table I).
Comparison of the putative soybean CHI with previously characterized CHIs and CHI orthologs from other species shows that these sequences fell into four different subfamilies (Fig. 3). CHIs in the first subfamily (CHI1) were found only in legumes and were at least 70% similar to MsaCHI1. Some members of this subfamily have previously been demonstrated to metabolize both naringenin chalcone, which is found in all plants, and isoliquiritigenin, which is found primarily in legumes (Dewick, 1993
Members of the CHI3 and CHI4 subfamilies also occur in a broad cross section of plant species (Fig. 3). These appear to be distantly related orthologs, containing only a subset of motifs that are conserved in the first two CHI subfamilies. None of these orthologs have been characterized biochemically; most were assembled from the Plant Genome Database based upon homology to MsaCHI1. Interestingly, all members of the CHI3 subfamily contain putative chloroplast targeting peptides.
To understand the physiological role of each CHI subfamily in flavonoid and isoflavonoid biosynthesis, the transcripts of CHI1A, CHI1B2, CHI2, CHI3, and CHI4A were analyzed in soybean and compared to those of IFS (AF195789) and F3H genes (see below). To study the transcriptional regulation of these genes, total RNA was extracted from the roots, shoots, flowers, and seeds of soybean and quantitative reverse transcription (RT)-PCR was performed (Fig. 4). Transcript levels were normalized against those of soybean ubiquitin (D28123) in each tissue. The IFS gene showed root-specific expression with approximately 32-fold higher transcript levels in roots than in shoots. IFS1 was much less expressed in flowers, with an expression level less than 0.1% of that in the root (Fig. 4A). IFS expression levels in seeds were comparable to those in the roots. These data agree with our earlier observations (Subramanian et al., 2004
The type I CHI, CHI2, showed high expression levels in the flowers. CHI2 exhibited 5-fold higher expression in flowers compared to shoots or roots (Fig. 4A). F3H showed higher expression level in flowers as well, with more than 11-fold higher expression than in the shoots (Fig. 4A). Both these genes showed highest expression in the seeds. Since F3H is an essential component of the anthocyanin pathway, the high expression of the gene in flowers is not surprising. The coordinate expression of CHI2 and F3H suggested that the type I CHI in soybean is indeed involved in flavonoid biosynthesis. However, F3H and CHI2 exhibited different expression patterns in the roots. CHI2 showed about 2-fold stronger expression in the roots than in the shoots, whereas F3H expression in the roots was barely detectable, suggesting that CHI2 may serve additional roles in the roots. Neither CHI3 nor CHI4A were expressed at significant levels in any plant tissues, suggesting these 2 CHIs might not play important roles in the phenylpropanoid pathway in soybean under normal conditions (data not shown).
To further delineate the function of different soybean CHIs, their expression in response to symbiotic bacteria (Bradyrhizobium japonicum) was measured by quantitative RT-PCR. The steady-state transcript levels of type II CHI1A and CHI1B2 increased significantly in roots 8 h after the inoculation (90- and 60-fold, respectively). Similar induction was observed for the IFS gene as well, with IFS transcript levels increasing 10-fold in the roots (Fig. 4B). A slight increase was observed for all three genes in the Bradyrhizobium-treated shoot tissue, which may reflect a general defense response during plant-microbe interactions. After Bradyrhizobium treatment, CHI1A and CHI1B2 expression levels in the shoots increased by 3-fold, and IFS1 expression increased by 2-fold. As a consequence, the ratios of shoot to root transcript levels were altered; without Bradyrhizobium treatment, the CHI1A had about 8-fold higher expression in the roots than in the shoots, and CHI1B2 is up to 7-fold higher. After the treatment, the ratio became 140-fold and 61-fold higher, respectively. In contrast, Bradyrhizobium did not induce the type I CHI2 in either roots or shoots, although F3H expression increased significantly in the shoots (Fig. 4B). The results suggest that during the early nodulation response, the type II CHI was highly induced while the type I was not. The IFS expression in response to Bradyrhizobium was similar to our earlier observations (Subramanian et al., 2004
The catalytic properties of representatives from each soybean CHI subfamily were assessed. The full-length coding sequences of CHI1A, CHI1B2, CHI2, and CHI4A were cloned into a bacterial expression vector and fused in-frame to a His8 epitope tag. CHI3 was truncated at its N terminus to remove its putative chloroplast targeting sequence, yielding CHI3
The steady-state kinetics data obtained for CHI1A, CHI1B2, and CHI2 is presented in Table I. Based on the comparison of kcat/Km values, the CHIs exhibited a marked preference for naringenin chalcone over the other chalcones. This is likely due to the functional equivalence of the 2'- and 6'-hydroxyl groups in the isomerization of the chalcone to the flavanone. Of these 3 enzymes, CHI2 most efficiently catalyzed the conversion of naringenin chalcone to naringenin, with a catalytic efficiency more than 6 times greater than CHI1B2 and almost 20 times greater than that of CHI1A (Table I). As mentioned above, CHI2 does not metabolize any of the remaining chalcones in vitro. CHI1A and CHI1B2 metabolize 6 of the remaining chalcones, exhibiting different catalytic efficiencies for each. CHI1A most efficiently metabolizes Ch3, followed by Ch1, Ch9, Ch8, Ch5, and Ch4, whereas CHI1B2 most efficiently metabolizes Ch5 and Ch8, followed by Ch1, Ch9, Ch4, and Ch3. Besides naringenin chalcone, isoliquiritigenin (Ch8) is the other naturally occurring CHI substrate in legumes. As such, the expectation would be that Ch8 would be isomerized more efficiently than the other chalcones. This is the case with CHI1B2, but not with CHI1A. CHI1B2 metabolizes Ch8 and Ch5 with almost equal efficiency. The only difference between these 2 chalcones is that Ch8 contains a hydroxyl group at C4, whereas Ch5 contains a methoxyl group at this position. Compared to earlier reports, CHI1B2 and MsaCHI have similar substrate preferences. Surprisingly, CHI1A preferred 4'-deoxychalcones (Ch3, Ch1, and Ch9) over 4'-hydroxychalcones (Ch8, Ch4, and Ch5). The 4'-hydroxyl residue has been postulated to hydrogen bond with Asn-113 and Thr-190 of MsaCHI to stabilize the substrate during catalysis. Although these 2 amino acids are conserved in all soybean type II CHIs, the hydroxyl group at C4' is not required to ensure high enzymatic activity.
As the first step in building the flavonoid/isoflavonoid pathway in yeast, we tested whether chalcones could be metabolized to flavanones in vivo using soybean CHIs. The full-length ORFs of CHI1A, CHI1B2, CHI2, and CHI4 and the truncated CHI3
Yeast transformed by CHI1A and CHI1B2 metabolized naringenin chalcone to naringenin (Fig. 6, B and C) and isoliquiritigenin to liquiritigenin (Fig. 6, F and G). CHI2 was able to metabolize naringenin chalcone to naringenin more efficiently than CHI1A or CHI1B2 (Fig. 6D), but it converted only a small amount of isoliquiritigenin to liquiritigenin in 12-h assays (Fig. 6H). Even the metabolism of this small amount of chalcone was unexpected, since the purified CHI2 was unable to metabolize isoliquiritigenin in vitro (see above kinetics data). The control yeast carrying the empty vector did not show any liquiritigenin accumulation, suggesting the automatic conversion of isoliquiritigenin did not contribute to liquiritigenin accumulation (Fig. 6E). This low level of conversion may have been the noncatalytic active-site conversion by the CHI (Bednar and Hadcock, 1988
When the other chalcones listed in Figure 5 were added to yeast cultures, CHI1A and CHI1B2 converted all chalcones to their corresponding flavanones, including Ch2, Ch6, and Ch7, which we did not observe in vitro activity (see Supplemental Fig. 1 for CHI1B2 results). The products were confirmed by HPLC based on retention time and UV spectra, and by GC-MS analysis as reported in supplemental materials (Supplemental Table III, except for Ch1 derived compounds, which cannot be derivatized by trimethylsilyl [TMS]). For CHI2, only isoliquiritigenin and naringenin chalcone were converted as shown above (Fig. 6). The isomerization of the unexpected chalcones may reflect a low CHI activity toward these compounds that cannot be detected in the 5-min in vitro assays.
We further tested the individual in vivo activity of IFS, FNSII, and F3H in yeast. All three enzymes use naringenin as a substrate. For isoflavonoid synthesis, the soybean IFS was cloned into pYeDP60 expression vector, under the control of a Gal-inducible promoter and transformed into the WAT11 yeast strain. The WAT11 strain contains an Arabidopsis Cyt P450: NADPH reductase (Pompon et al., 1996
We sought to demonstrate 2-hydroxyisoflavanones converts to isoflavones spontaneously in yeast. Microsomes were extracted from IFS-containing yeast. The in vitro IFS reaction produced 2-hydroxyisoflavanones and isoflavones. The addition of the specific Cyt P450 inhibitor ancymidol prevented further production of 2-hydroxyisoflavanone, but the dehydration to isoflavone from existing 2-hydroxyisoflavanone continues (Table II). When microsomes or soluble proteins extracted from wild-type yeast were added to the 2-hydroxyisoflavanone, the rate of conversion was not altered compared to solvent controls, supporting the nonenzymatic mechanism of this conversion (Table II).
For in vivo flavone synthesis, a Gerbera hybrida FNSII gene previously cloned into a yeast expression vector under a Gal-inducible promoter (Martens and Forkmann, 1999 ) was transformed into WAT11 cells. As observed by HPLC, FNSII metabolizes externally added naringenin and liquiritigenin to the flavones apigenin and 7,4'-dihydroxyflavone, respectively (data not shown; see below).
In addition, 1 complete soybean F3H contig, including ORF and 5'- and 3'-UTRs, was identified from the Plant Genome Database, based on its 83.0% similarity to the alfalfa F3H (X78994; Charrier et al., 1995
Having demonstrated the abilities of CHIs and other key flavonoid and isoflavonoid biosynthetic enzymes to metabolize chalcones and flavanones in vitro and in vivo, we tried to reconstitute the initiation points of the isoflavonoid and flavonoid pathways in vivo. When the chalcone substrates were added to the culture media of yeast cotransformed with IFS and CHI1A, the chalcones were first converted to flavanones by CHI and subsequently converted to 2-hydroxyisoflavanones and isoflavones by IFS. For example, when isoliquiritigenin was added, the 2, 7, 4'-trihydroxyisoflavanone product was readily detectable after 12 h (Fig. 8C). The 3, 7, 4'-trihydroxyflavanone side product was also detected (Sawada et al., 2002
The Gerbera FNSII or soybean F3H gene was independently cotransformed with CHI1A into yeast. In these yeasts, flavanones produced by CHI were converted to flavones by FNSII or to dihydroflavonols by F3H. For example, when yeast cotransformed with CHI1A and FNSII was fed isoliquiritigenin and naringenin chalcone, they produced 7, 4'-dihydroxyflavone (Fig. 8D) and apigenin (Fig. 8I), respectively. Similarly, when the same substrates were fed to the yeast cotransformed with CHI1A and F3H, 3, 7, 4'-trihydroxyflavonol (Fig. 8E) and dihydrokaempferol (Fig. 8J) were detected by HPLC and confirmed by GC-MS (Supplemental Table III).
Classification and Regulation of CHI
CHI catalyzes the stereospecific isomerization of chalcones into corresponding (2S)-flavanones. The in vitro enzyme kinetic assays indicate that CHI operates at the upper limit of the turnover rate for all enzymes, i.e. approaching the diffusion limit (Jez et al., 2000
CHIs have been divided into two classes based on substrate specificity, and their distribution is highly family specific. Type I CHIs are found in both legumes and nonlegumes and isomerize only naringenin chalcone to naringenin. Type II CHIs are found exclusively in leguminous plants and have activities toward both naringenin chalcone and isoliquiritigenin, yielding naringenin and liquiritigenin, respectively. Genes encoding both types of CHIs have been cloned from various plant species (Kimura et al., 2001
Type I and II CHIs have different expression patterns. In L. japonicus, type I and II CHIs are differentially regulated after fungal elicitor treatment (Shimada et al., 2003
Interestingly, not only type I and II CHIs seem to be conserved in M. truncatula; the homologs of soybean CHI3 and CHI4 were also discovered, suggesting physiological roles for these nonenzymatic CHIs may exist (Supplemental Fig. 4). Recent evidence suggests that some CHIs may have functions other than those of catalysts. Mutant maize CHIs with only 3% to 5% of wild-type CHI activity are able to complement the Arabidopsis tt5 mutant (Dong et al., 2001
In all higher plants, isomerization of naringenin chalcone into naringenin by CHI occurs rapidly. However, the conversion of isoliquiritigenin to liquiritigenin, has relatively slower kinetics, because of differences in hydrogen bonding between the active site of CHI and the substrate (Jez et al., 2002
Functional reconstruction of the pathways in vivo has been attempted in several heterologous systems with different pathways. For example, Phe ammonia lyase and C4H were functionally coexpressed in yeast recently and worked in concert to produce p-coumarate from endogenous Trp pools (Ro and Douglas, 2004 This in vivo system opens numerous possibilities for interesting biochemical studies. First, protein-protein interactions between pathway enzymes can be easily studied in yeast. Currently, we have coexpressed CHI, IFS, and FNSII/F3H to reconstitute the branching of phenylpropanoid pathway in yeast. This in vivo system allows us to easily measure and characterize the products of each enzyme. Thus, the flux of each pathway can be quantified, without interference from other flavonoid/isoflavonoid pathway enzymes. Second, the ease of generating and screening large numbers of yeast lines provides an opportunity to conduct mutagenesis of membrane-bound enzymes for structure-function analyses, or protein engineering. Third, since the consumption of flavonoid and isoflavonoid compounds is often associated with health benefits, introducing the biosynthetic capacity of these compounds into yeast may have direct economic impacts. It would allow for the production of specific flavonoid/isoflavonoid compounds in large scale. Additionally, producing novel flavonoids during fermentation may have many industrial applications, including the biosynthesis of novel pharmaceuticals. To demonstrate this point, yeast cotransformed with type II CHI1B2 and IFS were fed with each of the 10 chalcones mentioned above. CHI1B2 was found to convert many uncommon chalcones into their corresponding flavanones, thus providing uncommon substrates for IFS. In addition to Ch8 (isoliquiritigenin) and Ch10 (naringenin chalcone), the yeast produced isoflavonoids from all remaining chalcones (see Supplemental Fig. 1). This is the first time that IFS was shown to metabolize substrates other than isoliquiritigenin or naringenin chalcone. These results were subsequently confirmed by in vitro microsome assays (data not shown). Interestingly, the GC-MS data indicated that both 2-hydroxyisoflavanone and isoflavone products could be detected when Ch2, Ch6, Ch7, and Ch9 were the substrates. For the remaining Ch3, Ch4, and Ch5 reactions, only 2-hydroxyisoflavanones were detected (Supplemental Table III). It suggests that the dehydration of 2-hydroxyisoflavanones requires at least one hydroxyl group on both A and B rings of isoflavanones. Furthermore, a hydroxyl group at the C4' or C5' of chalcones appears to be important for IFS activity. Without any hydroxyl groups at these positions, Ch1 and Ch2 could not be efficiently converted to the corresponding isoflavonoids. Since the mechanism of IFS reaction is not clear, further studies are needed to illuminate the possible mechanism of this conversion in yeast. This yeast system also provided an opportunity to study the substrate specificity of FNSII and F3H. When the 10 chalcones were fed to the yeast cotransformed with CHI1B2 and FNSII, Ch1, Ch3, Ch4, Ch5, Ch8, and Ch10 were converted to their respective flavone compounds (see Supplemental Fig. 2). In the yeast cotransformed with CHI1B2 and F3H, only Ch3, Ch8, Ch9, and Ch10 were metabolized by the F3H (see Supplemental Fig. 3). In these assays, FNSII had a broader substrate spectrum than F3H. In conclusion, we have identified an entire family of CHIs in soybean. The kinetics and expression profiles suggest that the type I CHI is associated with flavonoid biosynthesis, while the type II CHIs are closely associated with isoflavonoid biosynthesis. For some CHIs, the 4'-hydoxyl residues of chalcones may not be essential for high enzyme activities. Functional reconstruction of flavonoid and isoflavonoid biosynthesis from a shared chalcone precursor proved successful with various enzyme combinations. The broad substrate specificity of soybean type II CHIs resulted in the production of several flavonoid/isoflavonoid compounds in yeast. This in vivo system provides multiple opportunities for research and industries.
EST Sequence Analysis and Cloning of cDNAs
CHI and F3H cDNA sequences were identified from the Plant Genome Database (http://www.plantgdb.org/; Dong et al., 2004
CHI1A and CHI4A were cloned previously (O. Yu and J.T. Odell, unpublished data). ESTs containing CHI1B2 (BQ786323), CHI2 (AW733840), CHI3 (BM885445), and F3H (BI894148) were obtained from the Washington University School of Medicine in St. Louis. All of these clones were sequenced to verify that they contained complete ORFs as well as 5'- and 3'-UTRs. Appropriate restriction sites for subsequent cloning steps were introduced immediately upstream of the ATG start codon and downstream of the stop codon via PCR, with the exception of CHI3. The 66 amino acid N-terminal extension was removed from CHI3 by PCR to create CHI3
RNA isolation and quantitative RT-PCR analysis were performed as described in Subramanian et al. (2004)
CHI1A, CHI1B2, CHI2, CHI3
Chalcones, flavonoids, and isoflavonoids were purchased from Indofine Chemical, Hillsborough, NJ. Naringenin chalcone was chemically synthesized from naringenin (Shimokoriyama, 1957
The in vitro IFS activity assay was carried out as described previously using microsomal extracts (Jung et al., 2000
For in vitro F3H activity assays, the F3H ORF was fused to an N-terminal FLAG tag and expressed using the in vitro Director PCR System (Sigma-Aldrich, St. Louis). The fusion protein was transcribed and translated in vitro and purified according to the manufacturer's protocol. Enzyme activity assays were carried out as described previously (Charrier et al., 1995
CHI1A, CHI1B2, CHI2, CHI3
HPLC samples were analyzed on a System Gold HPLC equipped with a photodiode-array detector (Beckman Coulter, Fullerton, CA), using a Luna C18 (2), 5-µm, 4.6- x 150-mm column (Phenomenex, Torrance, CA). Samples were diluted into ethyl acetate or methanol and then separated using an 18-min linear gradient from 20% methanol/80% 10 mM ammonium acetate, pH 5.6, to 100% methanol at a flow rate of 1 mL min–1. Elution of metabolites was monitored by photodiode array. Retention time and UV spectra were compared to those of authentic standards when available. GC-MS analysis was used to confirm the identities of the various chalcone, flavonoid, and isoflavonoid metabolites. Samples were first converted to TMS ether derivatives by adding N,O-bis(TMS)-trifluoroacetamide (Sigma-Aldrich) and incubating at 37°C for 1 h. Samples were then dried under nitrogen and dissolved in chloroform. GC-MS analysis was conducted using a ThermoFinnigan Trace 2000 GC (Thermoquest, San Jose, CA) equipped with an AS2000 autoinjector and a GCQ-plus Mass Spectrometer System. Samples were resolved using a 5-µm, 15-m x 0.25-mm i.d. DB-5ms column (J&W Scientific, Folsom, CA). The oven temperature was programmed from 200°C (3.5 min hold) to 300°C at a rate of 5°C min–1 with a column flow rate of 0.75 mL min–1 He. The ionization potential of the mass selective detector was 70 eV. TMS derivatives were identified by comparison of retention times and mass spectra with that of standards derivatized with N,O-bis(TMS)-trifluoroacetamide when available. Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers AY595413, AY595414, AY595419, AY595415, AY595416, AY595417, AY595418, and AY595420.
We thank Laura Walker and Chris Menne for technical assistance, Dr. Joe Jez for help on CHI in vitro assays and critical reading of the manuscript, and Drs. Jan Jaworski and Gene Guo for valuable discussions. We also thank Dr. Philip Urban for providing the WAT11 yeast strain, Dr. Stefan Martens for FNSII constructs, Dr. Gary Stacey for B. japonicum strains, Deana Pape for cDNAs, and Kristen Opper for in vitro expression of F3H. Received October 18, 2004; returned for revision January 25, 2005; accepted January 30, 2005.
1 This work was supported in part by the U.S. Department of Agriculture (postdoctoral fellowship no. 2002–35318–12593 to L.R.), by the Illinois-Missouri Biotechnology Alliance (grant no. 34346–13070 to O.Y.), and by the Missouri Soybean Merchandising Council (grant no. 03–242 to O.Y.). 
2 Present address: Sigma-Aldrich Biotechnology, 2909 Laclede Ave., St. Louis, MO 63103.
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.054502. * Corresponding author; e-mail oyu{at}danforthcenter.org; fax 314–587–1541.
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ABSTRACT
RESULTS
2-66. The His-tagged proteins were purified by affinity chromatography, and His-tags were subsequently removed by digestion with thrombin. All CHIs were expressed at high levels and could be isolated as soluble proteins in milligram quantities. The purified enzymes were then assayed for their ability to metabolize 10 different chalcones (
