2-hydroxyisoflavanone Synthase Research Articles

Integrated metabolomic and transcriptomic analysis of specialized metabolites and isoflavonoid biosynthesis in Sophora alopecuroides L. under different degrees of drought stress

Appropriate drought stress could promote the accumulation of specialized metabolites in plants, especially in medicinal plants. Sophora alopecuroides L. (Leguminosae), a medicinal plant with a variety of bioactive specialized metabolites, has an excellent drought resistance. In this study, drought stress-induced metabolomic and transcriptomic changes in S. alopecuroides were identified, to explore the mechanism of specialized metabolite changes in response to mild, moderate and severe drought stress. The metabolic analysis results showed that a total of 919 annotated metabolites were detected and quantified, of them, flavonoid metabolites were the main metabolites in S. alopecuroides. Moreover, a total of 411 differentially accumulated metabolites (DAMs) were identified, among them, 6-Hydroxydaidzein, 2-Hydroxy-2,3-dihydrogenistein, prunetin, and biochanin A significantly increased under mild drought stress but isoflavonoids significantly decreased under moderate and severe drought stress. Transcriptional analysis showed that a total of 21,307 differentially expressed genes (DEGs) were identified, and most of them were enriched in the biosynthesis of secondary metabolites pathway. The integrated metabolomic and transcriptomic analyses results proved that these DAMs and DEGs were most significantly enriched in isoflavonoid biosynthesis pathway (ko00943). In addition, some structural genes such as 2-hydroxyisoflavanone synthase (IFS), 2-hydroxyisoflavanone dehydratase (HID), isoflavone 7-O-glucosyltransferase (IF7GT), and isoflavone 7-O-glucoside-6''-O-malonyltransferase (IF7MAT) and seven MYB transcription factors (SaMYB1∼ SaMYB7) were identified, which played crucial regulatory roles in the isoflavonoid synthesis. This study provides useful insights into the molecular mechanism of S. alopecuroides in response to drought stress, and help develop biotechnological strategies to manipulate the levels of therapeutic specialized metabolites in S. alopecuroides.

The protein conformational basis of isoflavone biosynthesis

Isoflavonoids play important roles in plant defense and also exhibit a range of mammalian health-promoting activities. Their biosynthesis is initiated by two enzymes with unusual catalytic activities; 2-hydroxyisoflavanone synthase (2-HIS), a membrane-bound cytochrome P450 catalyzing a coupled aryl-ring migration and hydroxylation, and 2-hydroxyisoflavanone dehydratase (2-HID), a member of a large carboxylesterase family that paradoxically catalyzes dehydration of 2-hydroxyisoflavanones to isoflavone. Here we report the crystal structures of 2-HIS from Medicago truncatula and 2-HID from Pueraria lobata. The 2-HIS structure reveals a unique cytochrome P450 conformation and heme and substrate binding mode that facilitate the coupled aryl-ring migration and hydroxylation reactions. The 2-HID structure reveals the active site architecture and putative catalytic residues for the dual dehydratase and carboxylesterase activities. Mutagenesis studies revealed key residues involved in substrate binding and specificity. Understanding the structural basis of isoflavone biosynthesis will facilitate the engineering of new bioactive isoflavonoids.

Systems Metabolic Engineering of Escherichia coli Coculture for De Novo Production of Genistein.

Genistein is a plant-derived isoflavone possessing various bioactivities to prevent aging, carcinogenesis, and neurodegenerative and inflammation diseases. As a typical complex flavonoid, its microbial production from sugar remains to be completed. Here, we use systems metabolic engineering stategies to design and develop a three-strain commensalistic Escherichia coli coculture that for the first time realized the de novo production of genistein. First, we reconstituted the naringenin module by screening and incorporating chalcone isomerase-like protein, an auxiliary component to rectify the chalcone synthase promiscuity. Furthermore, we devised and constructed the genistein module by N-terminal modifications of plant P450 enzyme 2-hydroxyisoflavanone synthase and cytochrome P450 enzyme reductase. When naringenin-producing strain was cocultivated with p-coumaric acid-overproducing strain (a phenylalanine-auxotroph), two-strain coculture worked as commensalism through a unidirectional nutrient flow, which favored the efficient production of naringenin with a titer of 206.5 mg/L from glucose. A three-strain commensalistic coculture was subsequently engineered, which produced the highest titer to date of 60.8 mg/L genistein from a glucose and glycerol mixture. The commensalistic coculture is a flexible and versatile platform for the production of flavonoids, indicating a promising future for production of complex natural products in engineered E. coli.

Magnetized water: A way to enhance isoflavonoids in cultured Pueraria candollei var. mirifica cells

Magnetic fields can alter plant growth and development. In this study, the effect of magnetized water on the growth and secondary metabolite production of Pueraria candollei var. mirifica (PM) suspension cells was evaluated. Magnetized water was generated by passing water through a magnetic field (400 mT) at a 6 L/min flow rate. The suspension cells exposed to magnetized water for 10 (M2, 1.37 g) and 5 min (M1, 1.23 g) showed 21% and 9% higher biomass accumulation, respectively, compared with the control cells after 5 days. Compared with that in the control cells, the biomass was increased by approximately 43% when the cells were exposed to M2 (3.01 g) after 28 days. In addition, the use of magnetized water altered the accumulation pattern of isoflavonoids after 5 days of treatment. Isoflavonoid accumulation was the highest after treatment with M2 for 5 days and yielded 4.20 mg/g dry weight (DW) daidzein, 0.58 mg/g DW genistein, and 3.98 mg/g DW kwakhurin. When the cells were maintained in the treatment for a relatively long duration (28 days), a short span of magnetization (M1) resulted in the highest accumulation of isoflavonoids with the amounts of 3.51, 0.55, and 2.58 mg/g DW for daidzein, genistein, and kwakhurin, respectively. After 28 days of exposure to magnetized water, the expression of 2-hydroxyisoflavanone synthase and 2-hydroxyisoflavanone dehydratase increased, and this phenomenon coincided with the increased amount of isoflavonoids in the suspension cells. The magnetized water-treated PM cells also exhibited higher antioxidant activities than the control cells. The lowest half-maximal inhibitory concentration (37.93 mg/mL) was observed in the cells treated with magnetized water after 15 min (M3). The enhanced total antioxidant activity of the cells treated with M3 may be attributed to the presence of other compounds in addition to isoflavonoids. This study represents an innovative attempt at a low-cost and environmentally friendly approach that can enhance the growth, secondary metabolite production, and antioxidant activity of PM suspension cells.

Formation of Flavonoid Metabolons: Functional Significance of Protein-Protein Interactions and Impact on Flavonoid Chemodiversity.

Flavonoids are a class of plant specialized metabolites with more than 6,900 known structures and play important roles in plant survival and reproduction. These metabolites are derived from p-coumaroyl-CoA via the sequential actions of a variety of flavonoid enzymes, which have been proposed to form weakly bound, ordered protein complexes termed flavonoid metabolons. This review discusses the impacts of the formation of flavonoid metabolons on the chemodiversity of flavonoids. Specific protein-protein interactions in the metabolons of Arabidopsis thaliana and other plant species have been studied for two decades. In many cases, metabolons are associated with the ER membrane, with ER-bound cytochromes P450 hypothesized to serve as nuclei for metabolon formation. Indeed, cytochromes P450 have been found to be components of flavonoid metabolons in rice, snapdragon, torenia, and soybean. Recent studies illustrate the importance of specific interactions for the efficient production and temporal/spatial distribution of flavonoids. For example, in diverse plant species, catalytically inactive type-IV chalcone isomerase-like protein serves as an enhancer of flavonoid production via its involvement in flavonoid metabolons. In soybean roots, a specific isozyme of chalcone reductase (CHR) interacts with 2-hydroxyisoflavanone synthase, to which chalcone synthase (CHS) can also bind, providing a mechanism to prevent the loss of the unstable CHR substrate during its transfer from CHS to CHR. Thus, diversification in chemical structures and temporal/spatial distribution patterns of flavonoids in plants is likely to be mediated by the formation of specific flavonoid metabolons via specific protein-protein interactions.

Involvement of chalcone reductase in the soybean isoflavone metabolon: identification of GmCHR5, which interacts with 2-hydroxyisoflavanone synthase.

Soybean (Glycine max) 5-deoxyisoflavonoids (daidzein and its conjugates) are precursors of glyceollin phytoalexins. They are also converted to equol by microbes in the human intestine, resulting in health benefits. 5-Deoxyisoflavonoids accumulate in the roots (93% mol/mol of the total root isoflavonoids) and seeds of unstressed soybean plants. Chalcone reductase (CHR) is a key enzyme mediating 5-deoxyisoflavonoid biosynthesis because it catalyzes the production of 6'-deoxychalcone through its effects on the chalcone synthase (CHS)-catalyzed reaction. The soybean genome encodes at least 11 CHR-related homologs, but it is unclear which ones are functionally important for daidzein accumulation in unstressed plants. Among the CHR homologs, the temporal and spatial expression patterns of GmCHR5 were the most correlated with the distribution patterns of 5-deoxyisoflavonoids. The CHR activity of GmCHR5 was confirmed invitro and in planta. In the invitro assays, the ratio of CHR products (6'-deoxychalcone) to total CHS products (R value) was dependent on GmCHR5 and CHS concentrations, with higher concentrations resulting in higher R values (i.e. approaching 90%). Subcellular localization analyses revealed that GmCHR5 was present in the cytoplasm and nucleus. Protein-protein interaction assays indicated that GmCHR5, but not GmCHR1 and GmCHR6, interacted with 2-hydroxyisoflavanone synthase (IFS) isozymes. The CHS isozymes also interacted with IFS isozymes but not with GmCHR5. The proposed micro-compartmentalization of isoflavone biosynthesis through the formation of an IFS-mediated metabolon is probably involved in positioning GmCHR5 close to CHS, resulting in an R value that is high enough for the accumulation of abundant 5-deoxyisoflavonoids in soybean roots.

Draft genome assembly and annotation of Glycyrrhiza uralensis, a medicinal legume.

Chinese liquorice/licorice (Glycyrrhiza uralensis) is a leguminous plant species whose roots and rhizomes have been widely used as a herbal medicine and natural sweetener. Whole-genome sequencing is essential for gene discovery studies and molecular breeding in liquorice. Here, we report a draft assembly of the approximately 379-Mb whole-genome sequence of strain 308-19 of G. uralensis; this assembly contains 34445 predicted protein-coding genes. Comparative analyses suggested well-conserved genomic components and collinearity of gene loci (synteny) between the genome of liquorice and those of other legumes such as Medicago and chickpea. We observed that three genes involved in isoflavonoid biosynthesis, namely, 2-hydroxyisoflavanone synthase (CYP93C), 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase/isoflavone 4'-O-methyltransferase (HI4OMT) and isoflavone-7-O-methyltransferase (7-IOMT) formed a cluster on the scaffold of the liquorice genome and showed conserved microsynteny with Medicago and chickpea. Based on the liquorice genome annotation, we predicted genes in the P450 and UDP-dependent glycosyltransferase (UGT) superfamilies, some of which are involved in triterpenoid saponin biosynthesis, and characterised their gene expression with the reference genome sequence. The genome sequencing and its annotations provide an essential resource for liquorice improvement through molecular breeding and the discovery of useful genes for engineering bioactive components through synthetic biology approaches.

Identification of protein–protein interactions of isoflavonoid biosynthetic enzymes with 2-hydroxyisoflavanone synthase in soybean (Glycine max (L.) Merr.)

Metabolic enzymes, including those involved in flavonoid biosynthesis, are proposed to form weakly bound, ordered protein complexes, called “metabolons”. Some hypothetical models of flavonoid biosynthetic metabolons have been proposed, in which metabolic enzymes are believed to anchor to the cytoplasmic surface of the endoplasmic reticulum (ER) via ER-bound cytochrome P450 isozymes (P450s). However, no convincing evidence for the interaction of flavonoid biosynthetic enzymes with P450s has been reported previously. Here, we analyzed binary protein–protein interactions of 2-hydroxyisoflavanone synthase 1 (GmIFS1), a P450 (CYP93C), with cytoplasmic enzymes involved in isoflavone biosynthesis in soybean. We identified binary interactions between GmIFS1 and chalcone synthase 1 (GmCHS1) and between GmIFS1 and chalcone isomerases (GmCHIs) by using a split-ubiquitin membrane yeast two-hybrid system. These binary interactions were confirmed in planta by means of bimolecular fluorescence complementation (BiFC) using tobacco leaf cells. In these BiFC analyses, fluorescence signals that arose from the interaction of these cytoplasmic enzymes with GmIFS1 generated sharp, network-like intracellular patterns, which was very similar to the ER-localized fluorescence patterns of GmIFS1 labeled with a fluorescent protein. These observations provide strong evidence that, in planta, interaction of GmCHS1 and GmCHIs with GmIFS1 takes place on ER on which GmIFS1 is located, and also provide important clues to understand how enzymes and proteins form metabolons to establish efficient metabolic flux of (iso)flavonoid biosynthesis.

Expression quantitative trait loci infer the regulation of isoflavone accumulation in soybean (Glycine max L. Merr.) seed.

BackgroundMapping expression quantitative trait loci (eQTL) of targeted genes represents a powerful and widely adopted approach to identify putative regulatory variants. Linking regulation differences to specific genes might assist in the identification of networks and interactions. The objective of this study is to identify eQTL underlying expression of four gene families encoding isoflavone synthetic enzymes involved in the phenylpropanoid pathway, which are phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), 2-hydroxyisoflavanone synthase (IFS; EC1.14.13.136) and flavanone 3-hydroxylase (F3H; EC 1.14.11.9). A population of 130 recombinant inbred lines (F5:11), derived from a cross between soybean cultivar ‘Zhongdou 27’ (high isoflavone) and ‘Jiunong 20’ (low isoflavone), and a total of 194 simple sequence repeat (SSR) markers were used in this study. Overlapped loci of eQTLs and phenotypic QTLs (pQTLs) were analyzed to identify the potential candidate genes underlying the accumulation of isoflavone in soybean seed.ResultsThirty three eQTLs (thirteen cis-eQTLs and twenty trans-eQTLs) underlying the transcript abundance of the four gene families were identified on fifteen chromosomes. The eQTLs between Satt278-Sat_134, Sat_134-Sct_010 and Satt149-Sat_234 underlie the expression of both IFS and CHS genes. Five eQTL intervals were overlapped with pQTLs. A total of eleven candidate genes within the overlapped eQTL and pQTL were identified.ConclusionsThese results will be useful for the development of marker-assisted selection to breed soybean cultivars with high or low isoflavone contents and for map-based cloning of new isoflavone related genes.

Transcription analyses of GmICHG, a gene coding for a β-glucosidase that catalyzes the specific hydrolysis of isoflavone conjugates in Glycine max (L.) Merr

Isoflavone conjugate-hydrolyzing β-glucosidase (GmICHG) of soybeans [Glycine max (L.) Merr.] catalyzes the specific hydrolysis of isoflavone conjugates (β-7-O-(malonyl)glucosides of isoflavones) to produce free isoflavones. In this study, changes in the transcription levels of GmICHG in the individual organs of soybean seedlings (cv. Enrei) in response to microbial infection and abiotic stresses were analyzed and compared with those of genes coding for 2-hydroxyisoflavanone synthase (GmIFS) and isoflavone 7-O-glucosyltransferase (GmIF7GT). GmICHG was originally expressed in abundance only in the roots and at low levels only in the other organs. The transcription of GmICHG in the roots and other organs was suppressed upon infection of the roots by Phytophthora sojae. Upon wounding of the cotyledon, a transient long-distance up-regulation of GmICHG transcription in the roots was observed; upon fungal infection in the cotyledon, however, a delayed elevation of GmICHG transcription took place in the roots with the maximum at 10h after the infection. Such long-distance up-regulation patterns were not observed with either GmIFS or GmIF7GT. The transcription levels of GmICHG remained essentially unchanged upon treatment of the roots with Bradyrhizobium japonicum. The transcription of GmICHG in the roots was also sensitive to a variety of stresses on the roots, such as flooding, elicitation with yeast extract, drought, and treatment with plant hormones such as abscisic, salicylic, and jasmonic acids and ethylene.

Cyclic GMP acts as a common regulator for the transcriptional activation of the flavonoid biosynthetic pathway in soybean

Cyclic GMP (cGMP) is an important signaling molecule that controls a range of cellular functions. So far, however, only a few genes have been found to be regulated by cGMP in higher plants. We investigated the cGMP-responsiveness of several genes encoding flavonoid-biosynthetic enzymes in soybean (Glycine max L.) involved in legume-specific isoflavone, phytoalexin and anthocyanin biosynthesis, such as phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate:CoA ligase, chalcone synthase, chalcone reductase, chalcone isomerase, 2-hydroxyisoflavanone synthase, 2-hydroxyisoflavanone dehydratase, anthocyanidin synthase, UDP-glucose:isoflavone 7-O-glucosyltransferase, and isoflavone reductase, and found that the majority of these genes were induced by cGMP but not by cAMP. All cGMP-induced genes were also stimulated by sodium nitroprusside (SNP), a nitric oxide (NO) donor, and illumination of cultured cells with white light. The NO-dependent induction of these genes was blocked by 6-anilino-5,8-quinolinedione, an inhibitor of guanylyl cyclase. Moreover, cGMP levels in cultured cells were transiently increased by SNP. Consistent with the increases of these transcripts, the accumulation of anthocyanin in response to cGMP, NO, and white light was observed. The treatment of soybean cotyledons with SNP resulted in a high accumulation of isoflavones such as daidzein and genistein. Loss- and gain-of-function experiments with the promoter of chalcone reductase gene indicated the Unit I-independent activation of gene expression by cGMP. Together, these results suggest that cGMP acts as a second messenger to activate the expression of genes for enzymes involved in the flavonoid biosynthetic pathway in soybean.

2-Hydroxyisoflavanone Dehydratase is a Critical Determinant of Isoflavone Productivity in Hairy Root Cultures of Lotus japonicus

Hairy root cultures of a model legume, Lotus japonicus, were established to characterize two heterologous cDNAs encoding enzymes involved in isoflavone biosynthesis, i.e. licorice 2-hydroxyisoflavanone synthase (IFS) and soybean 2-hydroxyisoflavanone dehydratase (HID) catalyzing sequential reactions to yield isoflavones. While the control and the IFS overexpressor did not accumulate detectable isoflavones, the HID overexpressors did accumulate daidzein and genistein, showing that HID is a critical determinant of isoflavone productivity. Production of coumestrol in all the genotypes and isoliquiritigenin/liquiritigenin in IFS + HID-overexpressing lines was also noted. These results provide insight into the regulatory mechanism that controls isoflavonoid biosynthesis.

Cytochrome P450s in flavonoid metabolism

In this review, cytochrome P450s characterized at the molecular level catalyzing aromatic hydroxylations, aliphatic hydroxylations and skeleton formation in the flavonoid metabolism are surveyed. They are involved in the biosynthesis of anthocyanin pigments and condensed tannin (CYP75, flavonoid 3′,5′-hydroxylase and 3′-hydroxylase), flavones [CYP93B, (2S)-flavanone 2-hydroxylase and flavone synthase II], and leguminous isoflavonoid phytoalexins [CYP71D9, flavonoid 6-hydroxylase; CYP81E, isoflavone 2′-hydroxylase and 3′-hydroxylase; CYP93A, 3,9-dihydroxypterocarpan 6a-hydroxylase; CYP93C, 2-hydroxyisoflavanone synthase (IFS)]. Other P450s of the flavonoid metabolism include methylenedioxy bridge forming enzyme, cyclases producing glyceollins, flavonol 6-hydroxylase and 8-dimethylallylnaringenin 2′-hydroxylase. Mechanistic studies on the unusual aryl migration by CYP93C, regulation of IFS expression in plant organs and its biotechnological applications are introduced, and flavonoid metabolisms by non-plant P450s are also briefly discussed.

Multiple mutagenesis of P450 isoflavonoid synthase reveals a key active-site residue

The leguminous isoflavonoid skeleton is constructed by P450 2-hydroxyisoflavanone synthase (CYP93C). Two active-site residues of CYP93C2, Ser 310 and Lys 375, are critical for unusual aryl migration of the flavanone substrate. Leu 371 is located near the substrate in a homology model, and mutant proteins regarding this residue were expressed in recombinant yeast microsomes. The single mutant, L371V, yielded only inactive P420, but multiple mutants incorporating K375T restored the P450 fold: the S310T-L371V-K375T triple mutant showed four times higher P450 level than the wild type. L371V-K375T and S310T-L371V-K375T produced a mixture of major 3β-hydroxyflavanone and minor flavone, and 100% flavone, respectively, from a flavanone. Thus, Leu 371 appeared to control the substrate accommodation in favor of hydrogen abstraction from C-3 of the flavanone molecule and contribute to the P450 fold under the presence of Lys 375, the residue responsible for aryl migration. The molecular evolution of CYP93 enzymes is discussed.

Isoflavonoid biosynthesis and accumulation in developing soybean seeds.

Isoflavonoids are biologically active natural products that accumulate in soybean seeds during development. The amount of isoflavonoids present in soybean seed is variable, depending on genetic and environmental factors that are not fully understood. Experiments were conducted to determine whether isoflavonoids are synthesized within seed tissues during development, or made in other plant organs and transported to the seeds where they accumulate. An analysis of isoflavonoids by HPLC detected the compounds in all organs of soybean plant, but the amount of isoflavonoids present varied depending on the tissue and developmental stage. The greatest concentrations were found in mature seeds and leaves. The 2-hydroxyisoflavanone synthase genes IFS1 and IFS2 were studied to determine their pattern of expression in different tissues and developmental stages. The highest level of expression of IFS1 was observed in the root and seed coat, while IFS2 was most highly expressed in embryos and pods, and in elicitor-treated or pathogen-challenged tissues. Incorporation of radiolabel into isoflavonoids was observed when developing embryos and other plant organs were fed with [(14)C]phenylalanine. Embryos excised from developing soybean seeds also accumulated isoflavonoids from a synthetic medium. A maternal effect on seed isoflavonoid content was noted in reciprocal crosses between soybean cultivars that differ in seed isoflavonoids. From these results, we propose that developing soybean embryos have an ability to synthesize isoflavonoids de novo, but that transport from maternal tissues may in part contribute to the accumulation of these natural products in the seed.

A Cluster of Genes Encodes the Two Types of Chalcone Isomerase Involved in the Biosynthesis of General Flavonoids and Legume-Specific 5-Deoxy(iso)flavonoids in Lotus japonicus

Leguminous plants produce 5-deoxyflavonoids and 5-deoxyisoflavonoids that play essential roles in legume-microbe interactions. Together with chalcone polyketide reductase and cytochrome P450 2-hydroxyisoflavanone synthase, the chalcone isomerase (CHI) of leguminous plants is fundamental in the construction of these ecophysiologically active flavonoids. Although CHIs of nonleguminous plants isomerize only 6'-hydroxychalcone to 5-hydroxyflavanone (CHIs with this function are referred to as type I), leguminous CHIs convert both 6'-deoxychalcone and 6'-hydroxychalcone to 5-deoxyflavanone and 5-hydroxyflavanone, respectively (referred to as type II). In this study, we isolated multiple CHI cDNAs (cCHI1-cCHI3) from a model legume, Lotus japonicus. In contrast to previous observations, the amino acid sequence of CHI2 was highly homologous to nonleguminous CHIs, whereas CHI1 and CHI3 were the conventional leguminous type. Furthermore, genome sequence analysis revealed that four CHI genes (CHI1-3 and a putative gene, CHI4) form a tandem cluster within 15 kb. Biochemical analysis with recombinant CHIs expressed in Escherichia coli confirmed that CHI1 and CHI3 are type II CHIs and that CHI2 is a type I CHI. The occurrence of both types of CHIs is probably common in leguminous plants, and it was suggested that type II CHIs evolved from an ancestral CHI by gene duplication and began to produce 5-deoxy(iso)flavonoids along with the establishment of the Fabaceae.

Flavonoid 6-Hydroxylase from Soybean (Glycine maxL.), a Novel Plant P-450 Monooxygenase

Cytochrome P-450-dependent hydroxylases are typical enzymes for the modification of basic flavonoid skeletons. We show in this study that CYP71D9 cDNA, previously isolated from elicitor-induced soybean (Glycine max L.) cells, codes for a protein with a novel hydroxylase activity. When heterologously expressed in yeast, this protein bound various flavonoids with high affinity (1.6 to 52 microm) and showed typical type I absorption spectra. These flavonoids were hydroxylated at position 6 of both resorcinol- and phloroglucinol-based A-rings. Flavonoid 6-hydroxylase (CYP71D9) catalyzed the conversion of flavanones more efficiently than flavones. Isoflavones were hardly hydroxylated. As soybean produces isoflavonoid constituents possessing 6,7-dihydroxy substitution patterns on ring A, the biosynthetic relationship of flavonoid 6-hydroxylase to isoflavonoid biosynthesis was investigated. Recombinant 2-hydroxyisoflavanone synthase (CYP93C1v2) efficiently used 6,7,4'-trihydroxyflavanone as substrate. For its structural identification, the chemically labile reaction product was converted to 6,7,4'-trihydroxyisoflavone by acid treatment. The structures of the final reaction products for both enzymes were confirmed by NMR and mass spectrometry. Our results strongly support the conclusion that, in soybean, the 6-hydroxylation of the A-ring occurs before the 1,2-aryl migration of the flavonoid B-ring during isoflavanone formation. This is the first identification of a flavonoid 6-hydroxylase cDNA from any plant species.

Induction of isoflavonoid pathway in the model legume Lotus japonicus: molecular characterization of enzymes involved in phytoalexin biosynthesis

Treatment of the seedlings of Lotus japonicus, a model legume for molecular genetic studies, with reduced glutathione (GSH) resulted in the accumulation of an isoflavan phytoalexin, vestitol. Using PCR strategies based on the conserved amino acid sequences, full length P450 cDNAs were obtained from GSH-treated seedling roots. When the clones, LjCYP-1 (CYP93C family) and LjCYP-2 (CYP81E family), were heterologously expressed in yeast, the proteins exhibited 2-hydroxyisoflavanone synthase (IFS) and isoflavone 2′-hydroxylase (I2′H) activities, respectively. The transcription levels of LjCYP-1, LjCYP-2 and isoflavone reductase, which are all involved in vestitol biosynthesis, coordinately increased upon elicitation. Genomic Southern blot analysis indicated that the IFS gene forms a small gene family and a single copy of the I2′H gene is present in the L. japonicus genome. Molecular biological aspects of P450s involved in the isoflavonoid pathway and the genomic approach to flavonoid metabolism in this unique plant are discussed.

New scheme of the biosynthesis of formononetin involving 2,7,4'-trihydroxyisoflavanone but not daidzein as the methyl acceptor.

Glycyrrhiza echinata cell-free extract produced isoformononetin by the 7-O-transmethylation of daidzein from S-adenosyl-L-methionine (SAM). When the yeast microsome expressing 2-hydroxyisoflavanone synthase was mixed with the cell-free extract and incubated with liquiritigenin and SAM, formononetin emerged. Furthermore, the cell-free extract yielded formononetin on incubation with 2,7,4'-trihydroxyisoflavanone and SAM. We propose a novel pathway of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor.

Cloning and functional expression of a cytochrome P450 cDNA encoding 2-hydroxyisoflavanone synthase involved in biosynthesis of the isoflavonoid skeleton in licorice.

Isoflavonoids are distributed predominantly in leguminous plants and play critical roles in plant physiology. A cytochrome P450 (P450), 2-hydroxyisoflavanone synthase, is the key enzyme in their biosynthesis. In cultured licorice (Glycyrrhiza echinata L., Fabaceae) cells, the production of both an isoflavonoid-derived phytoalexin (medicarpin) and a retrochalcone (echinatin) is rapidly induced upon elicitation. In this study, we obtained a full-length P450 cDNA, CYP Ge-8 (CYP93C2), from the cDNA library of elicited G. echinata cells. When the flavanones liquiritigenin and naringenin were incubated with the recombinant yeast microsome expressing CYP93C2, major products emerged and were readily converted to the isoflavones daidzein and genistein by acid treatment. The chemical structures of the products from liquiritigenin (2-hydroxyisoflavanone and isoflavone) were confirmed by mass spectrometry. CYP93C2 was thus shown to encode 2-hydroxyisoflavanone synthase, which catalyzes the hydroxylation associated with 1,2-aryl migration of flavanones. Northern-blot analysis revealed that transcripts of CYP93C2, in addition to those of other P450s involved in phenylpropanoid/flavonoid pathways, transiently accumulate upon elicitation.