Probing the Deoxyflavonoid Biosynthesis: Naringenin Chalcone Is a Substrate for the Reductase Synthesizing Isoliquiritigenin.

Isoliquiritigenin formation is a key reaction during deoxyflavonoid biosynthesis, which is catalyzed by two enzymes, chalcone synthase (CHS) and reductase (CHR). The substrates for CHS are established. However, the substrate for CHR is unknown. In this study, an in vitro reaction was performed to confirm whether naringenin chalcone can be a substrate. Naringenin chalcone was used as a substrate during the CHR reaction. Analyzing the product revealed that isoliquiritigenin was produced from naringenin chalcone, indicating that naringenin chalcone is a substrate. This study is the first to identify a substrate for CHR, reveals that deoxyflavonoid biosynthesis diverges from naringenin chalcone, endorses the term "chalcone reductase," and answers the long-standing questions about doubly-labeled acetic acid uptake pattern in deoxyflavonoid biosynthesis.

Recombinant yeast as a functional tool for understanding bitterness and cucurbitacin biosynthesis in watermelon (Citrullus spp.).

Cucurbitacins are a group of bitter-tasting oxygenated tetracyclic triterpenes that are produced in the family Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defence against pathogens and pests. Cucurbitadienol, a triterpene synthesized from oxidosqualene, is the first committed precursor to cucurbitacins produced by a specialized oxidosqualene cyclase termed cucurbitadienol synthase. We explored cucurbitacin accumulation in watermelon in relation to bitterness. Our findings show that cucurbitacins are accumulated in bitter-tasting watermelon, Citrullus lanatus var. citroides, as well as in their wild ancestor, C. colocynthis, but not in non-bitter commercial cultivars of sweet watermelon (C. lanatus var. lanatus). Molecular analysis of genes expressed in the roots of several watermelon accessions led to the isolation of three sequences (CcCDS1, CcCDS2 and ClCDS1), all displaying high similarity to the pumpkin CpCPQ, encoding a protein previously shown to possess cucurbitadienol synthase activity. We utilized the Saccharomyces cerevisiae strain BY4743, heterozygous for lanosterol synthase, to probe for possible encoded cucurbitadienol synthase activity of the expressed watermelon sequences. Functional expression of the two sequences isolated from C. colocynthis (CcCDS1 and CcCDS2) in yeast revealed that only CcCDS2 possessed cucurbitadienol synthase activity, while CcCDS1 did not display cucurbitadienol synthase activity in recombinant yeast. ClCDS1 isolated from C. lanatus var. lanatus is almost identical to CcCDS1. Our results imply that CcCDS2 plays a role in imparting bitterness to watermelon. Yeast has been an excellent diagnostic tool to determine the first committed step of cucurbitacin biosynthesis in watermelon.

Cyclization of all-E- and 2Z-geranylfarnesols by a bacterial triterpene synthase: insight into sesterterpene biosynthesis in Aleuritopteris ferns.

Aleuritopteris ferns produce triterpenes and sesterterpenes with tricyclic cheilanthane and tetracyclic 18-episcalarane skeletons. The structural and mechanistic similarities between both classes of fern terpene suggest that their biosynthetic enzymes may be closely related. We investigate here whether a triterpene synthase is capable of recognizing geranylfarnesols as a substrate, and is able to convert them to cyclic sesterterpenes. We found that a bacterial triterpene synthase converted all-E-geranylfarnesol (1b) into three scalarane sesterterpenes with 18αH stereochemistry (5, 7 and 8), as well as mono- and tricyclic sesterterpenes (6 and 9). In addition, 2Z-geranylfarnesol (4) was converted into an 18-episcalarane derivative (10), whose skeleton can be found in sesterterpenes isolated from Aleuritopteris ferns. These results provide insight into sesterterpene biosynthesis in Aleuritopteris ferns.

Antimicrobial polyacetylenes from Panax ginseng hairy root culture.

Two new polyacetylenes, 1-hydroxydihydropanaxacol (3) and 17-hydroxypanaxacol (4), were isolated from Panax ginseng hairy root culture, along with dihydropanaxacol (1), panaxacol (2) and ginsenoyne D (5). Highly hydroxylated compounds 1-4 were isolated from the medium and compound 5, which was a biosynthetic precursor of compound 1, was isolated from the roots. Compounds 1-4 showed antimicrobial activity against Staphylococcus aureus, Bacillus subtilis, Cryptococcus neoformans and Aspergillus fumigatus. It is suggested that P. ginseng plants release antimicrobial polyacetylenes into the surrounding soil from the roots as defense compounds.

Identification and characterization of glycosyltransferases involved in the biosynthesis of soyasaponin I in Glycine max.

Triterpene saponins are a diverse group of compounds with a structure consisting of a triterpene aglycone and sugars. Identification of the sugar-transferase involved in triterpene saponin biosynthesis is difficult due to the structural complexity of triterpene saponin. Two glycosyltransferases from Glycine max, designated as GmSGT2 and GmSGT3, were identified and characterized. In vitro analysis revealed that GmSGT2 transfers a galactosyl group from UDP-galactose to soyasapogenol B monoglucuronide, and that GmSGT3 transfers a rhamnosyl group from UDP-rhamnose to soyasaponin III. These results suggest that soyasaponin I is biosynthesized from soyasapogenol B by successive sugar transfer reactions.

Molecular evolution of fern squalene cyclases.

Triterpenes, a diverse group of natural products comprising six isoprene units, are distributed across various organisms from bacteria to higher plants. Ferns are sporophytes that produce triterpenes and are lower on the evolutionary scale than higher plants. Among ferns that produce triterpenes analogous to bacterial hopanoids, Polypodiodes niponica produces migrated dammaranes and oleananes, which are also widely found in higher plants. Because the study of terpene-producing ferns could help us to understand the molecular basis of triterpene biosynthesis, cDNA cloning of squalene cyclases (SCs) from P. niponica was carried out. Two SCs (PNT and PNG) were obtained. The heterologously expressed PNT produces tirucalla-7,21-diene (67% major), and PNG produces germanicene (69%). Phylogenetic analysis revealed that PNT and PNG, which produce higher-plant-type migrated dammaranes and oleananes, are closely related to bacterial-type SCs. Furthermore, analysis of the minor products indicated that fern SCs gained the ability to directly form dammarenyl cations, which are key intermediates in oleanane formation during molecular evolution.

Protostadienol synthase from Aspergillus fumigatus: functional conversion into lanosterol synthase.

Oxidosqualene:protostadienol cyclase (OSPC) from the fungus Aspergillus fumigatus, catalyzes the cyclization of (3S)-2,3-oxidosqualene into protosta-17(20)Z,24-dien-3beta-ol which is the precursor of the steroidal antibiotic helvolic acid. To shed light on the structure-function relationship between OSPC and oxidosqualene:lanosterol cyclase (OSLC), we constructed an OSPC mutant in which the C-terminal residues (702)APPGGMR(708) were replaced with (702)NKSCAIS(708), as in human OSLC. As a result, the mutant no longer produced the protostadienol, but instead efficiently produced a 1:1 mixture of lanosterol and parkeol. This is the first report of the functional conversion of OSPC into OSLC, which resulted in a 14-fold decrease in the V(max)/K(M) value, whereas the binding affinity for the substrate did not change significantly. Homology modeling suggested that stabilization of the C-20 protosteryl cation by the active-site Phe701 through cation-pi interactions is important for the product outcome between protostadienol and lanosterol.

Biosynthesis of steroidal antibiotic fusidanes: functional analysis of oxidosqualene cyclase and subsequent tailoring enzymes from Aspergillus fumigatus.

Three putative oxidosqualene cyclase (OSC) genes exist in the genome of the fungus Aspergillus fumigatus that produces a steroidal antibiotic, helvolic acid. One of these genes, Afu4g14770, designated AfuOSC3, is clustered with genes of cytochrome P450 monooxygenases (P450s), a short-chain dehydrogenase/reductase (SDR), and acyltransferases, which presumably function in triterpene tailoring steps, suggesting that this gene cluster codes for helvolic acid biosynthesis. AfuOSC3 was PCR amplified from A. fumigatus IFO8866 genomic DNA and expressed in yeast. The yeast transformant accumulated protosta-17(20)Z,24-dien-3beta-ol, an established precursor for helvolic acid. Its structural isomer, (20R)-protosta-13(17),24-dien-3beta-ol, was also isolated from the transformed yeast. To further identify the function of triterpene tailoring enzymes, four P450 genes (CYP5081A1-D1) and a SDR gene (AfuSDR1) in the cluster were each coexpressed with AfuOSC3 in yeast. As a result, coexpression of AfuSDR1 gave a 3-keto derivative of protostadienol. On the other hand, coexpression with CYP5081A1 gave protosta-17(20)Z,24-diene-3beta,29-diol and protosta-17(20)Z,24-dien-3beta-ol-29-oic acid. These metabolites are in well accord with the oxidative modification involved in helvolic acid biosynthesis. AfuSDR1 and CYP5081A1 presumably function together to catalyze demethylation of C-29 methyl group. These results provided a firm ground for identification of the present gene cluster to be involved in helvolic acid biosynthesis.

Identification of a product specific beta-amyrin synthase from Arabidopsis thaliana.

Triterpene skeletons are produced by oxidosqualene cyclases (OSCs). The genome sequencing of Arabidopsis thaliana revealed the presence of thirteen OSC homologous genes including At1g78950, which has been revised recently as two independent ORFs, namely At1g78950 and At1g78955. The cDNA corresponding to the revised At1g78950 was obtained by RT-PCR, ligated into Saccharomyces cerevisiae expression vector pYES2, and expressed in a lanosterol synthase deficient S. cerevisiae strain. LC-MS and NMR analyses of the accumulated product in the host cells showed that the product of At1g78950 is beta-amyrin, indicating that At1g78950 encodes a beta-amyrin synthase (EC 5.4.99.-).

Biosynthesis of baccharis oxide, a triterpene with a 3,10-oxide bridge in the A-ring.

A new oxidosqualene cyclase (OSC) cDNA was cloned from the roots of Stevia rebaudiana. Functional expression in yeast and spectral analyses of the products established that the obtained OSC yields baccharis oxide as the major product. This is the first identification of an OSC yielding baccharis oxide. This result clearly demonstrated that baccharis oxide, a baccharane triterpene with a unique 3,10-oxide bridge in the A-ring, is a direct product from oxidosqualene by an OSC.

Dammaradiene synthase, a squalene cyclase, from Dryopteris crassirhizoma Nakai.

Ferns produce a variety of cyclic triterpene hydrocarbons in large amount. Squalene cyclases (SCs) are responsible enzymes for formation of cyclic triterpene hydrocarbon skeletons. Although more than ten bacterial SCs have been cloned and four of them characterized for their enzymatic products, the only example of a fern SC is ACH, from Adiantum capillus-veneris, which produces hydroxyhopane. To obtain a deeper understanding of the molecular evolution of SCs and the origin of the structural diversity of fern triterpenes, further cloning and characterization of SCs have been pursued. In this study, a SC cDNA, DCD, was cloned from Dryopteris crassirhizoma by homology-based RT-PCR. DCD contains a 2058-bp open reading frame that encodes a 685 amino acid polypeptide exhibiting 66% identity to the previously identified fern SC, ACH, and 35-40% identity to bacterial SCs. Heterologous expression of DCD in yeast established it to be a dammaradiene synthase affording dammara-18(28),21-diene, a tetracyclic triterpene hydrocarbon. Although neither this compound nor any derived metabolites have been previously reported from D. crassirhizoma, re-investigation of the leaflets demonstrated the presence of dammara-18(28),21-diene. DCD represents the first SC that produces a tetracyclic triterpene hydrocarbon.

Squalene cyclase and oxidosqualene cyclase from a fern.

Ferns are the most primitive vascular plants. The phytosterols of ferns are the same as those of higher plants, but they produce characteristic triterpenes. The most distinct feature is the lack of oxygen functionality at C-3, suggesting that the triterpenes of ferns may be biosynthesized by direct cyclization of squalene. To obtain some insights into the molecular bases for the biosynthesis of triterpenes in ferns, we cloned ACX, an oxidosqualene cyclase homologue, encoding a cycloartenol synthase (CAS) and ACH, a squalene cyclase homologue, encoding a 22-hydroxyhopane synthase from Adiantum capillus-veneris. Phylogenetic analysis revealed that ACH is located in the cluster of bacterial SCs, while ACX is in the cluster of higher plant CASs.

Production of triterpene acids by cell suspension cultures of Olea europaea.

Olive (Olea europaea) contains large quantity of triterpene acids including oleanolic acid (6) as a major one. Varieties of biological activities exhibited by triterpene acids attracted our attentions, especially from pharmaceutical viewpoints. Cell culture of olive plant was induced and its triterpene constituents were studied. From the cell suspension cultures, six ursane type triterpene acids; ursolic acid (9), pomolic acid (10), rotundic acid (11), tormentic acid (12), 2alpha-hydroxyursolic acid (13) and 19alpha-hydroxyasiatic acid (14), and two oleanane type acids; oleanolic acid and maslinic acid (7), have been isolated. Quantity of ursane type triterpene acids produced by cell cultures was larger than that of oleanane type. Further, a multifunctional oxidosqualene cyclase (OSC) named OEA was cloned by homology based PCRs from the same cultured cells. Major product of OEA is alpha-amyrin (ursane skeleton), showing good accordance to higher content of ursane-type triterpene acids in the cultured cells, and strongly suggesting OEA to be a major contributor OSC for their production.

Origin of structural diversity in natural triterpenes: direct synthesis of seco-triterpene skeletons by oxidosqualene cyclase.

At1g78500, one of the oxidosqualene cyclase (OSC) homologues from Arabidopsis thaliana, was expressed in a lanosterol synthase-deficient yeast strain and the products were analyzed. In addition to the known triterpenes, this OSC was found to produce two new triterpenes, the structures of which were determined by NMR and MS analyses. The new triterpenes are C-ring-seco-beta-amyrin (1) and C-ring-seco-alpha-amyrin (2) and named beta-seco-amyrin and alpha-seco-amyrin, respectively. beta-seco-Amyrin is produced from the oleanyl cation through bond cleavage between C8 and C14, and alpha-seco-amyrin is produced from the ursanyl cation in the same manner. Together with Grob fragmentation catalyzed by another OSC (marneral synthase) from A. thaliana, the formation of seco-amyrins by this OSC revealed that OSCs not only catalyze carbon-carbon bond formations and Wagner-Meerwein rearrangements but also cleave preformed ring systems in cationic intermediates. Based on this information, direct production of other natural seco-triterpenes by OSCs is proposed.

Stereochemical course in water addition during LUP1-catalyzed triterpene cyclization.

Arabidopsis thaliana LUP1 (At1g78970) catalyzes the cyclization of oxidosqualene into lupeol and 3beta,20-dihydroxylupane (lupanediol). The stereochemical course of water addition to the lupanyl cation was studied. The X-ray crystal structure of lupanylepoxide 3,5-dinitrobenzoate established the configuration of epoxide as 20S. LiAlD4 reduction of the epoxide enabled the chemical shift assignment of prochiral methyl groups at C20 of lupanediol. Correlation of these methyl groups with biosynthetic lupanediol from [1,2-(13)C(2)] acetate established the stereochemical course of water addition. [reaction: see text].

Molecular cloning and functional expression of a multifunctional triterpene synthase cDNA from a mangrove species Kandelia candel (L.) Druce.

Homology based PCRs with degenerate primers designed from the conserved sequences among the known oxidosqualene cylases (OSCs) have resulted in cloning of a triterpene synthase (KcMS) from the young roots of Kandelia candel (L.) Druce (Rhizophoraceae). KcMS consists of a 2286 bp open reading frame, which codes for 761 amino acids. The deduced amino acid sequence showed 79% homology to a lupeol synthase from Ricinus communis suggesting it to be a lupeol synthase of K. candel. KcMS was expressed in a lanosterol synthase deficient yeast with the expression vector pYES2 under the control of GAL1 promoter. GC-MS analysis showed that the transformant accumulated a mixture of lupeol, beta-amyrin and alpha-amyrin in a 2:1:1 ratio, indicating that KcMS encodes a multifunctional triterpene synthase, although it showed high sequence homology to a R. communis lupeol synthase. This is the first OSC cloning from mangrove tree species.

Dammarenediol-II synthase, the first dedicated enzyme for ginsenoside biosynthesis, in Panax ginseng.

Panax ginseng produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane type and oleanane type. Dammarane-type ginsenosides dominate over oleanane type not only in amount but also in structural varieties. However, their sapogenin structure is restricted to two aglycones, protopanaxadiol and protopanaxatriol. So far, the genes encoding oxidosqualene cyclase (OSC) responsible for formation of dammarane skeleton have not been cloned, although OSC yielding oleanane skeleton (beta-amyrin synthase) has been successfully cloned from this plant. In this study, cDNA cloning of OSC producing dammmarane triterpene was attempted from hairy root cultures of P. ginseng by homology based PCR method. A new OSC gene (named as PNA) obtained was expressed in a lanosterol synthase deficient (erg7) Saccharomyces cerevisiae strain GIL77. LC-MS and NMR analyses identified the accumulated product in the yeast transformant to be dammarenediol-II, demonstrating PNA to encode dammarenediol-II synthase.

A new triterpene synthase from Arabidopsis thaliana produces a tricyclic triterpene with two hydroxyl groups.

[structure: see text] Thirteen oxidosqualene cyclase homologues exist in the genome of Arabidopsis thaliana. One of these genes, At4g15340, was amplified by PCR and expressed in yeast. The yeast transformant accumulated tricyclic triterpene, (3S,13R)-malabarica-17,21-dien-3,14-diol (arabidiol), whose structure was determined by NMR and MS analyses. Its epoxide analogue, (3S,13R,21S)-malabarica-17-en-20,21-epoxy-3,14-diol (arabidiol 20,21-epoxide), was also isolated from the transformed yeast. This is the first example of a triterpene synthase that yields a tricyclic triterpene with two hydroxyl groups.

Lanosterol synthase in dicotyledonous plants.

Sterols are important as structural components of plasma membranes and precursors of steroidal hormones in both animals and plants. Plant sterols show a wide structural variety and significant structural differences from those of animals. To elucidate the origin of structural diversity in plant sterols, their biosynthesis has been extensively studied [Benveniste (2004) Annu. Rev. Plant. Biol. 55: 429, Schaller (2004) Plant Physiol. Biochem. 42: 465]. The differences in the biosynthesis of sterols between plants and animals begin at the step of cyclization of 2,3-oxidosqualene, which is cyclized to lanosterol in animals and to cycloartenol in plants. However, here we show that plants also have the ability to synthesize lanosterol directly from 2,3-oxidosqualene, which may lead to a new pathway to plant sterols. The Arabidopsis gene At3g45130, designated LAS1, encodes a functional lanosterol synthase in plants. A phylogenetic tree showed that LAS1 belongs to the previously uncharacterized branch of oxidosqualene cyclases, which differs from the cycloartenol synthase branch. Panax PNZ on the same branch was also shown to be a lanosterol synthase in a yeast heterologous expression system. The higher diversity of plant sterols may require two biosynthetic routes in steroidal backbone formation.

Identification of beta-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay.

Triterpenes exhibit a wide range of structural diversity produced by a sequence of biosynthetic reactions. Cyclization of oxidosqualene is the initial origin of structural diversity of skeletons in their biosynthesis, and subsequent regio- and stereospecific hydroxylation of the triterpene skeleton produces further structural diversity. The enzymes responsible for this hydroxylation were thought to be cytochrome P450-dependent monooxygenase, although their cloning has not been reported. To mine these hydroxylases from cytochrome P450 genes, five genes (CYP71D8, CYP82A2, CYP82A3, CYP82A4 and CYP93E1) reported to be elicitor-inducible genes in Glycine max expressed sequence tags (EST), were amplified by PCR, and screened for their ability to hydroxylate triterpenes (beta-amyrin or sophoradiol) by heterologous expression in the yeast Saccharomyces cerevisiae. Among them, CYP93E1 transformant showed hydroxylating activity on both substrates. The products were identified as olean-12-ene-3beta,24-diol and soyasapogenol B, respectively, by GC-MS. Co-expression of CYP93E1 and beta-amyrin synthase in S. cerevisiae yielded olean-12-ene-3beta,24-diol. This is the first identification of triterpene hydroxylase cDNA from any plant species. Successful identification of a beta-amyrin and sophoradiol 24-hydroxylase from the inducible family of cytochrome P450 genes suggests that other triterpene hydroxylases belong to this family. In addition, substrate specificity with the obtained P450 hydroxylase indicates the two possible biosynthetic routes from triterpene-monool to triterpene-triol.