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Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus
FEBS Letters 585 (2011) 1031–1036
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Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus Satoru Sawai 1, Hiroshi Uchiyama, Syuhei Mizuno, Toshio Aoki, Tomoyoshi Akashi, Shin-ichi Ayabe ⇑, Takeyoshi Takahashi Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan
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Article history: Received 31 January 2011 Revised 27 February 2011 Accepted 28 February 2011 Available online 4 March 2011 Edited by Ulf-Ingo Flügge Keywords: Asteraceae Evolution Oxidosqualene cyclase Shionone Triterpene Aster tataricus
a b s t r a c t Shionone is the major triterpenoid component of Aster tataricus possessing a unique all six-membered tetracyclic skeleton and 3-oxo-4-monomethyl structure. To clarify its biosynthetic process, an oxidosqualene cyclase cDNA was isolated from A. tataricus, and the function of the enzyme was determined in lanosterol synthase-deficient yeast. The cyclase yielded ca. 90% shionone and small amounts of b-amyrin, friedelin, dammara-20,24-dienol, and 4-epishionone and was designated as a shionone synthase (SHS). Transcripts of SHS were detected in A. tataricus organs, confirming its involvement in shionone biosynthesis. SHS was shown to have evolved in the Asteraceae from bamyrin synthase lineages and acquired characteristic species- and product-specificities. Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Plant triterpenes display a remarkable variety of skeletons. They contain mono- to pentacyclic structures, but the most frequently occurring are a tetracyclic system with three six- and one fivemembered rings (designated hereafter as the 6/6/6/5 system) and both pentacyclic 6/6/6/6/6 and 6/6/6/6/5 systems [1,2]. Shionone is the main triterpenoid component of an Asteraceae plant Aster tataricus L. (Japanese name: ‘shion’), the dried roots (Asteris Radix) of which have been used in a traditional oriental medicine to eliminate phlegm and relieve cough [3]. Shionone has a rare 6/6/ 6/6 tetracyclic skeleton and a C-3 carbonyl carbon (Fig. 1). The structure of the A/B/C ring portion of shionone is the same as that of friedelin, another triterpene ketone with a 6/6/6/6/6 pentacyclic system widely distributed in the plant kingdom. In spite of the
Abbreviations: BARS, baruol synthase; BAS, b-amyrin synthase; BOS, baccharis oxide synthase; CAS, cycloartenol synthase; FRS, friedelin synthase; LAS, lanosterol synthase; LUP, lupeol synthase; OSC, oxidosqualene cyclase; SHS, shionone synthase ⇑ Corresponding author. Address: Department of Applied Biological Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. Fax: +81 466 84 3704. E-mail address: [email protected] (S. Ayabe). 1 Present address: RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan. Deceased.
3-oxo structure, the molecular formula (C30H50O) of shionone as well as of friedelin is identical to that of many other cyclic 3hydroxytriterpenes (triterpene monoalcohols), which are biosynthesized directly from 2,3-oxidosqualene in single oxidosqualene cyclase (OSC) reactions. In the classical structural study of shionone done in the 1960s by Ourisson, Takahashi (an author of the present paper), and coworkers, the biogenetic consideration was adopted to elucidate a skeleton conforming to the friedelin type A/B ring and a C6 side chain attached to a tertiary carbon atom [4,5]: i.e., a unique intermediate, the baccharenyl cation [2], was hypothesized for the first time. The structure of shionone was then unambiguously established by chemico-physical methods [5,6]. However, no biochemical or molecular biological study of shionone biosynthesis has yet been published, and thus whether a single enzyme carries out the complex steps of shionone formation or multiple enzymes are involved in its biosynthesis remains unclear. Since the first molecular characterization of Arabidopsis thaliana cycloartenol synthase (CAS) [7], several dozen plant OSC cDNAs have been reported [1]. Single plants have multiple OSCs with various product specificities: for example, A. thaliana has 13 OSC genes in its genome, and their very diverse functions have now all been characterized [8]. The structural diversity of plant triterpenoid skeletons is attributed to both the presence of diverse OSC genes (proteins) and multiple products often observed in single OSC reactions. Molecular phylogenetic analysis has categorized most of the plant OSCs so far identified into four families:
0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.02.037
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Fig. 1. The predicted pathway for the SHS reaction (A) and biosynthesis of baccharis oxide and baruol from baccharenyl cation (B).
lanosterol synthase (LAS) and CAS families of the sterol biosynthetic pathway, lupeol synthase (LUP) family, and b-amyrin synthase (BAS) family [9,10]. The BAS family contains the most diverse OSCs, yielding mainly the pentacycles and multiple products. Until recently, the only plant OSC reported to yield a triterpene with a 6/6/6/6 tetracyclic system was A. thaliana At4g15370 (PEN2), which gives baruol as the major product together with 22 minor products (see Fig. 1B) [11], except that recombinant Pisum sativum BAS produced bacchar-12-en-3b-ol from the modified substrate 22,23-dihydro-2,3-oxidosqualene [12]. Subsequently, while our study on A. tataricus OSCs was in progress, baccharis oxide synthase (BOS) was cloned from Stevia rebaudiana, also as a multiproduct enzyme [13]. On the other hand, very recently, characterization of friedelin synthase (FRS) from Kalanchoe daigremontiana was reported [14]. In the FRS reaction, up to 10 rearrangement steps are presumed to yield friedelin from 2,3oxidosqualene. Shionone should also be synthesized by a very similar reaction mechanism, but the formation of shionone among the products of any OSC has never been documented. We have identified an A. tataricus OSC that yields shionone as the major product and report it here. The evolutionary aspects of this and related plant OSCs are discussed based on the mechanistic consideration and phylogenetic analysis. 2. Materials and methods 2.1. Cloning of Zw1 cDNA A. tataricus plants were grown in our University garden. mRNA was isolated from its underground parts using the Straight A’s
mRNA isolation system (Novagen). The preparation of cDNA templates and RACE PCR were performed using a Marathon cDNA Amplification Kit (Clontech). The Zw1 cDNA was obtained by a series of nested degenerate PCR, 50 - and 30 -RACE PCRs and a final amplification of the full ORF. The PCR primers and reaction conditions are listed in Supplementary Table S1. Briefly, after the two rounds of degenerate PCRs with the primer sets d111S/d610As and d158S/d561AS, the PCR product was cloned into a pT7-Blue T vector (Novagen). The RACE PCRs with the primer sets ZwAS2/ AP1 (50 -RACE) and ZwS2/AP1 (30 -RACE), respectively, were then done, and the nested RACE PCRs with ZwAS1/AP2 (50 -RACE) and ZwS1/AP2 (30 -RACE) were followed. The full ORF of Zw1 was amplified from the cDNA template, and cloned into the pT7-Blue T vector. This Zw1 ORF was transferred to HindIII and XhoI sites of the pYES2 (Invitrogen) by PCR subcloning with KOD plus DNA polymerase (Toyobo, Osaka, Japan) with a set of primers containing either a HindIII (Zw1_FUH1) or XhoI site (Zw1_FLX1). 2.2. Functional expression of Zw1 cDNA in yeast and analysis of triterpene products Saccharomyces cerevisiae GIL77 (gal2, hem3-6, erg7, ura3-167) strain [15] was transformed with Zw1 cDNA cloned into pYES2 using Frozen-EZ Yeast Transformation II (Zymo Research). The transformant was cultured in synthetic complete medium without uracil supplemented with 20 lg/ml ergosterol, 5 mg/ml Tween 80 and 13 lg/ml hemin for 2 days at 28 °C. The collected cells were resuspended in the same medium containing 2% galactose instead of glucose to induce the recombinant cDNA and cultured for 2 more days. Finally, the cells were incubated in 0.1 M K-Pi buffer
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(pH 7.0) supplemented with 3% glucose for 1 day before they were harvested. For the pilot experiment, 400 ml medium at each stage was employed, and for the identification of the Zw1 reaction products, a large-scale culture using 4.8 l of medium was performed. The cells harvested were refluxed with 20% KOH in 50% (v/v) EtOH for 1 h and extracted with n-hexane. The n-hexane extract from 70.3 g of transformed yeast cells was separated by silica gel (Wako gel C-200) column chromatography with n-hexane:ethyl acetate (9:1 v/v) as an eluent. The fractions were monitored by TLC using a silica gel 60 F254 TLC plate (Merck) with n-hexane:ethyl acetate (9:1 v/v) and visualized by spraying with 10% H2SO4 and heating at 100 °C. The major triterpene ketone fraction containing shionone and a minor fraction composed of 4-epishionone were purified by silica gel TLC. The triterpene monoalcohol fraction was purified by silica gel TLC and HPLC using a TSK-GEL ODS-80TM column (Tosoh, Tokyo, Japan) with 85% (v/v) acetonitrile for the eluent. The isolation yields of the triterpene ketone fraction containing shionone, the other ketone fraction containing 4-epishionone, and the triterpene monoalcohol fraction after the preparative TLC were 13.6, 0.2, and 0.2 mg, respectively. 2.3. Expression analysis of SHS (shionone synthase) and BAS-like2 genes in A. tataricus organs Total RNA from roots, stolons, rhizomes, radical leaves, stems, leaves, and flower heads of A. tataricus were isolated by a CTAB method and purified using an RNeasy Plant Mini Kit (QIAGEN), and contaminated genomic DNA was digested using an RNaseFree DNase Set (Qiagen). First strand cDNA synthesis was performed using a SuperScript III First-Strand Synthesis System (Life Technologies). Semi-quantitative RT-PCR was done with GoTaq Green Master Mix (Promega). For expression analysis of SHS, two different sets of gene-specific primers from the 30 -UTR region (AstSHS3Uf1/AstSHS3Ur1) and from the coding region (AstSHSf2/AstSHSr2) were designed (for the primer sequences and the PCR conditions, see Supplementary Table S1). Expression analysis of the BAS-like2 gene was also performed, and as an internal control, the actin gene was amplified. 2.4. Phylogenetic analysis
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an unusual partial sequence around the conserved DCTAE motif (including the catalytic Asp) of OSCs [18]: i.e., it showed the amino acid sequence of LSDCTTI in contrast to the [V/I]SDCTAE of the common plant OSCs (the changed amino acid residues are underlined). Because the catalytic Asp at the active site of OSC proteins is located proximal to the oxygen atom of 2,3oxidosqualene, the unusual sequence near this residue could affect the product structure around the A-ring. Given the characteristic 3-oxo-4-monomethyl A-ring structure of shionone, it seemed reasonable to assume that this sequence is part of the target OSC. Thus, we concentrated on this fragment and carried out further 50 - and 30 -RACE PCRs. As a result, a full length Zw1 cDNA of 2286 bp long encoding a polypeptide of 761 amino acid residues was cloned (Supplementary Fig. S1; database ID, AB609123 (AtaSHS); Zw named after ‘Ziwan,’ which is Chinese for the drug Asteris Radix). For the functional analysis of Zw1, the cells of yeast GIL77 [15] devoid of LAS were transformed with an expression vector harboring Zw1, and the constituents produced instead of lanosterol from 2,3-oxidosqualene by Zw1 protein were analyzed. Thin-layer chromatograms of the n-hexane extract of the transformed GIL77 after saponification showed a major spot of products that are less polar than triterpene monoalcohols and other minor spots including the spot of triterpene monoalcohols (Fig. 2A and Supplementary Fig. S2). The fraction (Fr. 1) from the silica gel column chromatography of the extract from the large-scale (4.8 l) culture of Zw1/ GIL77 giving the major spot was composed substantially of a single compound according to HPLC analysis (Fig. 2B). This product was identified as shionone by comparison with an authentic sample as well as by its electron ionization-MS [m/z (relative intensity) 426 (M+, 100), 411 (11), 341 (40), 273 (13)] [19], 1H NMR, and 13 C NMR data (Supplementary Table S3). The triterpene monoalcohol fraction (Fr. 3 in Fig. 2B) contained two major compounds at roughly equal levels by HPLC analysis, and they were identified as b-amyrin and dammara-20,24-dienol through comparison of spectral data of a standard b-amyrin sample and the record in the literature (Supplementary Table S3) [20,21]. The fraction eluted between the triterpene ketone and monoalcohol fractions (Fr. 2 in Fig. 2B) displayed a 1H NMR spectrum almost identical to the spectrum of shionone except that a secondary-CH3 at C-4 (CH3-23) appeared at d 1.12 and the CH3-24
Forty-seven nucleotide sequences of BAS and LUP (as outgroup) family genes of which the catalytic functions have been characterized were used for phylogenetic analysis (Supplementary Table S2). Phylogenetic and molecular evolutionary analyses were conducted using MEGA ver. 4 [16] and ver. 5-Beta. Multiple alignment of nucleotide sequences was generated from aligned amino acid sequences with Muscle [17] using default parameters. A neighbor-joining (NJ) tree was constructed using the maximum composite likelihood model and pairwise deletion of alignment gaps. The estimated reliability of the phylogenetic tree was tested by the bootstrap method (1000 replications).
3. Results 3.1. Cloning and characterization of Zw1 (SHS cDNA) from A. tataricus By PCR using nested degenerate primer sets designed from common sequences of known plant OSCs with the cDNA pool prepared from A. tataricus roots as the template, ca. 1.2 kb fragments were amplified, and cloned into the vector. Twenty transformed Escherichia coli colonies were picked up, and the inserted cDNA fragments were sequenced. They were composed of three BAS-like and one CAS-like sequences. The BAS-like sequence that was the most abundantly amplified (found in 17 colonies) had
Fig. 2. TLC of the hexane extract of yeast GIL77 transformed with Zw1 (A) and HPLC charts of the silica gel column chromatography fractions (B). Fr. 1 contained shionone, Fr. 2 eluted between Frs. 1 and 3 and consisted of 4-epishionone, and Fr. 3 was composed of triterpene monoalcohols. The HPLC conditions were as follows: Fr. 1, 100% acetonitrile at a flow rate of 1 ml/min; Fr. 2, 90% acetonitrile at a flow rate of 1.5 ml/min; Fr. 3, 85% acetonitrile at a flow rate of 1.5 ml/min.
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at C-5 was at d 0.92. These downfield shifts can be explained if the secondary-CH3 is located at 4a with an axial orientation, and this configuration was supported by the assignments of 13C and 1H signals for C-2 to C-4 parts by 2D-NMR techniques and particularly by the observation of NOE between CH3-23 and H-2a (ax) [d 2.46 (1H, m)] (Supplementary Fig. S3). Therefore, the product was determined to be 4-epishionone, which is not known as a natural product [22]. In addition, when examined carefully, the HPLC chromatogram of the triterpene ketone fraction (Fig. 2B, Fr. 1) exhibited a tiny peak at the same retention time (18.27 min) as the standard friedelin sample, and the 1H NMR spectrum of Fr. 1 displayed small signals assignable to protons of CH3-24, CH3-26, CH3-27, CH3-28, CH3-29, and CH3-30 of friedelin (Supplementary Fig. S4). Therefore, although it was not isolated, the Zw1/GIL77 very likely produced a small amount of a triterpene ketone friedelin together with a predominant amount of shionone. Deduced from the isolation yields of the column chromatography and HPLC fractions from a large scale culture of the yeast, the ratio of the products was roughly estimated as shionone:bamyrin:friedelin:dammara-20,24-dienol:4-epishionone = 90%:2%: 2%:1.5%:1.5%. None of these products was detected in the control yeast harboring the empty vector (Supplementary Fig. S2). Thus, the enzyme encoded by Zw1 was designated as a SHS that gives rise to small amounts of byproducts.
3.2. Phylogenetic relationship between SHS and other OSCs The SHS cDNA sequence exhibited the highest identity, 83% and 80% at the nucleotide and amino acid levels, with BOS cDNA of S. rebaudiana (Asteraceae) [13]. Also, it was almost equally identical, i.e., 79% and 77% at the nucleotide and amino acid levels, respectively, to BAS cDNAs of two other Asteraceae plants, Aster sedifolius [23] and Artemisia annua [24]. In contrast, only low identities (<70%) were demonstrated between SHS cDNA and A. thaliana baruol synthase (BARS1) cDNA [11] or K. daigremontiana FRS cDNA [14] (Supplementary Table S4). Fig. 3A shows the molecular phylogenetic tree based on the nucleotide sequences of SHS and related OSC genes from dicotyledonous plants using LUP family genes as the outgroup (see also Supplementary Fig. S5). SHS and BOS form a distinct clade together with two BASs of the Asteraceae plants. BASs from two other asterid plants, Panax ginseng and Gentiana straminea, are also positioned closely and form a monophyletic clade with Asteraceae OSCs. FRS is among the four OSCs cloned from K. daigremontiana, all of which are clustered with BASs and multifunctional OSCs of various plants (although none of the Kalanchoe OSCs produce b-amyrin as the main product). BARS1 (PEN2) is in the A. thaliana-specific branch composed of PENs, which are a unique group of enzymes of a rapidly evolving lineage containing those that yield tricyclic and A- and C-ring cleaved triterpenes.
Fig. 3. Molecular phylogenetic trees of SHS and related plant OSCs. (A) A tree of selected full length nucleotide sequences. (B) A tree of the nucleotide sequences of the degenerate RT-PCR products (ca. 1 kb) from A. tataricus roots and corresponding regions of known sequences.
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Fig. 4. RT-PCR detection of SHS and BAS-like2 sequences in different organs of A. tataricus. The patterns of band intensities from the two primer sets for SHS were almost identical, indicating that the fragments originating from the same SHS mRNA were amplified. Almost equal intensities of actin bands of all organs guarantees the semi-quantitative detection of the cDNA originating from each mRNA existed in the organs.
The nucleotide sequences (ca. 1 kb) of the fragments amplified by the degenerate RT-PCR on the mRNA of A. tataricus roots were also analyzed (Fig. 3B). In addition to the partial SHS sequence (SHS), two BAS-like sequences were obtained. One of these fragments (BAS-like1) was more closely related to SHS, while the other (BAS-like2) was more similar to A. sedifolius BAS, and the latter was estimated to be the partial sequence of an A. tataricus BAS. 3.3. Expression of SHS gene in A. tataricus organs Semi-quantitative RT-PCR was performed to examine the expression of SHS and the tentative A. tataricus BAS (BAS-like2) genes in different organs of mature A. tataricus. Two sets of primers for the SHS coding region and the 30 -UTR, respectively, were used in order to exclude the possibility of amplification of homologous gene transcripts. As shown in Fig. 4, transcripts of both SHS and BAS-like2 were detected in all organs, but the expression magnitudes were characteristically different. SHS was highly expressed in the aerial parts (stems, leaves, and flowers) except radical leaves, and moderately in the roots, whereas lower expression was demonstrated in stolons, rhizomes, and radical leaves. In contrast, the expression pattern of BAS-like2 was almost the same in all organs examined (see also Supplementary Fig. S6). 4. Discussion The foregoing results show that a single OSC, SHS, constructs the shionone structure from 2,3-oxidosqualene, clearly confirming the biosynthetic scheme for shionone more than 40 years after it was first proposed [4,5]. In the reaction catalyzed by SHS, an early intermediate 6/6/6/5 dammarenyl cation is converted to a 6/6/6/6 baccharenyl cation, from which extensive rearrangement steps, i.e., 1,2-shifts of three hydrides and four methyls (H-13b to C-18, CH314a to C-13, CH3-8b to C-14, H-9a to C-8, CH3-10b to C-9, H-5a to C-10, and CH3-4b to C-5), yield the direct shionone precursor with a positive charge at C-4, tentatively named the ‘C-4 cation’. Deprotonation of the C-4 cation from H-3a results in the enol form of shionone, and it may be isomerized non-enzymatically into the keto forms, shionone and 4-epishionone. While a hydride shift (H-3a to C-4) in the C-4 cation and deprotonation from the 3hydroxyl group can produce shionone, the observation of 4epishionone among the reaction products suggests that the shionone enol form is the direct enzyme product. The very high ratio of shionone compared to 4-epishionone should be due to the unfavorable diaxial interaction of CH3-4a and H-10a of 4-epishionone caused by the deformation of the A ring that is brought about by another 1,3-diaxial CH3-5b and CH3-9b (see Supplementary Fig. S3) [22].
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Fig. 5. Comparison of active-site residues (shaded) in human LAS [34] and corresponding residues of SHS and related OSCs (after [8]). Magenta and green shaded residues in human LAS indicate residues that interact with A- and B-rings, and C- and D-rings, respectively.
Shionone is the main triterpenoid constituent of dried A. tataricus roots, and very limited information regarding its occurrence in plants other than A. tataricus is available [25–27]. The observed accumulation of SHS transcript in the A. tataricus organs including the roots (Fig. 4) indicates that the SHS cDNA encodes the functional enzyme of this plant species. Baccharis oxide, which should be produced by the same pathway to shionone until two steps before the C-4 cation (Fig. 1B), has been reported from the Baccharis species (Asteraceae) [28,29], and a few other Asteraceae plants including S. rebaudiana appear to be its producer [13,30]. SHS and BOS should have evolved specifically in the Asteraceae, and SHS must have acquired a high product specificity, due to a rather strict controls of the reaction pathway at the branching points (see Fig. 1), yielding ca. 90% shionone. Baruol is also the product of the same pathway as SHS until the formation of the C-5 cation (Fig. 1B), and BARS1 was characterized among the OSCs of A. thaliana [11], but production of baruol in A. thaliana has not yet been reported. On the other hand, friedelin has been known since the 19th century as a constituent of a very wide variety of monocotyledonous and dicotyledonous plants with bryophytes and gymnosperms [31,32], and therefore, a ubiquitous occurrence of the OSC that yields friedelin can be predicted. Based on the proposed intrinsic versatility of the product formation in OSC reactions, i.e., the multiple products of OSC reactions due to the mechanistic diversity are the default state of triterpene synthesis [11], it may be reasonable to presume that the K. daigremontiana FRS is the first reported of a diverse array of OSCs that have acquired, or more likely retained, the activity of friedelin production [14]. One of the characteristic features of the SHS reaction, shared with BOS and BARS1, is the predominant formation of a baccharane skeleton rather than moving on to pentacyclic systems, and another, in common with FRS, is the extensive methyl and hydride rearrangements that are not terminated by trapping the intermediate cations until formation of the characteristic A-ring structure. When the amino acid sequences of these OSCs are compared, the conserved Val (most BAS family OSCs and LAS) or Ile (CAS) at the position two amino acids upstream to the catalytic Asp (included in the DCTAE motif) is changed to Leu in SHS and FRS or Thr in BOS (Fig. 5). While the hydrophilic Thr residue in BOS is implicated in flipping the A-ring of the immediate precursor of baccharis oxide up to enable the formation of an ether linkage between C-3–C-10 [13], the bulkiness and the shape of the residue here can be an important factor for the characteristic product formation [33]. Also, conserved Y237 and F521 (human LAS numbering) near the product C–D ring portion based on the human LAS stereostructure [34] in many BAS-like OSCs are changed into Phe and Ile in both SHS and BARS1. However, phylogenetically closer SHS and BOS do not share these characteristic amino acid substitutions, and, thus, subtle conformational changes of the proteins would also dynamically affect the reaction course in these versatile multiproduct OSCs. Further mechanistic studies by site-directed mutagenesis as well
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as in planta expressions of the wild type and mutated gene should be interesting from the viewpoints of the versatility of secondary metabolism and its possible ecological functions. Acknowledgments Dr. Takeyoshi Takahashi (Professor Emeritus, University of Tokyo) passed away in December 22, 2010, when the original manuscript of this paper was almost completed. We dedicate this paper to the memory of Professors Guy Ourisson (1926–2006) and Takeyoshi Takahashi (1926–2010). We thank Dr. Yoshihiko Moriyama (Professor Emeritus, Kyoto Prefectural University of Medicine) for the shionone sample and critical reading of the manuscript, Dr. Yutaka Ebizuka (Professor Emeritus, University of Tokyo) for the yeast strain GIL77 and information on baccharis oxide synthase, and Dr. Hiroshi Hirota (RIKEN) for useful discussions. This study was supported by a Grant-in-Aid for Scientific Research (No. 21510233) from Japan Society for the Promotion of Science to S.A.
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mechanisms: a challenge to prevailing concepts of triterpene biosynthesis. J. Am. Chem. Soc. 129, 11213–11222. Abe, I., Sakano, Y., Tanaka, H., Lou, W., Noguchi, H., Shibuya, M. and Ebizuka, Y. (2004) Enzymatic cyclization of 22, 23-dihydro-2,3-oxidosqualene into euph7-en-3b-ol and bacchar-12-en-3b-ol by recombinant b-amyrin synthase. J. Am. Chem. Soc. 126, 3426–3427. Shibuya, M., Sagara, A., Saitoh, A., Kushiro, T. and Ebizuka, Y. (2008) Biosynthesis of baccharis oxide, a triterpene with a 3, 10-oxide bridge in the A-ring. Org. Lett. 10, 5071–5074. Wang, Z., Yeats, T., Han, H. and Jetter, R. (2010) Cloning and characterization of oxidosqualene cyclases from Kalanchoe daigremontiana: enzymes catalyzing up to 10 rearrangement steps yielding friedelin and other triterpenoids. J. Biol. Chem. 285, 29703–29712. Kushiro, T., Shibuya, M. and Ebizuka, Y. (1998) b-Amyrin synthase – cloning of oxidosqualene cyclase that catalyzes the formation of the most popular triterpene among higher plants. Eur. J. Biochem. 256, 238–244. Tamura, K., Dudley, J., Nei, M. and Kumar, S. (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596–1599. Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797. Abe, I. and Prestwich, G.D. (1994) Active site mapping of affinity-labeled rat oxidosqualene cyclase. J. Biol. Chem. 269, 802–804. Hirota, H., Moriyama, Y., Tsuyuki, T., Tanahashi, Y., Takahashi, T., Katoh, Y. and Satoh, H. (1975) The high resolution mass spectra of shionane and friedelane derivatives. Bull. Chem. Soc. Jpn. 48, 1884–1888. Kushiro, T., Shibuya, M., Masuda, K. and Ebizuka, Y. (2000) Mutational studies on triterpene syntheses: engineering lupeol synthase into b-amyrin synthase. J. Am. Chem. Soc. 122, 6816–6824. Leong, Y.W. and Harrison, L.J. (1999) (20R, 23E)-Eupha-8, 23-diene-3b, 25-diol from Tripetalum cymosum. Phytochemistry 50, 849–857. Aoyagi, R., Yamada, S., Tsuyuki, T. and Takahashi, T. (1973) 4-Epifriedelin and 4-epishionone. Structure and reflex effect. Bull. Chem. Soc. Jpn. 46, 959–963. Cammareri, M., Consiglio, M.F., Pecchia, P., Corea, G., Lanzotti, V., Ibeas, J.I., Tava, A. and Conicella, C. (2008) Molecular characterization of b-amyrin synthase from Aster sedifolius L. and triterpenoid saponin analysis. Plant Sci. 175, 255–261. Kirby, J., Romanini, D.W., Paradise, E.M. and Keasling, J.D. (2008) Engineering triterpene production in Saccharomyces cerevisiae – b-amyrin synthase from Artemisia annua. FEBS J. 275, 1852–1859. Bohlmann, F., Grenz, M., Dahr, A.K. and Goodman, M. (1981) Labdane derivatives and flavones from Gutierrezia dracunculoides. Phytochemistry 20, 105–107. Tan, R.X., Hu, Y.H., Liu, Z.L. and Pan, X. (1993) New kaurane diterpenoids from Aster tongolensis. J. Nat. Prod. 56, 1917–1922. Hui, W.H., Lam, W.K. and Tye, S.M. (1971) Triterpenoid and steroid constituents of Aster baccharoides. Phytochemistry 10, 903–904. Anthonsen, T., Bruun, T., Hemmer, E., Holme, D., Lamvik, A., Sunde, E. and Sørensen, N.A. (1970) Baccharis oxide, a new triterpenoid from Baccharis halimifolia L. Acta Chem. Scand. 24, 2479–2488. Nurnberg, V., Fujiwara, F.Y. and Marsaioli, A.J. (1998) Total NMR chemical shift assignments of baccharis oxide via 2D-INADEQUATE. Magn. Reson. Chem. 36, 766–768. Bohlmann, F., Zdero, C., King, R.M. and Robinson, H. (1984) Gutierrezial and further diterpenes from Gutierrezia sarothrae. Phytochemistry 23, 2007–2012. Chandler, R.F. and Hooper, S.N. (1979) Friedelin and associated triterpenoids. Phytochemistry 18, 711–724. Plouvier, V., Brouard, J.P. and Martin, M.T. (1992) n-Alkanes, friédéline et neooleane-3(5)12 diene chez les végétaux. Bull. Soc. Bot. Fr., Lettres Bot. 139, 307–319. Segura, M.J.R., Jackson, B.E. and Matsuda, S.P.T. (2003) Mutagenesis approaches to deduce structure-function relationships in terpene synthases. Nat. Prod. Rep. 20, 304–317. Thoma, R., Schulz-Gasch, T., D’Arcy, B., Benz, J., Aebi, J., Dehmlow, H., Hennig, M., Stihle, M. and Ruf, A. (2004) Insight into steroid scaffold formation from the structure of human oxidosqualene cyclase. Nature 432, 118–122.
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Molecular characterization of an oxidosqualene cyclase that yields shionone, a unique tetracyclic triterpene ketone of Aster tataricus Satoru Sawai 1, Hiroshi Uchiyama, Syuhei Mizuno, Toshio Aoki, Tomoyoshi Akashi, Shin-ichi Ayabe ⇑, Takeyoshi Takahashi Department of Applied Biological Sciences, Nihon University, Fujisawa, Kanagawa 252-0880, Japan
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Article history: Received 31 January 2011 Revised 27 February 2011 Accepted 28 February 2011 Available online 4 March 2011 Edited by Ulf-Ingo Flügge Keywords: Asteraceae Evolution Oxidosqualene cyclase Shionone Triterpene Aster tataricus
a b s t r a c t Shionone is the major triterpenoid component of Aster tataricus possessing a unique all six-membered tetracyclic skeleton and 3-oxo-4-monomethyl structure. To clarify its biosynthetic process, an oxidosqualene cyclase cDNA was isolated from A. tataricus, and the function of the enzyme was determined in lanosterol synthase-deficient yeast. The cyclase yielded ca. 90% shionone and small amounts of b-amyrin, friedelin, dammara-20,24-dienol, and 4-epishionone and was designated as a shionone synthase (SHS). Transcripts of SHS were detected in A. tataricus organs, confirming its involvement in shionone biosynthesis. SHS was shown to have evolved in the Asteraceae from bamyrin synthase lineages and acquired characteristic species- and product-specificities. Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction Plant triterpenes display a remarkable variety of skeletons. They contain mono- to pentacyclic structures, but the most frequently occurring are a tetracyclic system with three six- and one fivemembered rings (designated hereafter as the 6/6/6/5 system) and both pentacyclic 6/6/6/6/6 and 6/6/6/6/5 systems [1,2]. Shionone is the main triterpenoid component of an Asteraceae plant Aster tataricus L. (Japanese name: ‘shion’), the dried roots (Asteris Radix) of which have been used in a traditional oriental medicine to eliminate phlegm and relieve cough [3]. Shionone has a rare 6/6/ 6/6 tetracyclic skeleton and a C-3 carbonyl carbon (Fig. 1). The structure of the A/B/C ring portion of shionone is the same as that of friedelin, another triterpene ketone with a 6/6/6/6/6 pentacyclic system widely distributed in the plant kingdom. In spite of the
Abbreviations: BARS, baruol synthase; BAS, b-amyrin synthase; BOS, baccharis oxide synthase; CAS, cycloartenol synthase; FRS, friedelin synthase; LAS, lanosterol synthase; LUP, lupeol synthase; OSC, oxidosqualene cyclase; SHS, shionone synthase ⇑ Corresponding author. Address: Department of Applied Biological Sciences, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-0880, Japan. Fax: +81 466 84 3704. E-mail address: [email protected] (S. Ayabe). 1 Present address: RIKEN Plant Science Center, Yokohama, Kanagawa 230-0045, Japan. Deceased.
3-oxo structure, the molecular formula (C30H50O) of shionone as well as of friedelin is identical to that of many other cyclic 3hydroxytriterpenes (triterpene monoalcohols), which are biosynthesized directly from 2,3-oxidosqualene in single oxidosqualene cyclase (OSC) reactions. In the classical structural study of shionone done in the 1960s by Ourisson, Takahashi (an author of the present paper), and coworkers, the biogenetic consideration was adopted to elucidate a skeleton conforming to the friedelin type A/B ring and a C6 side chain attached to a tertiary carbon atom [4,5]: i.e., a unique intermediate, the baccharenyl cation [2], was hypothesized for the first time. The structure of shionone was then unambiguously established by chemico-physical methods [5,6]. However, no biochemical or molecular biological study of shionone biosynthesis has yet been published, and thus whether a single enzyme carries out the complex steps of shionone formation or multiple enzymes are involved in its biosynthesis remains unclear. Since the first molecular characterization of Arabidopsis thaliana cycloartenol synthase (CAS) [7], several dozen plant OSC cDNAs have been reported [1]. Single plants have multiple OSCs with various product specificities: for example, A. thaliana has 13 OSC genes in its genome, and their very diverse functions have now all been characterized [8]. The structural diversity of plant triterpenoid skeletons is attributed to both the presence of diverse OSC genes (proteins) and multiple products often observed in single OSC reactions. Molecular phylogenetic analysis has categorized most of the plant OSCs so far identified into four families:
0014-5793/$36.00 Ó 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2011.02.037
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Fig. 1. The predicted pathway for the SHS reaction (A) and biosynthesis of baccharis oxide and baruol from baccharenyl cation (B).
lanosterol synthase (LAS) and CAS families of the sterol biosynthetic pathway, lupeol synthase (LUP) family, and b-amyrin synthase (BAS) family [9,10]. The BAS family contains the most diverse OSCs, yielding mainly the pentacycles and multiple products. Until recently, the only plant OSC reported to yield a triterpene with a 6/6/6/6 tetracyclic system was A. thaliana At4g15370 (PEN2), which gives baruol as the major product together with 22 minor products (see Fig. 1B) [11], except that recombinant Pisum sativum BAS produced bacchar-12-en-3b-ol from the modified substrate 22,23-dihydro-2,3-oxidosqualene [12]. Subsequently, while our study on A. tataricus OSCs was in progress, baccharis oxide synthase (BOS) was cloned from Stevia rebaudiana, also as a multiproduct enzyme [13]. On the other hand, very recently, characterization of friedelin synthase (FRS) from Kalanchoe daigremontiana was reported [14]. In the FRS reaction, up to 10 rearrangement steps are presumed to yield friedelin from 2,3oxidosqualene. Shionone should also be synthesized by a very similar reaction mechanism, but the formation of shionone among the products of any OSC has never been documented. We have identified an A. tataricus OSC that yields shionone as the major product and report it here. The evolutionary aspects of this and related plant OSCs are discussed based on the mechanistic consideration and phylogenetic analysis. 2. Materials and methods 2.1. Cloning of Zw1 cDNA A. tataricus plants were grown in our University garden. mRNA was isolated from its underground parts using the Straight A’s
mRNA isolation system (Novagen). The preparation of cDNA templates and RACE PCR were performed using a Marathon cDNA Amplification Kit (Clontech). The Zw1 cDNA was obtained by a series of nested degenerate PCR, 50 - and 30 -RACE PCRs and a final amplification of the full ORF. The PCR primers and reaction conditions are listed in Supplementary Table S1. Briefly, after the two rounds of degenerate PCRs with the primer sets d111S/d610As and d158S/d561AS, the PCR product was cloned into a pT7-Blue T vector (Novagen). The RACE PCRs with the primer sets ZwAS2/ AP1 (50 -RACE) and ZwS2/AP1 (30 -RACE), respectively, were then done, and the nested RACE PCRs with ZwAS1/AP2 (50 -RACE) and ZwS1/AP2 (30 -RACE) were followed. The full ORF of Zw1 was amplified from the cDNA template, and cloned into the pT7-Blue T vector. This Zw1 ORF was transferred to HindIII and XhoI sites of the pYES2 (Invitrogen) by PCR subcloning with KOD plus DNA polymerase (Toyobo, Osaka, Japan) with a set of primers containing either a HindIII (Zw1_FUH1) or XhoI site (Zw1_FLX1). 2.2. Functional expression of Zw1 cDNA in yeast and analysis of triterpene products Saccharomyces cerevisiae GIL77 (gal2, hem3-6, erg7, ura3-167) strain [15] was transformed with Zw1 cDNA cloned into pYES2 using Frozen-EZ Yeast Transformation II (Zymo Research). The transformant was cultured in synthetic complete medium without uracil supplemented with 20 lg/ml ergosterol, 5 mg/ml Tween 80 and 13 lg/ml hemin for 2 days at 28 °C. The collected cells were resuspended in the same medium containing 2% galactose instead of glucose to induce the recombinant cDNA and cultured for 2 more days. Finally, the cells were incubated in 0.1 M K-Pi buffer
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(pH 7.0) supplemented with 3% glucose for 1 day before they were harvested. For the pilot experiment, 400 ml medium at each stage was employed, and for the identification of the Zw1 reaction products, a large-scale culture using 4.8 l of medium was performed. The cells harvested were refluxed with 20% KOH in 50% (v/v) EtOH for 1 h and extracted with n-hexane. The n-hexane extract from 70.3 g of transformed yeast cells was separated by silica gel (Wako gel C-200) column chromatography with n-hexane:ethyl acetate (9:1 v/v) as an eluent. The fractions were monitored by TLC using a silica gel 60 F254 TLC plate (Merck) with n-hexane:ethyl acetate (9:1 v/v) and visualized by spraying with 10% H2SO4 and heating at 100 °C. The major triterpene ketone fraction containing shionone and a minor fraction composed of 4-epishionone were purified by silica gel TLC. The triterpene monoalcohol fraction was purified by silica gel TLC and HPLC using a TSK-GEL ODS-80TM column (Tosoh, Tokyo, Japan) with 85% (v/v) acetonitrile for the eluent. The isolation yields of the triterpene ketone fraction containing shionone, the other ketone fraction containing 4-epishionone, and the triterpene monoalcohol fraction after the preparative TLC were 13.6, 0.2, and 0.2 mg, respectively. 2.3. Expression analysis of SHS (shionone synthase) and BAS-like2 genes in A. tataricus organs Total RNA from roots, stolons, rhizomes, radical leaves, stems, leaves, and flower heads of A. tataricus were isolated by a CTAB method and purified using an RNeasy Plant Mini Kit (QIAGEN), and contaminated genomic DNA was digested using an RNaseFree DNase Set (Qiagen). First strand cDNA synthesis was performed using a SuperScript III First-Strand Synthesis System (Life Technologies). Semi-quantitative RT-PCR was done with GoTaq Green Master Mix (Promega). For expression analysis of SHS, two different sets of gene-specific primers from the 30 -UTR region (AstSHS3Uf1/AstSHS3Ur1) and from the coding region (AstSHSf2/AstSHSr2) were designed (for the primer sequences and the PCR conditions, see Supplementary Table S1). Expression analysis of the BAS-like2 gene was also performed, and as an internal control, the actin gene was amplified. 2.4. Phylogenetic analysis
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an unusual partial sequence around the conserved DCTAE motif (including the catalytic Asp) of OSCs [18]: i.e., it showed the amino acid sequence of LSDCTTI in contrast to the [V/I]SDCTAE of the common plant OSCs (the changed amino acid residues are underlined). Because the catalytic Asp at the active site of OSC proteins is located proximal to the oxygen atom of 2,3oxidosqualene, the unusual sequence near this residue could affect the product structure around the A-ring. Given the characteristic 3-oxo-4-monomethyl A-ring structure of shionone, it seemed reasonable to assume that this sequence is part of the target OSC. Thus, we concentrated on this fragment and carried out further 50 - and 30 -RACE PCRs. As a result, a full length Zw1 cDNA of 2286 bp long encoding a polypeptide of 761 amino acid residues was cloned (Supplementary Fig. S1; database ID, AB609123 (AtaSHS); Zw named after ‘Ziwan,’ which is Chinese for the drug Asteris Radix). For the functional analysis of Zw1, the cells of yeast GIL77 [15] devoid of LAS were transformed with an expression vector harboring Zw1, and the constituents produced instead of lanosterol from 2,3-oxidosqualene by Zw1 protein were analyzed. Thin-layer chromatograms of the n-hexane extract of the transformed GIL77 after saponification showed a major spot of products that are less polar than triterpene monoalcohols and other minor spots including the spot of triterpene monoalcohols (Fig. 2A and Supplementary Fig. S2). The fraction (Fr. 1) from the silica gel column chromatography of the extract from the large-scale (4.8 l) culture of Zw1/ GIL77 giving the major spot was composed substantially of a single compound according to HPLC analysis (Fig. 2B). This product was identified as shionone by comparison with an authentic sample as well as by its electron ionization-MS [m/z (relative intensity) 426 (M+, 100), 411 (11), 341 (40), 273 (13)] [19], 1H NMR, and 13 C NMR data (Supplementary Table S3). The triterpene monoalcohol fraction (Fr. 3 in Fig. 2B) contained two major compounds at roughly equal levels by HPLC analysis, and they were identified as b-amyrin and dammara-20,24-dienol through comparison of spectral data of a standard b-amyrin sample and the record in the literature (Supplementary Table S3) [20,21]. The fraction eluted between the triterpene ketone and monoalcohol fractions (Fr. 2 in Fig. 2B) displayed a 1H NMR spectrum almost identical to the spectrum of shionone except that a secondary-CH3 at C-4 (CH3-23) appeared at d 1.12 and the CH3-24
Forty-seven nucleotide sequences of BAS and LUP (as outgroup) family genes of which the catalytic functions have been characterized were used for phylogenetic analysis (Supplementary Table S2). Phylogenetic and molecular evolutionary analyses were conducted using MEGA ver. 4 [16] and ver. 5-Beta. Multiple alignment of nucleotide sequences was generated from aligned amino acid sequences with Muscle [17] using default parameters. A neighbor-joining (NJ) tree was constructed using the maximum composite likelihood model and pairwise deletion of alignment gaps. The estimated reliability of the phylogenetic tree was tested by the bootstrap method (1000 replications).
3. Results 3.1. Cloning and characterization of Zw1 (SHS cDNA) from A. tataricus By PCR using nested degenerate primer sets designed from common sequences of known plant OSCs with the cDNA pool prepared from A. tataricus roots as the template, ca. 1.2 kb fragments were amplified, and cloned into the vector. Twenty transformed Escherichia coli colonies were picked up, and the inserted cDNA fragments were sequenced. They were composed of three BAS-like and one CAS-like sequences. The BAS-like sequence that was the most abundantly amplified (found in 17 colonies) had
Fig. 2. TLC of the hexane extract of yeast GIL77 transformed with Zw1 (A) and HPLC charts of the silica gel column chromatography fractions (B). Fr. 1 contained shionone, Fr. 2 eluted between Frs. 1 and 3 and consisted of 4-epishionone, and Fr. 3 was composed of triterpene monoalcohols. The HPLC conditions were as follows: Fr. 1, 100% acetonitrile at a flow rate of 1 ml/min; Fr. 2, 90% acetonitrile at a flow rate of 1.5 ml/min; Fr. 3, 85% acetonitrile at a flow rate of 1.5 ml/min.
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at C-5 was at d 0.92. These downfield shifts can be explained if the secondary-CH3 is located at 4a with an axial orientation, and this configuration was supported by the assignments of 13C and 1H signals for C-2 to C-4 parts by 2D-NMR techniques and particularly by the observation of NOE between CH3-23 and H-2a (ax) [d 2.46 (1H, m)] (Supplementary Fig. S3). Therefore, the product was determined to be 4-epishionone, which is not known as a natural product [22]. In addition, when examined carefully, the HPLC chromatogram of the triterpene ketone fraction (Fig. 2B, Fr. 1) exhibited a tiny peak at the same retention time (18.27 min) as the standard friedelin sample, and the 1H NMR spectrum of Fr. 1 displayed small signals assignable to protons of CH3-24, CH3-26, CH3-27, CH3-28, CH3-29, and CH3-30 of friedelin (Supplementary Fig. S4). Therefore, although it was not isolated, the Zw1/GIL77 very likely produced a small amount of a triterpene ketone friedelin together with a predominant amount of shionone. Deduced from the isolation yields of the column chromatography and HPLC fractions from a large scale culture of the yeast, the ratio of the products was roughly estimated as shionone:bamyrin:friedelin:dammara-20,24-dienol:4-epishionone = 90%:2%: 2%:1.5%:1.5%. None of these products was detected in the control yeast harboring the empty vector (Supplementary Fig. S2). Thus, the enzyme encoded by Zw1 was designated as a SHS that gives rise to small amounts of byproducts.
3.2. Phylogenetic relationship between SHS and other OSCs The SHS cDNA sequence exhibited the highest identity, 83% and 80% at the nucleotide and amino acid levels, with BOS cDNA of S. rebaudiana (Asteraceae) [13]. Also, it was almost equally identical, i.e., 79% and 77% at the nucleotide and amino acid levels, respectively, to BAS cDNAs of two other Asteraceae plants, Aster sedifolius [23] and Artemisia annua [24]. In contrast, only low identities (<70%) were demonstrated between SHS cDNA and A. thaliana baruol synthase (BARS1) cDNA [11] or K. daigremontiana FRS cDNA [14] (Supplementary Table S4). Fig. 3A shows the molecular phylogenetic tree based on the nucleotide sequences of SHS and related OSC genes from dicotyledonous plants using LUP family genes as the outgroup (see also Supplementary Fig. S5). SHS and BOS form a distinct clade together with two BASs of the Asteraceae plants. BASs from two other asterid plants, Panax ginseng and Gentiana straminea, are also positioned closely and form a monophyletic clade with Asteraceae OSCs. FRS is among the four OSCs cloned from K. daigremontiana, all of which are clustered with BASs and multifunctional OSCs of various plants (although none of the Kalanchoe OSCs produce b-amyrin as the main product). BARS1 (PEN2) is in the A. thaliana-specific branch composed of PENs, which are a unique group of enzymes of a rapidly evolving lineage containing those that yield tricyclic and A- and C-ring cleaved triterpenes.
Fig. 3. Molecular phylogenetic trees of SHS and related plant OSCs. (A) A tree of selected full length nucleotide sequences. (B) A tree of the nucleotide sequences of the degenerate RT-PCR products (ca. 1 kb) from A. tataricus roots and corresponding regions of known sequences.
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Fig. 4. RT-PCR detection of SHS and BAS-like2 sequences in different organs of A. tataricus. The patterns of band intensities from the two primer sets for SHS were almost identical, indicating that the fragments originating from the same SHS mRNA were amplified. Almost equal intensities of actin bands of all organs guarantees the semi-quantitative detection of the cDNA originating from each mRNA existed in the organs.
The nucleotide sequences (ca. 1 kb) of the fragments amplified by the degenerate RT-PCR on the mRNA of A. tataricus roots were also analyzed (Fig. 3B). In addition to the partial SHS sequence (SHS), two BAS-like sequences were obtained. One of these fragments (BAS-like1) was more closely related to SHS, while the other (BAS-like2) was more similar to A. sedifolius BAS, and the latter was estimated to be the partial sequence of an A. tataricus BAS. 3.3. Expression of SHS gene in A. tataricus organs Semi-quantitative RT-PCR was performed to examine the expression of SHS and the tentative A. tataricus BAS (BAS-like2) genes in different organs of mature A. tataricus. Two sets of primers for the SHS coding region and the 30 -UTR, respectively, were used in order to exclude the possibility of amplification of homologous gene transcripts. As shown in Fig. 4, transcripts of both SHS and BAS-like2 were detected in all organs, but the expression magnitudes were characteristically different. SHS was highly expressed in the aerial parts (stems, leaves, and flowers) except radical leaves, and moderately in the roots, whereas lower expression was demonstrated in stolons, rhizomes, and radical leaves. In contrast, the expression pattern of BAS-like2 was almost the same in all organs examined (see also Supplementary Fig. S6). 4. Discussion The foregoing results show that a single OSC, SHS, constructs the shionone structure from 2,3-oxidosqualene, clearly confirming the biosynthetic scheme for shionone more than 40 years after it was first proposed [4,5]. In the reaction catalyzed by SHS, an early intermediate 6/6/6/5 dammarenyl cation is converted to a 6/6/6/6 baccharenyl cation, from which extensive rearrangement steps, i.e., 1,2-shifts of three hydrides and four methyls (H-13b to C-18, CH314a to C-13, CH3-8b to C-14, H-9a to C-8, CH3-10b to C-9, H-5a to C-10, and CH3-4b to C-5), yield the direct shionone precursor with a positive charge at C-4, tentatively named the ‘C-4 cation’. Deprotonation of the C-4 cation from H-3a results in the enol form of shionone, and it may be isomerized non-enzymatically into the keto forms, shionone and 4-epishionone. While a hydride shift (H-3a to C-4) in the C-4 cation and deprotonation from the 3hydroxyl group can produce shionone, the observation of 4epishionone among the reaction products suggests that the shionone enol form is the direct enzyme product. The very high ratio of shionone compared to 4-epishionone should be due to the unfavorable diaxial interaction of CH3-4a and H-10a of 4-epishionone caused by the deformation of the A ring that is brought about by another 1,3-diaxial CH3-5b and CH3-9b (see Supplementary Fig. S3) [22].
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Fig. 5. Comparison of active-site residues (shaded) in human LAS [34] and corresponding residues of SHS and related OSCs (after [8]). Magenta and green shaded residues in human LAS indicate residues that interact with A- and B-rings, and C- and D-rings, respectively.
Shionone is the main triterpenoid constituent of dried A. tataricus roots, and very limited information regarding its occurrence in plants other than A. tataricus is available [25–27]. The observed accumulation of SHS transcript in the A. tataricus organs including the roots (Fig. 4) indicates that the SHS cDNA encodes the functional enzyme of this plant species. Baccharis oxide, which should be produced by the same pathway to shionone until two steps before the C-4 cation (Fig. 1B), has been reported from the Baccharis species (Asteraceae) [28,29], and a few other Asteraceae plants including S. rebaudiana appear to be its producer [13,30]. SHS and BOS should have evolved specifically in the Asteraceae, and SHS must have acquired a high product specificity, due to a rather strict controls of the reaction pathway at the branching points (see Fig. 1), yielding ca. 90% shionone. Baruol is also the product of the same pathway as SHS until the formation of the C-5 cation (Fig. 1B), and BARS1 was characterized among the OSCs of A. thaliana [11], but production of baruol in A. thaliana has not yet been reported. On the other hand, friedelin has been known since the 19th century as a constituent of a very wide variety of monocotyledonous and dicotyledonous plants with bryophytes and gymnosperms [31,32], and therefore, a ubiquitous occurrence of the OSC that yields friedelin can be predicted. Based on the proposed intrinsic versatility of the product formation in OSC reactions, i.e., the multiple products of OSC reactions due to the mechanistic diversity are the default state of triterpene synthesis [11], it may be reasonable to presume that the K. daigremontiana FRS is the first reported of a diverse array of OSCs that have acquired, or more likely retained, the activity of friedelin production [14]. One of the characteristic features of the SHS reaction, shared with BOS and BARS1, is the predominant formation of a baccharane skeleton rather than moving on to pentacyclic systems, and another, in common with FRS, is the extensive methyl and hydride rearrangements that are not terminated by trapping the intermediate cations until formation of the characteristic A-ring structure. When the amino acid sequences of these OSCs are compared, the conserved Val (most BAS family OSCs and LAS) or Ile (CAS) at the position two amino acids upstream to the catalytic Asp (included in the DCTAE motif) is changed to Leu in SHS and FRS or Thr in BOS (Fig. 5). While the hydrophilic Thr residue in BOS is implicated in flipping the A-ring of the immediate precursor of baccharis oxide up to enable the formation of an ether linkage between C-3–C-10 [13], the bulkiness and the shape of the residue here can be an important factor for the characteristic product formation [33]. Also, conserved Y237 and F521 (human LAS numbering) near the product C–D ring portion based on the human LAS stereostructure [34] in many BAS-like OSCs are changed into Phe and Ile in both SHS and BARS1. However, phylogenetically closer SHS and BOS do not share these characteristic amino acid substitutions, and, thus, subtle conformational changes of the proteins would also dynamically affect the reaction course in these versatile multiproduct OSCs. Further mechanistic studies by site-directed mutagenesis as well
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as in planta expressions of the wild type and mutated gene should be interesting from the viewpoints of the versatility of secondary metabolism and its possible ecological functions. Acknowledgments Dr. Takeyoshi Takahashi (Professor Emeritus, University of Tokyo) passed away in December 22, 2010, when the original manuscript of this paper was almost completed. We dedicate this paper to the memory of Professors Guy Ourisson (1926–2006) and Takeyoshi Takahashi (1926–2010). We thank Dr. Yoshihiko Moriyama (Professor Emeritus, Kyoto Prefectural University of Medicine) for the shionone sample and critical reading of the manuscript, Dr. Yutaka Ebizuka (Professor Emeritus, University of Tokyo) for the yeast strain GIL77 and information on baccharis oxide synthase, and Dr. Hiroshi Hirota (RIKEN) for useful discussions. This study was supported by a Grant-in-Aid for Scientific Research (No. 21510233) from Japan Society for the Promotion of Science to S.A.
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