FEBS Letters 582 (2008) 310–318 Squalene cyclase and oxidosqualene cyclase from a fern Junichi Shinozakia,1, Masaaki Shibuyaa, Kazuo Masudab, Yutaka Ebizukaa,* a Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan b Showa Pharmaceutical University, 3-3165 Higashi-Tamagawagakuen, Machida, Tokyo 194-8543, Japan Received 16 November 2007; revised 7 December 2007; accepted 9 December 2007 Available online 26 December 2007 Edited by Ulf-Ingo Flu¨gge diploptene (hop-22(29)-ene, 2), and fernene (fern-9(11)-ene, 3) Abstract Ferns are the most primitive vascular plants. The phy- tosterols of ferns are the same as those of higher plants, but they (Fig. 1). They are found in almost all ferns with a few excep- produce characteristic triterpenes. The most distinct feature is tions [1]. These triterpenes lack oxygen functionality at C-3, the lack of oxygen functionality at C-3, suggesting that the tri- which is ubiquitously found in triterpenes of flowering plants. terpenes of ferns may be biosynthesized by direct cyclization of In higher plants, cyclic triterpene skeletons are formed at the squalene. To obtain some insights into the molecular bases for cyclization step of the common substrate (3S)-oxidosqualene the biosynthesis of triterpenes in ferns, we cloned ACX, an (4) by the enzyme termed oxidosqualene cyclase (OSC). Pro- oxidosqualene cyclase homologue, encoding a cycloartenol syn- tonation and opening of epoxide (Fig. 2A) initiate polyene thase (CAS) and ACH, a squalene cyclase homologue, encoding cyclization and subsequent electrophilic additions to the neigh- a 22-hydroxyhopane synthase from Adiantum capillus-veneris. boring double bonds, followed by Wagner–Meerwein rear- Phylogenetic analysis revealed that ACH is located in the cluster rangements to construct varieties of triterpene skeleton. of bacterial SCs, while ACX is in the cluster of higher plant CASs. Thus, all the cyclization products, such as cycloaretenol (5), Ó 2007 Federation of European Biochemical Societies. Published b-amyrin (8), and lupeol (9), retain epoxide oxygen as the b-hy- by Elsevier B.V. All rights reserved. droxyl group at C-3. In contrast, most triterpenes from ferns, with a few exceptions, are of the hydrocarbon type and lack Keywords: Hydroxyhopane synthase; Squalene cyclase; oxygen functionality at C-3. The 3-deoxy-structure is reminis- Oxidosqualene cyclase; Triterpene; Ferns; Adiantum cent of bacterial hopanoids produced from squalene (10) [2–4]. capillus-veneris Some prokaryotes utilize squalene, and not oxidosqualene, as the cyclization substrate to yield 3-deoxy triterpene products (Fig. 2B). Tetrahymanol (11), a pentacyclic gammacerane-type triterpene from the protozoa Tetrahymena pyriformis, also lacks 3-oxygenation and is also a cyclization product of squa- 1. Introduction lene (Fig. 2C). These findings imply that the triterpenes of ferns are formed by direct cyclization of squalene, and thus Ferns are evolutionarily the most primitive vascular plants squalene cyclases (SCs) are present in ferns. and have flourished on earth for more than 400 million years. Prokaryotic SCs yielding pentacyclic triterpenes have been Some 12000 species are widely distributed throughout the extensively studied and their genes were cloned from some bac- world, mostly in temperate to subtropical regions. Differing terial species including Alicyclobacillus acidocaldarius [5], from higher plants (gymnosperms and angiosperms), ferns Zymomonas mobilis [6], Bradyrhizobium japonicum [7], and reproduce via spores and do not flower, making the botanical Methylococcus capsulatus [8]. These SCs show high sequence identification of species rather difficult. This may be one reason identities with each other (34–56%) and lower, but still signif- why only a limited number of species of ferns have been uti- icant, sequence identities (10–20%) with OSCs from eukaryotic lized for medicinal purposes, despite their abundance of un- organisms, suggesting that both SCs and OSCs evolved from a ique secondary metabolites that cannot be found in higher common ancestor. plants. It is interesting to note that prokaryotic SCs accept a non- Triterpenes are isoprenoidal natural products comprising six physiological substrate, oxidosqualene, as well. A protozoan isoprene units. To date, more than 200 triterpene derivatives cyclase from T. pyriformis, responsible for tetrahymanol bio- have been reported from ferns which have structures distinct synthesis from squalene, converted (3S)-oxidosqualene into from those found in higher plants. The most commonly occur- gammacerane-3b,21a-diol and (3R)-enantiomer into 3a-epi- ring triterpenes in ferns are diplopterol (22-hydroxyhopane, 1), mer in vitro [9]. A cell-free preparation of M. capsulatus, a met- hanotrophic bacterium, converted not only squalene into a mixture of hop-22(29)-ene and hydroxyhopane but also *Corresponding author. Fax: +81 3 5841 4744. (RS)-oxidosqualene into a mixture of lanosterol, 3-epi-lanos- E-mail address: [email protected] (Y. Ebizuka). terol, and 3a- and 3b-hydroxyhop-22(29)-ene, respectively [10]. This bacterium is a rare example, in that it produces both 1Present address: Showa Pharmaceutical University, Japan. hopanoids and sterols. One SC clone from this bacterium re- Abbreviations: OSC, oxidosqualene cyclase; SC, squalene cyclase; acted only with squalene, and not with oxidosqualene, when SHC, squalene-hopene cyclase; CAS, cycloartenol synthase expressed in Escherichia coli, suggesting the presence of an 0014-5793/$32.00 Ó 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.12.023 J. Shinozaki et al. / FEBS Letters 582 (2008) 310–318 311 22 22 11 OH 9 29 3 3 3 diplopterol (22-hydroxyhopane, 1) diploptene (hop-22(29)-ene, 2) fernene (fern-9(11)-ene, 3) Fig. 1. Constituents of ferns. (A) Higher plants 3 3 HO HO β-sitosterol (6) cycloartenol (5) + O 3 H HO 2,3(S)-oxidosqualene (4) lanosterol (7) H 3 3 HO HO β-amyrin (8) lupeol (9) (B) Bacteria (Methylococcus capsulatus, Alicyclobacillus acidocaldarius, etc. ) 22 22 OH + 29 3 3 H+ squalene (10) 22-hydroxyhopane (1) hop-22(29)-ene (2) (C) Protozoa (Tetrahymena thermophila ) OH 21 10 3 tetrahymanol (11) (gammaceran-21α-ol ) Fig. 2. Biosynthesis of triterpenes in higher plants, some bacteria, and protozoa. 312 J. Shinozaki et al. / FEBS Letters 582 (2008) 310–318 H squalene (10) H isohopanyl cation (15) hopanyl cation (12) OH filic-3-ene (16) 22-hydroxyhopane (1) hop-22(29)-ene (2) 21 H O adiantone (13) O adiantoxide (17) H H OH isodiantol B (14) Fig. 3. Proposed biosynthesis of triterpenes in A. capills-veneris L. OSC that accepts oxidosqualene as the substrate. Quite re- the potential to react with oxidosqualene, since the proton- cently, the identification of lanosterol synthase has been re- ation of an epoxide is more facile than that of an olefinic dou- ported in this bacterium [11]. So far, none of the plant OSCs ble bond. During the course of evolution, bacterial SCs may has been reported to cyclize squalene to produce 3-deoxy-type have gained the ability to accommodate (3S)-oxidosqualene products. as the substrate to evolve into OSCs. Bode et al. reported the presence of a cycloartenol synthase Bacterial hopanoids, typically a bacteriohopanetetrol, are (CAS) in Stigmatella aurantiaca, a myxobacterium producing indispensable metabolites as they serve as membrane reinforc- sterols [12], and suggested that higher plant OSCs evolved ers controlling the rigidity and fluidity of bacterial membranes, from bacterial SCs via myxobacterial OSC (CAS) because thus acting like a surrogate for eukaryotic sterols [13]. Sterols the CAS clone from S. aurantiaca showed higher sequence of ferns, including b-sitosterol (6), are the same as those from identity with plant CASs (38–40%) than with bacterial SCs higher plants. The isolation of cycloartenol (5) [14], an estab- (18–22%) [12]. From a chemical point of view, SCs should have lished precursor of phytosterol biosynthesis, from some fern J. Shinozaki et al. / FEBS Letters 582 (2008) 310–318 313 species suggests their derivation from (3S)-oxidosqualene via GCCCADATIGGRAA-30). PCR was carried out for 30 cycles with cycloartenol in the same manner as in higher plants. During the following protocol: 94 °C for 1 min; 42 °C for 2 min; 72 °C for the course of evolution, ferns may have acquired OSC (CAS) 3 min; and final extension at 72 °C for 10 min. The PCR product of ex- pected size (ca. 450 bp) was subcloned into pT7 Blue T-Vector (Nova- for sterol biosynthesis to be utilized for eukaryotic membrane gen, Merck Biosciences, Darmstadt, Germany) and propagated in construction, leaving SCs, which were no longer needed to be E. coli DH5a (Takara Biochemicals, Ciga, Japan). Plasmid DNA specific to produce hopanoids, to evolve to produce a diverse was prepared from 22 individual transformants and sequenced. All array of 3-deoxy triterpene structures. If so, the question here were identical to each other, and the corresponding full-length cDNA was named ACX. Based on the sequence obtained above, specific oli- is whether OSC (CAS) of ferns for sterol biosynthesis is similar gonucleotide primers were synthesized for 30- and 50-RACE. That to bacterial SCs or more similar to OSCs of higher plants. for 30-RACE was carried out with the primers ACX-573S (50- Adiantum capillus-veneris L. (Parkeriaceae) is one of the CTTTGGCAGCTTTCAAAAAG-30), ACX-584S (50-CGGACA- most widely distributed ferns. In China, the dried whole plants GAGGAAGTCAATGCT-30), RACE32, and anchor primer RACE17 0 0 have been used as an antipyretic and diuretic and in the treat- (5 -GACTCGAGTCGACATCG-3 ). PCR conditions were the same as described above, but the annealing temperature was 52 °C instead ment of bronchitis in traditional medicine. Our extensive phy- of 42 °C. To obtain the 50-end sequence, stepwise 50-RACE was per- tochemical studies of the fresh fronds collected in Japan formed. For the first-step, nested PCR was performed under the same resulted in the isolation of 56 triterpene derivatives, including conditions as for 30-RACE, first with 162S and ACX-505A (50-GAT- 0 0 13 novel compounds [15,16].