Squalene Cyclase and Oxidosqualene Cyclase from a Fern

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), 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 methanotrophic 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 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)

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)

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

squalene (10)

H isohopanyl cation (15)

hopanyl cation (12)

filic-3-ene (16)

22-hydroxyhopane (1) hop-22(29)-ene (2)

21 H

adiantone (13) O

adiantoxide (17)

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 expected 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]. Among them, adiantone (13), TCACCAACAATGTCAGA-3 ) and then with 162S and 495A (5 - CAATGCTAGTGCAGCCTTAAA-30), and yielded a 1.0-kb DNA isoadiantol B (14), and adiantoxide (17) are the major constit- fragment. Based on the sequence obtained in the first-step PCR, spe- uents. Adiantone is (21S)-22-oxo-adiantane, while isoadiantol cific oligonucleotide primers, ACX-191A (50-GTTCTTGCCCCAAT- B is (21R)-22(R)-hydroxy-adiantane. Their biosynthetic rela- AGTCTTAG-30), ACX-229A (50-TGTACCAAGCACAGATAGC- tion can be summarized as shown in Fig. 3. Cyclization of CA-30), and ACX-312A (50-TACAAATCTTCCTTAGCACA-30), squalene via hopanyl cation (12) yields hop-22(29)-ene (2) were synthesized. Prior to the second-step, total RNA was reverse- transcribed with the ACX-312A primer, followed by poly (A) tail addi- and/or 22-hydroxyhopane (1). Oxidative C-1 removal from tion at the 30-terminus using terminal deoxynucleotidyl transferase the side chain of either 1 or 2 yields adiantone (13), and further (Toyobo, Tokyo, Japan). The resultant cDNA was used as the tem- epimerization at C-21 and reduction lead to the production of plate for nested PCR. PCR conditions were the same as above, but isoadiantol B. On the other hand, adiantoxide is an epoxida- the annealing temperature for the second-step PCR was 55 C. The primer sets were RACE 32 and ACX-229A for the first PCR and RACE tion product of filic-3-ene (16), which is derived from squalene 17 and ACX-191A for the second. via isohopanyl cation (15) with extensive Wagner–Meerwein rearrangements, strongly suggesting the presence of a distinct 2.4. Expression of ACX in the yeast mutant GIL77 filic-3-ene synthase, in addition to hop-22(29)-ene and/or ho- The full-length cDNA of ACX was obtained using nested PCR. SacI pan-22-ol synthase, in A. capillus-veneris. and XbaI sites were introduced immediately upstream from the ATG To obtain the molecular bases for the biosynthesis of triter- codon and downstream from the stop codon, respectively. The primers used were: ACX-50 (50-ACTTCAAGGATCTATTGAGAGAG-30) penes and sterols in ferns, cDNA cloning of SCs and OSCs and ACX-30 (50-TCTCAAGAGGTGCATGCATGTCA-30) for the from A. capillus-veneris was attempted using the homology- first PCR, and Sac-ACX-N (50-GAGAGAGCTCAGAATGTGGAC- based PCR in this study. CTTGAAGATAGCA-30) and Xba-ACX-C (50-AAATTCTAGATC- AATGGCTCAAGACCCGCCG-30) for the second PCR (underlined: start and stop codons, bold: restriction enzyme site). The first PCR with 100 pmol each of primers ACX-50 and ACX-30 and the second 2. Materials and methods PCR with 100 pmol each of primers Sac-ACX-N and Xba-ACX-C were carried out under the same conditions as described above, except 2.1. Chemicals that the annealing temperature was 58 C. The 2.3-kb PCR product Authentic cycloartenol was prepared by chemical hydrolysis of its was digested with SacI and XbaI and ligated into the sites of pYES2 acetate isolated from Goniophlebium mengtzeense [14], and hydroxyho- (Invitrogen) to construct the plasmid pYES2-ACX. Following the pane was isolated from Adiantum pedatum [17]. Other analytical-grade same method reported previously [20], the mutant yeast strain chemicals were purchased commercially. Synthetic oligonucleotides for GIL77 [18] was transformed with the resultant plasmid pYES2- PCR primers were obtained from Nihon Bioservice (Saitama, Japan). ACX. The culture conditions of the transformed yeasts, induction protocol, preparation of cell extracts, and analysis using TLC were the same as reported previously [18]. 2.2. RNA and cDNA preparation The aerial parts of A. capillus-veneris L. were collected from the medicinal botanical garden of Showa Pharmaceutical University (To- 2.5. LC-APCIMS analysis of the ACX product kyo, Japan). Total RNA was extracted from the aerial parts (1.5 g) LC-APCIMS was measured with LCQ (Thermo Fisher Scientific, using RNAqueous-Midi (Ambion, TX, USA). Poly (A)+ RNA was MA, USA) equipped with a HP1100 series LC system (Agilent Tech- purified from the total RNA using an mRNA Purification Kit (GE nologies, CA, USA) under the same conditions as reported previously Healthcare UK Ltd., England) according to the manufacturerÕs proto- [19]. The spot corresponding to triterpene monoalcohol on TLC was col. A single-stranded cDNA pool was prepared using SuperScript II subjected to LC–MS. HPLC was carried out using a SUPER-ODS col- 0 (Invitrogen, CA, USA) with 50 lM of RACE32 (5 -GAC- umn (/ 4.6 · 200 mm) (Tosoh, Tokyo, Japan) with 95% CH3CN aq. TCGAGTCGACATCGATTTTTTTTTTTTTT-30) primer in a total (flow rate 1.0 ml/min, detection UV 202 nm) at 40 C. + volume of 20 ll. In addition, a double-stranded cDNA mixture was Cycloartenol: MS m/z 409 [M+HH2O] , MS/MS (precursor ion at also synthesized using the Marathon cDNA Amplification Kit (Clon- m/z 409) 299 (30%), 285 (25%), 271 (20%), 257 (25%), 231 (30%), 217 tech, CA, USA). (100%), 203 (60%), 191 (90%).

2.3. cDNA cloning of OSC 2.6. cDNA cloning of SC Following the conditions reported in previous papers [18,19], nested Based on the highly conserved amino acid regions of bacterial SCs, PCR was carried out with the single-stranded cDNA mixture as a tem- four degenerate oligonucleotide primers were designed. The nucleotide plate and the degenerate primers 162S (50-GAYGGIGGITGGGGIY- sequences of these primers were: SC-306S-1 (50-GCIWSIATHWSIC- TICA-30), 463S (50-MGICAYATHWSIAARGGIGCITGG-30), 467S CIRTITGGGAYAC-30), SC-306S-2 (50-SCITGYBTIWSICCIRTIT- (50-AARGGIGCITGGCCITTYWSIAC-30), 623A (50-CCCAISWIC- GGGAYAC-30), SC-494A (50-WARTTIRYICCCCAICKICCRW- CITMCCAISWICCRTC-30), and 711A (50-CKRTAYTCICCIARI- ACCA-30), and SC-537A (50-KYYTCICCCCAICCICCRTC-30). The 314 J. Shinozaki et al. / FEBS Letters 582 (2008) 310–318

first PCR with either primers SC-306S-1 and SC-537A or SC-306S-2 clones were sequenced. All were identical to each other. To ob- and SC-537A was carried out under the same conditions as described tain the full-length sequence, both 30- and 50-RACE [22] were above. The PCR product was applied to SUPREC-02 (Takara Bio- performed. The full-length sequence obtained was named chemicals) to remove the primers, and the volume was adjusted to 100 ll. Three microliters of the solution was used as a template for ACX. The open reading frame (ORF) of ACX consists of the second PCR under the same conditions as for the first PCR. The 2277 nucleotides that encodes a 758-amino acid polypeptide.2 primer sets of the second PCR were either SC-306S-1 and SC-494A ACX exhibits high (49–72%) amino acid identities to known or SC-306S-2 and SC-494A. The PCR product of expected size (ca. plant OSCs, especially to CASs (greater than 68%), suggesting 550 bp) was subcloned into the plasmid vector as described above. Twenty-two independent clones were sequenced. All were identical to that ACX is a cDNA encoding CAS. each other, and the corresponding full-length cDNA was named ACH.30-RACE was carried out with the primers ACH–386S (50- 3.2. Expression of ACX in the yeast mutant GIL77 0 0 AATGCTATCAAGCTACAAGATGAT-3 ), ACH-466S (5 -TCGG- The PCR product (ca. 2.3 kb in length) with the primers cor- CCATTTTGCCGCCAAGG-30), RACE32, and RACE17. PCR conditions were the same as described above, but the annealing tem- responding to the N- and C-termini was ligated downstream peratures were 50 C for the first PCR and 52 C for the second from the GAL1 promoter of pYES2 to construct the plasmid PCR instead of 42 C. 50-RACE was carried out using the Marathon pYES2-ACX. S. cerevisiae strain GIL77, which is deficient in cDNA Amplification Kit (Clontech) with the specific primer ACH- lanosterol synthase ERG7, was transformed by the resultant 0 0 353A (5 -GTAAAACCAGTTCCCATGCTGAGTGGC-3 ). plasmid. The transformant was cultured, and ACX protein expression was induced. Cells were harvested and extracted 2.7. Expression of ACH in yeast with hexane after alkaline treatment. The hexane extract ob- To obtain the full-length cDNA of ACH, nested PCR was carried out using following primers: ACH-50 (50-ACTTGGCTCGGAGG- tained was subjected to TLC analysis. The extract of the trans- GCACTTCACTA-30), N-ACH-Eco (50-CAGCCGAATTCAATATG- formant harboring pYES2-ACX gave two new spots that were CTGCCATACAACCAAGAT-30), C-ACH-Xho (50-GGTAGCTC- not found in the void vector control. The less polar spot with 0 0 0 GAGTCATTTTGCCATTTTGCCAGCTCG-3 ), and ACH-3 (5 - an Rf value corresponding to that of triterpene monoalcohol 0 TGGGAAATCATGCGGTAGATG-3 ). The first PCR with primers was extracted with hexane and analyzed using LC–MS ACH-50 and ACH-30 was performed using KOD Plus DNA Polymer- ase (Toyobo) in a final volume of 50 ll following the manufacturerÕs (Fig. 4). The retention time of the product of ACX was the protocol. The PCR was carried out for 30 cycles of amplification using same as that of cycloartenol, and the MS/MS spectrum of a stepped program (15 s at 94 C, 30 s at 60 C, and 2 min at 68 C) in the precursor ion at m/z 409 was also identical to that of Robocycler Gradient 96 (Stratagene, Agilent Technologies). The first authentic cycloartenol. The polar spot on TLC, which was al- PCR product was applied on SUPREC-02 (Takara Biochemicals) to remove the primers, and the volume was adjusted to 100 ll. The sec- ways detected in our previous experiments on CAS expression ond PCR was carried out with primers N-ACH-Eco and C-ACH- in yeast, was identified as a mixture of 31-norcycloartenol and Xho and 1 ll of the first PCR solution as a template under the same cycloeucalenol, the metabolites of cycloartenol in the host conditions as for the first PCR. The 2.0-kb PCR product was digested yeast (our unpublished data). No triterpene hydrocarbon was with EcoRI and XhoI and ligated into the corresponding sites of detected on TLC with phosphomolybdic acid staining. These pYES2 (Invitrogen) to construct the plasmid pYES2-ACH. Saccharo- myces cerevisiae strain INVSc2 (Invitrogen) was transformed with the results confirm that ACX encodes a CAS of A. capillus-veneris. resultant plasmid pYES2-ACH, following the previously reported method [20]. The transformant was grown in 3 ml of synthetic com- 3.3. cDNA cloning of SC plete medium without uracil (SC-U) at 30 C with shaking overnight. As mentioned above, the genes encoding SCs have been A portion (100 ll) of the overnight culture was inoculated in 20 ml of SC-U and incubated at 30 C with shaking. After 2 days, cells were cloned from four bacterial species, A. acidocaldarius [5], Z. collected and resuspended in 20 ml of SC-U without glucose, supple- mobilis [6], B. japonicum [7], and M. capsulatus [8], and the mented with 50 lg/ml of terbinafine [21], 2% galactose was added for products of these SCs were identified as 22-hydroxyhopane induction, and the mixture was further incubated at 30 C for 24 h. (1) and hop-22(29)-ene (2). In addition to these clones, several Cells were collected and refluxed with 2 ml of 15% KOH/MeOH for nucleotide sequences are available in the DDBJ/GenBank/ 5 min. After extraction with the same volume of hexane, the extract was concentrated and subjected to GC–MS analysis. EMBL databank as putative SCs from bacteria and cyanobac- teria [23,24], although their functions have not been deter- mined. All these clones showed 33–77% amino acid 2.8. GC–MS analysis of the ACH product identities. Based on the conserved sequences among these GC–MS was measured with GCMS-QP2010 (Shimadzu, Kyoto, Ja- pan). Ionization was by electron impact at 70 eV. An Rtx-5MS column SCs, four degenerate primers were designed. With these prim- (30 m · 0.25 mm, Restek, PA, USA) was used for GC. The tempera- ers, two sets of nested PCRs were carried out. The resultant ture program was: holding at 250 C for 2 min, increasing in incre- 550-bp DNA fragments were subcloned into the plasmid vec- ments of 20 C/min to 330 C, and holding at 330 C for 5 min. tor, and 22 independent clones were sequenced. Sequencing Hydroxyhopane: Retention time, 9.18 min. m/z 191 (fragment peak from A/B ring, 100%), 189 (fragment peak from D/E ring, 90%). showed that all the clones were identical. The sequence of this fragment shared 38–42% amino acid identities with bacterial SCs and included the DXDD motif [25,26], strongly suggesting it to be a part of the cDNA encoding SC. To obtain the full- 3. Results and discussion length clone, both 30- and 50-RACE were carried out. The ORF of the full-length clone consisted of 2088-bp nucleotides, 3.1. cDNA cloning of OSC encoding 695 amino acids. It showed 35–39% amino acid iden- For OSC cloning, degenerate primers were designed from the highly conserved regions of the known OSCs from higher plants. RNA was prepared from the aerial part of A. capillus-veneris. Nested PCR with the degenerate primers and a 2Nucleotide sequence data are available in the DDBJ/EMBL/GenBank cDNA pool yielded 420-bp DNA fragments. After subcloning databases under the accession numbers AB368375 (ACX) and of the fragments into the plasmid vector, 22 independent AB368376 (ACH). J. Shinozaki et al. / FEBS Letters 582 (2008) 310–318 315

20.32 tion time of 9.18 min under this GC condition is longer than

20.38 that of triterpene hydrocarbons, indicating that this ACH product is not a hydrocarbon but a triterpene alcohol. The EI-MS spectrum of this peak showed strong fragment ions at m/z 191 and m/z 189 (Fig. 5B), both of which are known to be the characteristic fragmentation ions of hydroxyhopane (Fig. 6) [27]. These results strongly suggest that this ACH product is 22-hydroxyhopane, and rigorous identification was achieved by direct comparison of its retention time (Fig. 5A(b)) and fragmentation pattern (Fig. 5B) with those a of an authentic specimen (Fig. 5A(a) and C). A squalene-hopene cyclase (SHC)3 from A. acidocaldarius b was reported to produce a mixture of hop-22(29)-ene (2) and 10 15 20 25 22-hydroxyhopane (1) in a ca. 5:1 ratio (Fig. 2B), the former min produced from hopanyl cation by deprotonation and the latter by nucleophilic attack of water [3,28]. In contrast, ACH specif- 191.1 100 217.1 ically produces 22-hydroxyhopane exclusively. Kolesnikova 90 et al. discussed that in oxidosqualene cyclization catalyzed 80 by OSCs, termination by deprotonation is the more favored 70 process over quenching of carbocation by water addition, be- 60 cause hydroxylation needs efficient replenishment of a water 50 molecule and an attack on the empty 2p orbital along the axis 40 231.1 299.1 285.2 [29]. The same discussion could be applied here, and as ACH 30 271.2 Relative intensity (%) 20 exclusively produces 22-hydroxyhopane, a water addition 10 product, it must precisely control the position and supply of 0 a water molecule in its active site. 100 150 200 250 300 350 400 450 m/z Regarding substrate specificity, we examined whether ACH could cyclize oxidosqualene (4) as a substrate when expressed 191.1 in a lanosterol synthase-deficient yeast mutant GIL77. Despite 100 217.1 an extensive search for products, no product from oxidosqua- 90 80 lene was detected at a significant level (data not shown). To- 70 gether with the fact that no hopane derivative with an 60 oxygen function at C-3 has been isolated from A. capillus-vene- 50 ris, ACH seems to have specificity for squalene and not to ac- 40 299.2 231.1 cept oxidosqualene. To establish its substrate specificity, 30 285.1

Relative intensity (%) 271.1 however, in vitro assay by using the puriﬁed enzyme is needed, 20 as a protozoan SC from T. pyriformis is reported to convert 10 0 oxidosqualene, a nonphysiological substrate, into gammacera- 100 150 200 250 300 350 400 450 ne-3,21a-diol in vitro [9]. m/z

Fig. 4. LC–MS analysis of the ACX product. HPLC profiles of 3.5. Molecular evolution of SC and OSC in ferns authentic cycloartenol (5) (a) and the ACX product (b) monitored by We have successfully cloned the cDNAs of an OSC and a SC UV absorption at 202 nm are shown in (A). (B) and (C) indicate MS/ MS spectra of authentic cycloartenol and the ACX product with from A. capillus-veneris. To the best of our knowledge, this is + precursor ion at m/z 409 [M+HH2O] , respectively. the first gene cloning of OSC and SC from any fern species. Furthermore, ACH is the first example of a product-specific hydroxyhopane synthase from any organism including bacte- tities with bacterial SCs so far reported. This cDNA was ria. named ACH. ACH shows 35–39% amino acid identities with bacterial SCs producing 22-hydroxyhopane (1) and hop-22(29)-ene (2), 3.4. Expression of ACH in yeast which show 37–58% amino acid identities with each other. Expression plasmid pYES2-ACH was constructed using the ACX (CAS) shows high amino acid identities (68–72%) to same method as that for ACX. The resultant plasmid was those from higher plants. The phylogenetic relationship of introduced into the yeast strain INVSc2 (Invitrogen), which ACH and ACX, together with the known OSCs and SCs from has no deficiency in ergosterol biosynthesis. The transformant other organisms, was analyzed. As shown in Fig. 7 (i) bacterial was cultured for three days, and then terbinafine (50 lg/ml), a SCs and eukaryotic OSCs form their respective clusters, (ii) squalene epoxidase inhibitor, was added for accumulation of CASs and triterpene synthases of higher plants form the endogenous squalene in the host yeast, together with galactose respective subclusters in the eukaryotic OSC cluster, (iii) a for induction. The cells were harvested and extracted with hex- CAS of a myxobacterium (S. aurantiaca) and a lanosterol syn- ane after alkaline treatment. The extract was then subjected to GC–MS analysis. A product peak, which was absent in a void vector control (Fig. 5A(b)), was detected at 9.18 min. No other 3Enzymes: cycloartenol synthase (EC 5.4.99.8); squalene-hopene significant peak was detected as the ACH product. The reten- cyclase (EC 5.4.99.17). 316 J. Shinozaki et al. / FEBS Letters 582 (2008) 310–318

Fig. 5. GC–MS analysis of the ACH product. Gas chromatograms of authentic 22-hydroxyhopane (1) (a) and the ACH product (b) with total ion monitoring are shown in (A). MS spectra of the ACH product and authentic 22-hydroxyhopane (1) are shown in (B) and (C), respectively.

a 4

+ - H2O OH m/z 189 +

m/z 191 m/z 207

Fig. 6. Fragmentation of 22-hydroxyhopane (1) in EI-MS analysis. thase of a methanotrophic-bacterium (M. capsulatus) are lo- CASs. From these results, one might argue that OSCs of ferns cated closer than other bacterial SCs to the cluster of eukary- have evolved along the same path as those of higher plant otic OSCs, (iv) ACH (SC) is in the cluster of bacterial SCs, and OSCs, possibly from bacterial SCs via bacterial OSCs, or they (v) ACX (CAS) is not located around the cluster of bacterial were laterally transferred from higher plants at some evolu- SCs but is found in the subcluster of CASs in the cluster of tionary stage. As ACH and ACX are the ﬁrst examples of higher plant OSCs. These clearly indicate that fern SC is at SC and OSC from ferns, however, it is still diﬃcult to draw the almost same evolutionary stage as bacterial SCs, whereas any conclusive answer to the intriguing question of how these fern CAS is at the same evolutionary stage as higher plant enzymes evolved. J. Shinozaki et al. / FEBS Letters 582 (2008) 310–318 317

Eukaryotic ACH OSCs Bacterial SCs CASs 1 8 9

Triterpene 7 ACX synthases 11 12 13 14 2 15

17 18 3 19 20

Higher plant OSCs 4 6 5 Bacterial OSCs 10

Fig. 7. Phylogenetic analysis of SCs and OSCs. Distances between each clone were calculated with the program CLUSTAL W. The indicated scale represents 0.1 amino acid substitution per site. Each number indicates, 1: Alicyclobacillus acidocaldarius SHC (accession number of the sequence on EMBL, GenBank, and DDBJ sequence databanks: AB007002), 2: Bradyrhizobium japonicum SHC (X86552-4), 3: Zymomonas mobilis SHC (AJ001401-6), 4: Methylococcus capsulatus SHC (Y09978-3), 5: Methylococcus capsulatus lanosterol synthase (YP115266), 6: Stigmatella aurantiaca CAS (AJ494839), 7: Homo sapiens lanosterol synthase (D63807), 8: Saccharomyces cerevisiae lanosterol synthase (U04841), 9: Candida albicans lanosterol synthase (XM717378), 10: Trypanosoma brucei brucei lanosterol synthase (AF226705), 11: Oryza sativa CAS (AF169966), 12: Arabidopsis thaliana CAS (U02555), 13: Pisum sativum CAS (D89619), 14: Panax ginseng CAS (AB009029), 15: Betula platyphylla lupeol synthase (AB055511), 16: Olea europaea lupeol synthase (AB025343), 17: Taraxacum oﬃcinale lupeol synthase (AB025345), 18: Panax ginseng b-amyrin synthase (AB009030), 19: Glycyrrhiza glabra b-amyrin synthase (AB037203), 20: Pisum sativum b-amyrin synthase (AB034802).

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