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Dual Biosynthetic Pathways to Phytosterol Via Cycloartenol and Lanosterol in Arabidopsis

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Dual Biosynthetic Pathways to Phytosterol Via Cycloartenol and Lanosterol in Arabidopsis Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis Kiyoshi Ohyamaa, Masashi Suzukia, Jun Kikuchia,b,c, Kazuki Saitoa,d, and Toshiya Muranakaa,e,1 aRIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan; bGraduate School of Bioagriculture Sciences, Nagoya University, 1-1 Fro-cho, Chikusa-ku, Nagoya, Aichi, 464-8601, Japan; cInternational Graduate School of Integrated Sciences, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama, 230-0045, Japan; dGraduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba, 263-8522, Japan; and eKihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama, Kanagawa, 244-0813, Japan Edited by Charles J. Arntzen, Arizona State University, Tempe, AZ, and approved November 21, 2008 (received for review August 5, 2008) The differences between the biosynthesis of sterols in higher HO acetyl-CoA -OOC plants and yeast/mammals are believed to originate at the cycliza- OH tion step of oxidosqualene, which is cyclized to cycloartenol in mevalonate higher plants and lanosterol in yeast/mammals. Recently, lanos- terol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants pos- CAS sess dual biosynthetic pathways to phytosterols via lanosterol, and HO O through cycloartenol. To identify the biosynthetic pathway to parkeol phytosterol via lanosterol, and to reveal the contributions to 2,3-oxido- CAS squalene LAS phytosterol biosynthesis via each cycloartenol and lanosterol, we 13 2 performed feeding experiments by using [6- C H3]mevalonate with Arabidopsis seedlings. Applying 13C-{1H}{2H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of HO ? HO HO cycloartenol lanosterol lanosterol phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic phytosterols pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol HO ‘‘the lanosterol pathway.’’ LAS1 expression is reported to be HO HO HO induced by the application of jasmonate and is thought to have campesterol sitosterol ergosterol cholesterol evolved from an ancestral cycloartenol synthase to a triterpenoid plants yeasts mammals synthase, such as ␤-amyrin synthase and lupeol synthase. Consid- ering this background, the lanosterol pathway may contribute to Fig. 1. Cyclization step of oxidosqualene in yeasts, mammals, and plants. the biosynthesis of not only phytosterols, but also steroids as Recently, lanosterol synthase genes were identified from several plants, Ara- secondary metabolites. bidopsis thaliana, Panax ginseng, and Lotus japonica (6–8). However, no clear data confirm the existence of that biosynthetic pathway of phytosterol via lanosterol in the plant kingdom. CAS, cycloartenol synthase; LAS, lanosterol ͉ ͉ ͉ ͉ metabolic diversity mevalonate sterol tracer experiment triterpene synthase. terols are indispensable compounds in all eukaryotes, be- Scause they are structural components of plasma membranes. Both cycloartenol and lanosterol are biosynthesized from oxi- In mammals, insects, and higher plants, sterols are also con- dosqualene after the formation of the protosteryl cation. Both verted to steroidal hormones. Because of their chemical signif- H-19 and H-9 protons of the protosteryl cation are deprotonated icance, the main pathway of sterol biosynthesis has been studied to afford cycloartenol and lanosterol, respectively (Fig. 2) (9). extensively, and is believed to be fully understood (1). The We noted the difference of this cyclization mechanism, and 13 2 differences in the biosynthesis of sterols between higher plants designed a tracer experiment by using [6- C H3]mevalonate 13 2 and yeast/mammals are generally accepted to begin at the ([6- C H3]MVL) (Fig. 2). Each of the labeled atom positions, cyclization step of 2,3-oxidosqualene, a common precursor. C-18, C-19, C-21, and C-27 (Pro-R), of phytosterol are biosyn- Ergosterol and cholesterol are biosynthesized via lanosterol, thetically equivalent to C-6 of MVL (10). Elucidation of the catalyzed by lanosterol synthase (LAS), in yeast and in mammals deuterium on C-19 behavior of phytosterol made it possible to (2, 3), respectively. Phytosterols, such as campesterol and sitos- clarify whether the phytosterol is biosynthesized via either terol, are biosynthesized via cycloartenol and catalyzed by cycloartenol synthase (CAS) in higher plants (Fig. 1) (4, 5). Recently, however, three different laboratories have identified Author contributions: K.O., K.S., and T.M. designed research; K.O. and M.S. performed LAS genes from dicotyledonous plant species including Arabi- research; K.O. and J.K. contributed new reagents/analytic tools; K.O. and M.S. analyzed dopsis thaliana (6, 7), Panax ginseng (6), and Lotus japonica (8) data; and K.O. and M.S. wrote the paper. using a yeast expression system. The existence of LAS genes in The authors declare no conflict of interest. the plant kingdom led us to address the question of whether the This article is a PNAS Direct Submission. biosynthetic pathway of phytosterols via lanosterol as the first Freely available online through the PNAS open access option. cyclic intermediate exists. 1To whom correspondence should be addressed. E-mail: [email protected]. To overturn the common knowledge, one must clarify that This article contains supporting information online at www.pnas.org/cgi/content/full/ endogenous lanosterol is metabolized to phytosterols by using 0807675106/DCSupplemental. BIOCHEMISTRY upstream substrates such as acetate and mevalonate (MVL). © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0807675106 PNAS ͉ January 20, 2009 ͉ vol. 106 ͉ no. 3 ͉ 725–730 Downloaded by guest on September 28, 2021 O Br O OEt , Zn OH O LiAlH4 OH TBSCl, DMAP, Et3N HO 13CD 13CD 3 3 H OEt OH -OOC Et2O Et2O CH2Cl2 OH 13 1 66% 2 85% [6- CD3]MVL for 2 steps O 13 : CD 13 2 HO 13 2 oxidosqualene 3 OH TPAP, NMO O C H3MgI C H3 TBAF OTBS OTBS CH2Cl2 Et2O OTBS 345 Enz-B: 66% 75% 99% D 13 2 H 1) K2CO3 HO C H3 13 13 2 AcO 13 2 AcO 13 2 CD2 HO C H3 Ac O, DMAP C H RuCl , NaIO C H 2 3 3 4 O 3 2) HCl OH HO CH2Cl2 OAc CCl4, CH3CN, HO OAc phosphate buffer O O 67 8 [6-13C2H ]MVL protosteryl cation 96% 92% 84% 3 13 2 D D Fig. 3. Total synthesis of [6- C H3]MVL. D D D 13C 13C HO HO to increase the incorporation ratio of the labeled MVL into cycloartenol lanosterol phytosterol (13). The isolated sterol fraction was analyzed initially using gas chromatography-mass spectrometry (GC-MS). R R D The mass spectra for major phytosterols, such as campesterol, H D D D D 13C 13C stigmasterol, and sitosterol, are shown in Fig. 4A. Nonlabeled campesterol, stigmasterol, and sitosterol have a molecular ion at HO HO m/z ϭ 400, 412, and 414, respectively. The extracted phytosterols phytosterol had major molecular ions at m/z ϭ 415, 427, and 429, corre- 13 2 Fig. 2. Label pattern of phytosterol in the feeding experiment using sponding to those molecules with four C atoms and 11 H 13 [6- CD3]MVL. C-19 of phytosterol biosynthesized via cycloartenol and lanos- atoms per molecule. Phytosterols were biosynthesized effectively 13 13 13 2 terol are labeled as CD2H and CD3, respectively. from exogenous [6- C H3]MVL in this experiment. GC-MS results suggested that three deuteriums at C-6 of MVL were retained in the C-18, -21, and -27 positions, whereas a deuterium cycloartenol or lanosterol. If the phytosterol retained two deu- at the C-19 position was substituted with a hydrogen during terium labels at the C-19 position, then it was biosynthesized via sterol biosynthesis. Phytosterol with three deuteriums retained cycloartenol. In the case when three deuteriums are detected on at the C-19 position could not be determined by the analyses of C-19, the results would indicate that the phytosterol was bio- MS data. Next, the major phytosterol, sitosterol, was analyzed by synthesized via lanosterol. The number of deuterium labels was using NMR after the separation of sterol fraction by HPLC analyzed by proton and deuterium double-decoupled 13C- (High Performance Liquid Chromatography). The labeling pat- nuclear magnetic resonance (NMR) (13C-{1H}{2H}NMR). Be- tern of the sitosterol was determined by 13C-{1H}{2H}NMR. cause the signal for a carbon connected to a deuterium would be The 13C-{1H}{2H}NMR spectrum and chemical shift values for shifted theoretically by approximately 0.3 ppm upfield because C-18, -19, -21, and -27 are shown in Fig. 5B and Table 1, of isotope effects (11), C-19 carbon peaks connected to two and three deuteriums were shifted to 0.6 ppm and 0.9 ppm upfield, respectively. Resonances at 10.98 (C-18), 17.84 (C-21), 18.08 respectively, compared with the unshifted 13C spectrum. By (C-27), and 18.81 (C-19) ppm were identified with 0.88, 0.94, using this strategy, one can determine whether phytosterols are biosynthesized via lanosterol. The contribution to phytosterol biosynthesis via lanosterol can also be determined by comparing A WT B LAS1 over C las1 mutant 13 100 the area of two C peaks, namely, the signals shifted 0.6 and 0.9 c b c c a b a ab ppm upfield. 0 8121410 8121410 81216 13 2 In this study, [6- C H3]MVL was synthesized and fed to the a Retention time (min) seedlings of the wild type (WT), LAS1 overexpressing plant, and 100 415 415 415 a las1 mutant of Arabidopsis.
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