Functional characterization of CYP71D443, a cytochrome P450 catalyzing C-22 hydroxylation in the 20-hydroxyecdysone biosynthesis of Ajuga hairy roots
Abstract
20-Hydroxyecdysone (20HE), a molting hormone of insects, is also distributed among a variety of plant families. 20HE is thought to play a role in protecting plants from insect herbivores. In insects, biosynthesis of 20HE from cholesterol proceeds via 7-dehydrocholesterol and 3β,14α-dihydroxy-5β-cholest-7-en-6-one (5β-ketodiol), the latter being converted to 20HE through sequential hydroxylation catalyzed by four P450 enzymes, which have been cloned and identified. In contrast, little is known about plant 20HE biosynthesis, and no biosynthetic 20HE gene has been reported thus far. We recently proposed involvement of 3β-hydroxy-5β-cholestan-6-one (5β-ketone) in 20HE biosynthesis in the hairy roots of Ajuga reptans var. atropurpurea (Lamiaceae). In this study, an Ajuga EST library was generated from the hairy roots and P450 genes were deduced from the library. Five genes with a high expression level (CYP71D443, CYP76AH19, CYP76AH20, CYP76AH21 and CYP716D27) were screened for a possible involvement in 20HE biosynthesis. As a result, CYP71D443 was shown to have C-22 hydroxylation activity for the 5β-ketone substrate using a yeast expression system. The hydroxylated product, 22-hydroxy-5β-ketone, had a 22R configuration in agreement with that of 20HE. Furthermore, labeling experiments indicated that (22R)-22-hydroxy-5β-ketone was converted to 20HE in Ajuga hairy roots. Based on the present results, a possible 20HE biosynthetic pathway in Ajuga plants involved CYP71D443 is proposed.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Most enzymes of ecdysteroid biosynthesis, except 14α-hydroxylase and 22-hydroxylase, have been identified only in insects/arthropods.
“Moreover, most of the enzymes, except 14α-hydroxylase [179] and 22-hydroxylase [36], have been identified only in insects/arthropods.”
From Ecdysteroids (2021) · cites this paper as [36]
Cites
Cytochrome P-450: Structure, Mechanism, and Biochemistry
de Montellano, O., & Paul, R. (1986) · Medical Entomology and Zoology · Cited by 2,667 · see citing works
Cytochrome P450: Structure, mechanism, and biochemistry
PRYOR, W. A. (1996) · Free Radical Biology and Medicine · doi:10.1016/0891-5849(96)83807-x · Cited by 1,610 · see citing works
Brassinolide, a plant growth-promoting steroid isolated from Brassica napus pollen
Grove, M. D., Spencer, G. F., Rohwedder, W. K., Mandava, N. B., Worley, J. F., Warthen, J. D., Steffens, G. L., Flippen-Anderson, J. L., & Cook, J. C. (1979) · Nature · doi:10.1038/281216a0 · Cited by 1,024 · see citing works
Choe, S., Dilkes, B. P., Fujioka, S., Takatsuto, S., Sakurai, A., & Feldmann, K. A. (1998) · The Plant Cell · doi:10.1105/tpc.10.2.231 · Cited by 484 · see citing works
Seki, H., Ohyama, K., Sawai, S., et al. (2008) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0803876105 · Cited by 389 · see citing works
Self-citationWarren, J. T., Petryk, A., Marqués, G., Jarcho, M. P., Parvy, J.-P., Dauphin‐Villemant, C., O’Connor, M. B., & Gilbert, L. I. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.162375799 · Cited by 335 · see citing works
Chávez, V. M., Marqués, G., Delbecque, J. P., Kobayashi, K., Hollingsworth, M., Burr, J., Natzle, J. E., & O’Connor, M. B. (2000) · Development · doi:10.1242/dev.127.19.4115 · Cited by 297 · see citing works
Enzymes for ecdysteroid biosynthesis: Their biological functions in insects and beyond
Niwa, R., & Niwa, Y. S. (2014) · Bioscience, Biotechnology, and Biochemistry · doi:10.1080/09168451.2014.942250 · Cited by 249 · see citing works
The DIMINUTO gene of Arabidopsis is involved in regulating cell elongation.Preprint
Takahashi, T., Gasch, A., Nishizawa, N. K., & Chua, N. (1995) · Genes & Development · doi:10.1101/gad.9.1.97 · Cited by 239 · see citing works
Rewitz, K., O’Connor, M. B., & Gilbert, L. I. (2007) · Insect Biochemistry and Molecular Biology · doi:10.1016/j.ibmb.2007.02.012 · Cited by 234 · see citing works
Cytochrome P450 CYP307A1/Spook: A regulator for ecdysone synthesis in insects
Namiki, T., Niwa, R., Sakudoh, T., Shirai, K.-I., Takeuchi, H., & Kataoka, H. (2005) · Biochemical and Biophysical Research Communications · doi:10.1016/j.bbrc.2005.09.043 · Cited by 189 · see citing works
Niwa, R., Namiki, T., Ito, K., Shimada‐Niwa, Y., Kiuchi, M., Kawaoka, S., Kayukawa, T., Banno, Y., Fujimoto, Y., Shigenobu, S., et al. (2010) · Development · doi:10.1242/dev.045641 · Cited by 185 · see citing works
Self-citationEcdysone: Structures and Functions
(2009) · doi:10.1007/978-1-4020-9112-4 · Cited by 160 · see citing works
Ralston, L., Kwon, S. T., Schoenbeck, M. A., et al. (2001) · Archives of Biochemistry and Biophysics · doi:10.1006/abbi.2001.2483 · Cited by 144 · see citing works
New players in the regulation of ecdysone biosynthesis
Huang, X., Warren, J. T., & Gilbert, L. I. (2008) · Journal of genetics and genomics/Journal of Genetics and Genomics · doi:10.1016/s1673-8527(08)60001-6 · Cited by 136 · see citing works
On the distribution of phytoecdysteroids in plants
Dinan, L., Savchenko, T., & Whiting, P. (2001) · Cellular and Molecular Life Sciences · doi:10.1007/pl00000926 · Cited by 126 · see citing works
Krokida, A., Delis, C., Geisler, K., et al. (2013) · New Phytologist · doi:10.1111/nph.12414 · Cited by 116 · see citing works
Wang, E., & Wagner, G. J. (2003) · Planta · doi:10.1007/s00425-002-0904-4 · Cited by 97 · see citing works
Insights into the function and evolution of P450s in plant steroid metabolism
Ohnishi, T., Yokota, T., & Mizutani, M. (2009) · Phytochemistry · doi:10.1016/j.phytochem.2009.09.015 · Cited by 95 · see citing works
Biosynthesis and distribution of insect-molting hormones in plants—A review
Adler, J. H., & Grebenok, R. J. (1995) · Lipids · doi:10.1007/bf02537830 · Cited by 79 · see citing works
Burstein, S., Middleditch, B., & Gut, M. (1975) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)40689-3 · Cited by 77 · see citing works
Phytoecdysteroids: Diversity, biosynthesis and distribution
Dinan, L., Harmatha, J., Volodin, V., & Lafont, R. (2009) · Ecdysone: Structures and Functions · doi:10.1007/978-1-4020-9112-4_1 · Cited by 77 · see citing works
Production of phytoecdysteroids by hairy root cultures of Ajuga reptans var. atropurpurea.
Matsumoto, T., & Tanaka, N. (1991) · Agricultural and Biological Chemistry · doi:10.1271/bbb1961.55.1019 · Cited by 72 · see citing works
Biosynthesis and potential functions of the ecdysteroid 20-hydroxyecdysone — a review
Festucci-Buselli, R. A., Contim, L. A. S., Barbosa, L. C. A., Stuart, J. J., & Otoni, W. C. (2008) · Botany · doi:10.1139/b08-049 · Cited by 63 · see citing works
Ecdysteroids in Spinach (Spinacia oleracea L.): Biosynthesis, Transport and Regulation of Levels
Bakrim, A., Maria, A., Sayah, F., Lafont, R., & Takvorian, N. (2008) · Plant Physiology and Biochemistry · doi:10.1016/j.plaphy.2008.06.002 · Cited by 63 · see citing works
Wüst, M., Little, D. B., Schalk, M., & Croteau, R. (2001) · Archives of Biochemistry and Biophysics · doi:10.1006/abbi.2000.2248 · Cited by 60 · see citing works
Occurrence, biosynthesis, and putative role of ecdysteroids in plants
Adler, J. H., & Grebenok, R. J. (1999) · Critical Reviews in Biochemistry and Molecular Biology · doi:10.1080/10409239991209282 · Cited by 51 · see citing works
Mechanism of formation of the A/B cis ring junction of ecdysteroids in Polypodium vulgare
Davies, T. G. E., Lockley, W. J. S., Boid, R., Rees, H. H., & Goodwin, T. W. (1980) · Biochemical Journal · doi:10.1042/bj1900537 · Cited by 38 · see citing works
Phytoecdysteroids: Structure, Sources, and Biosynthesis in Plants
Baltaev, U. A. (2000) · Russian Journal of Bioorganic Chemistry · doi:10.1023/a:1026662505403 · Cited by 34 · see citing works
The first cytochrome P450 in ferns
Canals, D., Irurre‐Santilari, J., & Casas, J. (2005) · FEBS Journal · doi:10.1111/j.1742-4658.2005.04897.x · Cited by 32 · see citing works
Nagakari, M., Kushiro, T., Matsumoto, T., Tanaka, N., Kakinuma, S., & Fujimoto, Y. (1994) · Phytochemistry · doi:10.1016/s0031-9422(00)90461-8 · Cited by 32 · see citing works
Self-citationThe Function and Evolution of the Halloween Genes: The Pathway to the Arthropod Molting Hormone
Gilbert, L. I., & Rewitz, K. (2009) · Springer eBooks · doi:10.1007/978-1-4020-9112-4_9 · Cited by 30 · see citing works
Ecdysone 20-monooxygenase, a cytochrome P450 enzyme from spinach, Spinacia oleracea
Grebenok, R. J., Galbraith, D. W., Benveniste, I., & Feyereisen, R. (1996) · Phytochemistry · doi:10.1016/0031-9422(96)00094-5 · Cited by 29 · see citing works
Biosynthesis of ecdysteroids in Zea mays
Devarenne, T. P., Sen-Michael, B., & Adler, J. H. (1995) · Phytochemistry · doi:10.1016/0031-9422(95)00435-a · Cited by 28 · see citing works
Nagakari, M., Kushiro, T., Yagi, T., Tanaka, N., Matsumoto, T., Kakinuma, S., & Fujimoto, Y. (1994) · Journal of the Chemical Society Chemical Communications · doi:10.1039/c39940001761 · Cited by 21 · see citing works
Self-citationHyodo, R., & Fujimoto, Y. (2000) · Phytochemistry · doi:10.1016/s0031-9422(00)00018-2 · Cited by 19 · see citing works
Self-citationLockley, W. J. S., Boid, R., Lloyd-Jones, G., Rees, H. H., & Goodwin, T. W. (1975) · Journal of the Chemical Society Chemical Communications · doi:10.1039/c39750000346 · Cited by 19 · see citing works
Reixach, N., Lafont, R., Camps, F., & Casas, J. (1999) · European Journal of Biochemistry · doi:10.1046/j.1432-1327.1999.00905.x · Cited by 18 · see citing works
Okuzumi, K., Hara, N., Fujimoto, Y., Yamada, J., Nakamura, A., Takahashi, K., & Morisaki, M. (2003) · Tetrahedron Letters · doi:10.1016/s0040-4039(02)02565-0 · Cited by 16 · see citing works
Self-citationFujimoto, Y., Kushiro, T., & Nakamura, K. (1997) · Tetrahedron Letters · doi:10.1016/s0040-4039(97)00432-2 · Cited by 16 · see citing works
Self-citationFujimoto, Y., Hiramoto, M., Kakinuma, S., et al. (1989) · Steroids · doi:10.1016/0039-128x(89)90026-3 · Cited by 12 · see citing works
Self-citationFujimoto, Y., Maeda, I., Ohyama, K., Hikiba, J., & Kataoka, H. (2015) · Phytochemistry · doi:10.1016/j.phytochem.2014.12.019 · Cited by 12 · see citing works
Self-citationEcdysone 20-Monooxygenase Activity of Cytochrome P450 in Ajuga reptans L. Plants and Cell Culture
Alekseeva, L. I. (2004) · Applied Biochemistry and Microbiology · doi:10.1023/b:abim.0000018915.75810.a7 · Cited by 11 · see citing works
Phytoecdysteroid C2‐hydroxylase is microsomal in spinach, Spinacia oleracea L.
Bakrim, A., Crilat, E., Maria, A., de Virville, J. D., Lafont, R., & Takvorian, N. (2009) · Archives of Insect Biochemistry and Physiology · doi:10.1002/arch.20330 · Cited by 5 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Lafont, R., Balducci, C., & Dinan, L. (2021) · Encyclopedia · doi:10.3390/encyclopedia1040096 · Cited by 49 · see citing works
Felipe, S. H. S., Batista, D. S., Vital, C. E., Chagas, K., Silva, P. O., Silva, T. D., Fortini, E. A., Correia, L. N. D. F., Ávila, R. T., Maldaner, J., et al. (2019) · Plant Physiology and Biochemistry · doi:10.1016/j.plaphy.2019.05.002 · Cited by 23 · see citing works
Fortini, E. A., Batista, D. S., de Castro, K. M., Silva, T. D., Felipe, S. H. S., Correia, L. N. F., Chagas, K., Farias, L. M., Leite, J. P. V., & Otoni, W. C. (2020) · Plant Cell Tissue and Organ Culture (PCTOC) · doi:10.1007/s11240-020-01886-3 · Cited by 14 · see citing works
Ecdysteroids as defensive chemicals
(2022) · Advances in Insect Physiology · doi:10.1016/bs.aiip.2022.09.001 · Cited by 11 · see citing works
- Authors
- Tsukagoshi, Y., Ohyama, K., Seki, H., Akashi, T., Muranaka, T., Suzuki, H., & Fujimoto, Y.
- Affiliations
- Tokyo Institute of Technology (JP)
- The University of Osaka (JP)
- Nihon University (JP)
- Kazusa DNA Research Institute (JP)
- Corresponding author
- Fujimoto, Yoshinori
- Venue
- Phytochemistry
- Publisher
- Elsevier BV
- Published
- 2016-07
- Volume
- 127
- Pages
- 23-28
- ISSN
- 0031-9422 (ISSN-L)
- OpenAlex
- W2310379724
- Cited by
- 27
- References
- 44
- Citations by year
- 2026: 3 · 2025: 4 · 2024: 2 · 2023: 3 · 2022: 3 · 2021: 1 · 2020: 3 · 2019: 2 · 2018: 5 · 2016: 1
- FWCI
- 1.39
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Phytochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- Ministry of Education, Culture, Sports, Science and Technology
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant tissue culture and regeneration
- Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Molecular Biology Research
- Keywords
- 20-hydroxyecdysone, cytochrome P450, hairy roots, ecdysteroid biosynthesis, insect molting hormone, yeast expression system, stereochemistry
- MeSH terms
- Cholestanones, Cholesterol, Cytochrome P-450 Enzyme System, Dehydrocholesterols, Ecdysterone, Molecular Structure, Plant Roots, Cytochrome P-450 CYP1A1, Lamiaceae, Ecdysteroids, Ajuga
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
