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J. Biol. Chem., Vol. 279, Issue 34, 35942-35949, August 20, 2004

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CYP306A1, a Cytochrome P450 Enzyme, Is Essential for Ecdysteroid Biosynthesis in the Prothoracic Glands of Bombyx and Drosophila^*

Ryusuke Niwa, A research fellow of the Japan Society for the Promotion of Science., Takahiro Matsuda¶, Takuji Yoshiyama, Toshiki Namiki, Kazuei Mita||, Yoshinori Fujimoto¶, and Hiroshi Kataoka**

From the Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, the ^¶Department of Chemistry and Materials Science, Tokyo Institute of Technology, Meguro, Tokyo 152-8551, and the ^||Laboratory of Insect Genome, National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8643, Japan

Ecdysteroids mediate a wide variety of developmental and physiological events in insects. In the postembryonic development of insects, ecdysone is synthesized in the prothoracic gland (PG). Although many studies have revealed the biochemical and physiological properties of the enzymes for ecdysteroid biosynthesis, most of the molecular identities of these enzymes have not been elucidated. Here we describe an uncharacterized cytochrome P450 gene, designated Cyp306a1, that is essential for ecdysteroid biosynthesis in the PGs of the silkworm Bombyx mori and fruit fly Drosophila melanogaster. Using the microarray technique for analyzing gene expression profiles in PG cells during Bombyx development, we identified two PG-specific P450 genes whose temporal expression patterns are correlated with changes in ecdysteroid titer during development. Amino acid sequence analysis showed that one of the Bombyx P450 genes belongs to the CYP306A1 subfamily. The temporal and spatial expression pattern of the Drosophila Cyp306a1 homolog is essentially the same as that of Bombyx Cyp306a1. We also found that Drosophila Cyp306a1 is disrupted in the phantom (phm) mutant, known also as the Halloween mutant. The morphological defects and decreased expression of ecdysone-inducible genes in phm suggest that this mutant cannot produce a high titer of ecdysone. Finally we demonstrate that S2 cells transfected with Cyp306a1 convert ketodiol to ketotriol via carbon 25 hydroxylation. These results strongly suggest that CYP306A1 functions as a carbon 25 hydroxylase and has an essential role in ecdysteroid biosynthesis during insect development.

Received for publication, April 23, 2004 , and in revised form, June 14, 2004.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AB162964 (for Bombyx Cyp306a1) and AB167737 (for Bombyx Cyp315a1/sad).

^* This work was supported by grants from Research for the Future Program of the Japan Society for Promotion of Sciences, Japan (to H. K.) and from the Program for Promotion of Basic Research Activities for Innovative Biosciences and the Insect Technology Project of the Ministry of Agriculture, Forestry and Fisheries of Japan (to K. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1 and 2.

To whom correspondence may be addressed: Dept. of Integrated Biosciences, Rm. 201, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan. Tel.: 81-4-7136-3622; Fax: 81-4-7136-3623; E-mail: rniwa{at}k.u-tokyo.ac.jp. ^** To whom correspondence may be addressed: Dept. of Integrated Biosciences, Rm. 201, The University of Tokyo, Kashiwa, Chiba 277-8562, Japan. Tel.: 81-4-7136-3622; Fax: 81-4-7136-3623; E-mail: kataoka{at}k.u-tokyo.ac.jp.

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