Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: A P450 enzyme critical in ecdysone biosynthesis

James T. Warren, Anna Petryk, Guillermo Marqués, Jean Philippe Parvy, Tetsuro Shinoda, Kyo Itoyama, Jun Kobayashi, Michael Jarcho, Yutai Li, Michael B O'Connor, Chantal Dauphin-Villemant, Lawrence I. Gilbert

Research output: Contribution to journalArticlepeer-review

195 Scopus citations

Abstract

We have reported recently the identification and characterization of the last three mitochondrial cytochrome P450 enzymes (CYP) controlling the biosynthesis of 20-hydroxyecdysone, the molting hormone of insects. These are encoded by the following genes: disembodied (dib, Cyp302a1, the 22-hydroxylase); shadow (sad, Cyp315a1, the 2-hydroxylase); and shade (shd, Cyp314a1, the 20-hydroxylase). Employing similar gene identification and transfection techniques and subsequent biochemical analysis of the expressed enzymatic activity, we report the identity of the Drosophila gene phantom (phm), located at 17D1 of the X chromosome, as encoding the microsomal 25-hydroxylase (Cyp306a1). Similar analysis following differential display-based gene identification has also resulted in the characterization of the corresponding 25-hydroxylase gene in Bombyx mori. Confirmation of 2,22,25-trideoxyecdysone (3β,5β-ketodiol) conversion to 2,22-dideoxyecdysone (3β,5β-ketodiol) mediated by either Phm enzyme employed LC, MS and definitive NMR analysis. In situ developmental gene analysis, in addition to northern, western and RT-PCR techniques during Drosophila embryonic, larval and adult development, are consistent with this identification. That is, strong expression of phm is restricted to the prothoracic gland cells of the Drosophila larval ring gland, where it undergoes dramatic changes in expression, and in the adult ovary, but also in the embryonic epidermis. During the last larval-larval transition in Bombyx, a similar expression pattern in the prothoracic gland is observed, but as in Drosophila, slight expression is also present in other tissues, suggesting a possible additional role for the phantom enzyme.

Original languageEnglish (US)
Pages (from-to)991-1010
Number of pages20
JournalInsect Biochemistry and Molecular Biology
Volume34
Issue number9
DOIs
StatePublished - Sep 2004

Bibliographical note

Funding Information:
We thank Susan Whitfield for graphics and David Harris and Marc Ter Horst at the University of North Carolina NMR facility and Agnes Mesneau (Université P. et M. Curie) for technical assistance. The SHO-3 antibody was a gift of Dr. Sho Sakurai (Kanazawa University, Japan). The Drosophila embryonic library was a gift of Nick Brown (Cambridge University, UK). The University of North Carolina MS facility is supported in part by National Institutes of Health (NIH) Grants P30 CA16086 and P30 ES10126. This work was supported by National Science Foundation Grant IBN0130825 (to L.I.G. and J.T.W.). A.P. was supported by NIH Training Grant HD33692. M.B.O. is an Investigator of the Howard Hughes Medical Institute. C.D.-V. and J.-P.P. are supported by the Université Pierre et Marie Curie and the Ministère de la Recherche Scientifique.

Keywords

  • 20-hydroxyecdysone
  • Microsomal CYP enzyme
  • Prothoracic gland
  • Ring gland
  • Steroid molting hormone

Fingerprint Dive into the research topics of 'Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: A P450 enzyme critical in ecdysone biosynthesis'. Together they form a unique fingerprint.

Cite this