Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster

doi:10.1073/pnas.162375799

Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophilamelanogaster

^*Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280; ^‡Department of Pediatrics, University of Minnesota, 516 Delaware Street Southeast, Minneapolis, MN 55455;^§ Department of Genetics, Cell Biology, and Development and^¶ Howard Hughes Medical Institute, University of Minnesota, 321 Church Street Southeast, Minneapolis, MN 55455; and^∥ Université P. et M. Curie, Laboratoire Endocrinologie Moléculaire et Évolution, Bâtiment 5A, 5ème Étage, Case 29, 7 Quai St. Bernard, 75252 Paris Cedex 05, France

Communicated by William S. Bowers, University of Arizona, Tucson, AZ (received for review May 12, 2002)

Abstract

Five different enzymatic activities, catalyzed by both microsomal and mitochondrial cytochrome P450 monooxygenases (CYPs), are strongly implicated in the biosynthesis of ecdysone (E) from cholesterol. However, none of these enzymes have been characterized completely. The present data show that the wild-type genes of two members of the Halloween family of embryonic lethals, disembodied (dib) and shadow (sad), code for mitochondrial cytochromes P450 that mediate the last two hydroxylation reactions in the ecdysteroidogenic pathway in Drosophila, namely the C₂₂- and C₂-hydroxylases. When sad (CYP315A1) is transfected into Drosophila S2 cells, the cells metabolize 2-deoxyecdysone (2dE) to E and the [³H]ketotriol (2,22-dideoxyecdysone) to 22-deoxyecdysone. In contrast, dib (CYP302A1) is responsible for the conversion of the [³H]ketotriol to [³H]2dE. When cells are transfected with both dib and sad, they metabolize the [³H]ketotriol to [³H]E in high yield. The expression of sad and dib is concentrated within the individual segments of the developing epidermis when there is a surge of ecdysteroid midway through embryogenesis. This result occurs before the ring gland has developed and suggests that the embryonic epidermis is a site of ecdysteroid biosynthesis. This pattern then diminishes, and, during late embryogenesis, expression of both genes is concentrated in the prothoracic gland cells of the developing ring gland. Expression of dib and sad continues to be localized in this endocrine compartment during larval development, being maximal in both the late second and third instar larvae, about the time of the premolt peaks in the ecdysteroid titer.

Footnotes

↵ † J.T.W. and A.P. contributed equally to this work.
↵ ** To whom reprint requests should be addressed. E-mail: lgilbert{at}unc.edu.
Data deposition: The sequence reported in this paper has been deposited in the GenBank database [accession no. (sad)].
Abbreviations:
1. 2dE, 2-deoxyecdysone
2. 22dE, 22-deoxyecdysone
3. 2,22dE, 2,22-dideoxyecdysone (ketotriol)
4. dib, disembodied
5. E, ecdysone
6. 20E, 20-hydroxyecdysone
7. sad, shadow
8. CYP, cytochrome P450 monooxygenase
9. GFP, green fluorescent protein
10. ESI, electrospray ionization