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

Abstract

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.

Ligand-independent requirements of steroid receptors EcR and USP for cell survival

“…The phm-Gal4 driver is expressed in early embryo. 40 However, the two receptors do not appear to cause deleterious effects, further supporting the pre-metamorphosis time requirement for cell survival. By contrast, experiments with bsk and Br-C had to be carried out in the L2 switch-on mode to bypass their early lethality.…”

Discrete pulses of molting hormone, 20‐hydroxyecdysone, during late larval development of Drosophila melanogaster: Correlations with changes in gene activity

“…To address this issue, we studied the expression of Phm and Dib, two cytochrome P450 enzymes specific to the prothoracic gland cells of the larval ring gland that are required for E biosynthesis (Warren et al, 2002(Warren et al, , 2004Niwa et al, 2004). By Western blot analysis, the significant, although low, expression of both proteins in these same early third instar larval samples is indicative of a rather constant capacity to synthesize E during this period (Fig.…”

“…4) (Warren et al, 2002(Warren et al, , 2004Petryk et al, 2003;Niwa et al, 2004). Quantitative Western blot analysis, performed on the insoluble protein remaining after ecdysteroid extraction (see Experimental Procedures section), reveals significant expression throughout the third instar of both Phantom (Phm, the terminal 25-hydroxylase) and Disembodied (Dib, the subsequent 22-hydroxylase) (Fig.…”

Comparative genomics of insect juvenile hormone biosynthesis☆

“…However, little is known on the molecular aspects of hormone biosynthesis and regulation, so that high-throughput screens of such endocrine targets cannot be deployed. Recently, several genes coding for enzymes of the ecdysteroid biosynthetic pathway in prothoracic glands have been identified in Drosophila melanogaster through the study of Halloween mutants, and in Bombyx mori (Warren et al, 2002(Warren et al, , 2004Niwa et al, 2004). Fewer molecular tools are available for the analysis of JH biosynthesis even though the early steps of JH biosynthesis follow the mevalonate pathway from acetyl-CoA to farnesyl pyrophosphate (Bellé s et al, 2005).…”