26‐Hydroxylation of Ecdysteroids is Catalyzed by a Typical Cytochrome P‐450‐Dependent Oxidase and Related to Ecdysteroid Resistance in an Insect Cell Line

Abstract

The epithelial cell line from the dipteran Chironomus tentwzLy responds to the insect steroid hormone 20-hydroxyecdysone and the non-steroidal analogue tebufenozide by undergoing a morphogenetic and biochemical differentiation program. Long-term culture in the presence of 20-hydroxyecdysone has resulted in the selection of subclones that are resistant to the steroid but respond normally to the nonsteroidal analogue. In the present study, several subclones that were resistant to the steroid hormone have been compared with steroid-sensitive subclones with respect to their capability to metabolize 20-hydroxyecdysone. Homogenates of both types of cells, when incubated with 'H-labelled steroid in the presence of NADPH, produced 20,26-dihydroxyecdysone, which was further metabolized to two compounds, which behaved less polar than 20-hydroxyecdysone o n reverse-phase HPLC. Ecdysone, a lessactive hormone precursor, provided 26-hydroxyecdysone as the only product. The metabolites were identified by mass spectrometry coupled to HPLC, chromatography with authentic samples, and forination of acetonides. The structure of 20,26-dihydroxyecydsone was confirmed by 'H-NMR.The enzyme responsible for the synthesis of 20,26-dihydroxyecdysone in the Chirononius cell preparations has been characterized as a typical cytochrome P-450-dependent monooxygenase. It was a strictly microsomal enzyme, sensitive to inhibition by carbon monoxide and imidazole/triazole-based fungicides, and required NADPH for maximal activity. NADH could partly replace NADPH. The Michaelis constant ( K J for 20-hydroxyecdysone was 0.96 yM, and the maximal enzyme velocity (V,,,,,) was 50 pmol substrate metabolized . mg protein-' . min..'. 26-Hydroxylation of 20-hydroxyecdysone was inhibited by ecdysone, an alternative substrate, and by inokosterone, a product analogue, to 50% at 1.4 pM and 0.73 pM, respectively. When various subclones were compared with respect to their in vitro rate of 20-hydroxyecdysone metabolization, those clones known to be resistant to the steroid were 'high metabolizers' ( > 7 0 % relative rate), whereas the sensitive clones were 'poor metabolizers' (< 30% relative rate). Hence, it is tempting to conclude that ecdysteroid resistance of the Chiroricimu.s cell clones is due to metabolic inactivation of the steroid hormone.

BAS1 : A gene regulating brassinosteroid levels and light responsiveness in Arabidopsis

“…As is the case with BAS1, CYP450-mediated C26-hydroxylation inactivates the insect hormones ecdysteroids, although the precise enzyme has not been identified (36)(37)(38). Though ecdysteroids are inactivated in a similar manner to BRs, these insect hormones do not induce BR responses in plants (39).…”

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

“…However, in contrast to the findings of Berreur et al (1984), little if any free E was detected in any of these early third instar extracts. Yet these repetitive, discrete elevations of 20E and 26E are the likely result of the increased secretion of E by the prothoracic gland cells of the ring gland, followed by its very rapid and efficient conversion to 20E by peripheral 20-hydroxylase (ecdysone 20-monooxygenase) activity (E20MO) (see Petryk et al, 2003) and/or to 26E by tissue 26-hydroxylase activity (Kayser et al, 1997;Williams et al, 2000) (Fig. 4).…”

“…2). In turn, endogenous or exogenous 20E can up-regulate 26-hydroxylase activity Williams et al, 1997;Kayser et al, 1997). In addition to direct changes in P450 expression levels, the intrinsic activity of both of these enzymes may be directly modulated by reversible phosphorylation (Hoggard et al, 1989;Williams et al, 2000).…”

Variations in ecdysteroid levels and Cytochrome p450 expression during moult and reproduction in male shore crabsCarcinus maenas

“…The haemolymph E level is tightly correlated to E production in the Y-organ in relation to moulting (Lachaise et al 1989). In peripheral tissues including the hepatopancreas, E is converted into 20E, which is further metabolised into inactive forms such as 20, 26-OH-E, excreted primarily in urine (Snyder & Chang 1991, Kayser et al 1997, Williams et al 2000. The low ecdysteroid levels in the tissues just prior to and after moulting indicate that all active ecdysteroids must be removed in order to allow chitinisation of the new exoskeleton (Freeman 1980).…”