RRC ID 47240
Author Saito J, Kimura R, Kaieda Y, Nishida R, Ono H.
Title Characterization of candidate intermediates in the Black Box of the ecdysone biosynthetic pathway in Drosophila melanogaster: Evaluation of molting activities on ecdysteroid-defective larvae.
Journal J Insect Physiol
Abstract Early steps of the biosynthetic pathway of the insect steroid hormone ecdysone remains the "Black Box" wherein the characteristic ecdysteroid skeleton is built. 7-Dehydrocholesterol (7dC) is the precursor of uncharacterized intermediates in the Black Box. The oxidation step at C-3 has been hypothesized during conversion from 7dC to 3-oxo-2,22,25-trideoxyecdysone, yet 3-dehydroecdysone is undetectable in some insect species. Therefore, we first confirmed that the oxidation at C-3 occurs in the fruitfly, Drosophila melanogaster using deuterium-labeled cholesterol. We next investigated the molting activities of candidate intermediates, including oxidative products of 7dC, by feeding-rescue experiments for Drosophila larvae in which an expression level of a biosynthetic enzyme was knocked down by the RNAi technique. We found that the administration of cholesta-4,7-dien-3-one (3-oxo-Δ4,7C) could overcome the molting arrest of ecdysteroid-defective larvae in which the expression level of neverland was reduced. However, feeding 3-oxo-Δ4,7C to larvae in which the expression levels of shroud and Cyp6t3 were reduced inhibited molting at the first instar stage, suggesting that this steroid could be converted into an ecdysteroid-antagonist in loss of function studies of these biosynthetic enzymes. Administration of the highly conjugated cholesta-4,6,8(14)-trien-3-one, oxidized from 3-oxo-Δ4,7C, did not trigger molting of ecdysteroid-defective larvae. These results suggest that an oxidative product derived from 7dC is converted into ecdysteroids without the formation of this stable conjugated compound. We further found that the 14α-hydroxyl moiety of Δ4-steroids is required to overcome the molting arrest of larvae in loss of function studies of Neverland, Shroud, CYP6T3 or Spookier, suggesting that oxidation at C-14 is indispensable for conversion of these Δ4-steroids into ecdysteroids via 5β-reduction.
Volume 93-94
Pages 94-104
Published 2016-10-1
DOI 10.1016/j.jinsphys.2016.09.012
PII S0022-1910(16)30311-0
PMID 27662806
MeSH Animals Biosynthetic Pathways Drosophila melanogaster / growth & development Drosophila melanogaster / physiology* Ecdysone / biosynthesis* Ecdysteroids / metabolism* Larva / growth & development Larva / physiology Metamorphosis, Biological Oxidation-Reduction
IF 2.862
Times Cited 5
Drosophila DGRC#105669