Publications

• Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone.

  Petryk A., Warren J.T., Marques G., Jarcho M.P., Gilbert L.I., Kahler J., Parvy J.-P., Li Y., Dauphin-Villemant C., O'Connor M.B.

  The steroid 20-hydroxyecdysone (20E) is the primary regulatory hormone that mediates developmental transitions in insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdystero ... >> More

  The steroid 20-hydroxyecdysone (20E) is the primary regulatory hormone that mediates developmental transitions in insects and other arthropods. 20E is produced from ecdysone (E) by the action of a P450 monooxygenase that hydroxylates E at carbon 20. The gene coding for this key enzyme of ecdysteroidogenesis has not been identified definitively in any insect. We show here that the Drosophila E-20-monooxygenase (E20MO) is the product of the shade (shd) locus (cytochrome p450, CYP314a1). When shd is transfected into Drosophila S2 cells, extensive conversion of E to 20E is observed, whereas in sorted homozygous shd embryos, no E20MO activity is apparent either in vivo or in vitro. Mutations in shd lead to severe disruptions in late embryonic morphogenesis and exhibit phenotypes identical to those seen in disembodied (dib) and shadow (sad) mutants, two other genes of the Halloween class that code for P450 enzymes that catalyze the final two steps in the synthesis of E from 2,22-dideoxyecdysone. Unlike dib and sad, shd is not expressed in the ring gland but is expressed in peripheral tissues such as the epidermis, midgut, Malpighian tubules, and fat body, i.e., tissues known to be major sites of E20MO activity in a variety of insects. However, the tissue in which shd is expressed does not appear to be important for developmental function because misexpression of shd in the embryonic mesoderm instead of the epidermis, the normal embryonic tissue in which shd is expressed, rescues embryonic lethality. << Less

  Proc. Natl. Acad. Sci. U.S.A. 100:13773-13778(2003) [PubMed] [EuropePMC]

• Ecdysome 20-hydroxylase from the midgut of the tobacco hornworm (Manduca sexta L.).

  Nigg H.N., Svoboda J.A., Thompson M.J., Dutky S.R., Kaplanis J.N., Robbins W.E.

  An ecdysone 20-hydroxylase enzyme system that converts alpha-ecdysone to 20-hydroxyecdysone was prepared from the midgut of the tobacco hornworm prepupa. This partially purified enzyme is NADPH dependent and is localized in the mitochondrial fraction of the midgut tissue.

  Experientia 32:438-439(1976) [PubMed] [EuropePMC]

• The mechanism of C-20 hydroxylation of alpha-ecdysone in the desert locust, Schistocerca gregaria.

  Johnson P., Rees H.H.

  1. The C-20 hydroxylation of alpha-ecdysone to produce beta-ecdysone was investigated in the desert locust, Schistocerca gregaria. 2. alpha-Ecdysone C-20 hydroxylase activity was located primarily in the fat-body and Malpighian tubules. The properties of the hydroxylation system from Malpighian tu ... >> More

  1. The C-20 hydroxylation of alpha-ecdysone to produce beta-ecdysone was investigated in the desert locust, Schistocerca gregaria. 2. alpha-Ecdysone C-20 hydroxylase activity was located primarily in the fat-body and Malpighian tubules. The properties of the hydroxylation system from Malpighian tubules investigated further. 3. The enzyme system was mitochondrial, had a pH optimum of 6.5, an apparent Km of 12.5 micron and required O2 and NADPH. 4. The activity of the hydroxylation system showed developmental variation within the fifth instar, the maximum activity corresponding to the maximum tire of endogenous moulting hormone. The significance of these results is assessed in relation to the control of the endogenous titre of beta-ecdysone. 5. The mechanism of the hydroxylation system was investigated by using known inhibitors of hydroxylation reactions such as CO, metyrapone and cyanide. 6. The CO difference spectrum of the reduced mitochondrial preparation indicated the presence of cytochrome P-450 in the preparation. 7. It concluded that the alpha-ecdysone C-20 hydroxylase system is a cytochrome P-450-deendent mono-oxygenase. << Less

  Biochem J 168:513-520(1977) [PubMed] [EuropePMC]

• Ecdysone 20-monooxygenase: characterization of an insect cytochrome p-450 dependent steroid hydroxylase.

  Smith S.L., Bollenbacher W.E., Cooper D.Y., Schleyer H., Wielgus J.J., Gilbert L.I.

  Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone at C-20 of the side-chain to form ecdysterone, has been characterized in the fat body of early last instar larvae of the tobacco hornworm, Manduca sexta, using a radioenzymological assay. Ecdysterone was demonstrated to be the ... >> More

  Ecdysone 20-monooxygenase, the enzyme system that hydroxylates ecdysone at C-20 of the side-chain to form ecdysterone, has been characterized in the fat body of early last instar larvae of the tobacco hornworm, Manduca sexta, using a radioenzymological assay. Ecdysterone was demonstrated to be the product of the enzyme system by high-pressure liquid chromatography, gas-liquid chromatography and mass spectrometry. Differential centrifugation, sucrose-gradient centrifugation, electron microscopy and organelle-marker enzyme analysis revealed that ecdysone 20-monooxygenase activity is associated with the mitochondria. The enzymatic properties of ecdysone 20-monooxygenase are that it is most active in a 0.05 M phosphate buffer, is inhibited by Mg2+ and exhibits pH and temperature optima at 7.5 and 30 degrees C, respectively. The enzyme complex has an apparent Km for ecdysone of 1.60 x 10(-7) M and is competitively inhibited by its product, ecdysterone, with an apparent Ki of 2.72 x 10(-5) M. The cytochrome P-450 nature of this insect steroid hydroxylase was initially suggested by its obligate requirement for NADPH and its inhibition by carbon monoxide, p-chloromercuribenzoate, metyrapone and p-aminoglutethimide but not by cyanide. Difference spectroscopy revealed the presence of cytochrome P-450 in the fat-body mitochondrial fraction. A photochemical action spectrum of ecdysone 20-monooxygenase activity confirmed the involvement of cytochrome P-450 in this monooxygenase system. << Less

  Mol Cell Endocrinol 15:111-133(1979) [PubMed] [EuropePMC]