Ecdysone 20-monooxygenase: Characterization of an insect cytochrome P-450 dependent steroid hydroxylase

Smith, Stan L.; Bollenbacher, Walter E.; Cooper, David Y.; Schleyer, Heinz; Wielgus, John J.; Gilbert, Lawrence I.

doi:10.1016/0303-7207(79)90033-9

Cited by 92 publications

(39 citation statements)

References 26 publications

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“…For Dib and Sad, this is consistent with prior biochemical fractionation data showing that both the 22-and 2-monooxygenase activities are mitochondrial (29). Prior cell fractionation data for the E20MO is more ambiguous, with a microsomal and͞or mitochondrial localization identified, depending on the insect, tissue, and developmental stage being examined (4,5,7,46,47). The N terminus of the Shd protein contains both hydrophobic signal-type sequences typical of microsomal P450s (48) as well as a charged segment containing sequences characteristic of typical mitochondrial enzymes (49).…”

Section: Discussionsupporting

confidence: 75%

“…Although it has been known for several decades that the E20MO is a P450 enzyme that is localized in the endoplasmic reticulum (4) and͞or mitochondria (5,6), depending on the insect, tissue, and developmental stage (7,8), it has not been purified to homogeneity nor has the gene coding for this enzyme been cloned. Here, we report the cloning of shade (CYP314a1), a member of the Halloween gene family (9), and demonstrate that its gene product codes for the Drosophila E20MO.…”

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confidence: 99%

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

Petryk

Warren

Marqués

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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410

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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.

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Section: Discussionsupporting

confidence: 75%

mentioning

confidence: 99%

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

Petryk

Warren

Marqués

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

410

373

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“…The fat body enzyme is located in the mitochondria (Bollenbacher et al, 1977;Smith et al, 1979Smith et al, ,1980. The midgut contains two different E20MOs, one in the mitochondria and one in the microsomes.…”

Section: Introductionmentioning

confidence: 99%

Ecdysone 20-hydroxylation inManduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

Weirich

Williams

Feldlaufer

1996

Arch. Insect Biochem. Physiol.

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The larval midgut of the tobacco hornworm, Manduca sexta, has high ecdysone 20‐monooxygenase (E20MO) activity, located both in the mitochondria and in the microsomes. The apparent kinetic parameters for E20MO in mitochondria and microsomes were determined. The Km5 (for ecdysone) of the mitochondrial and microsomal enzymes were 1.63 × 10−5 and 3.67 × 10−7 M, respectively. The Vmax was 82.7 pmol/min/mg protein for mitochondria and 32.0 pmol/min/mg protein for microsomes. Although the mitochondrial E20MO has the higher Vmax, at physiological ecdysone concentrations (10−7 − 10−8 M) it is only one‐eighth to one‐tenth as active as the microsomal enzyme. It is concluded that the microsomal E20MO is the primary, if not the only, enzyme involved in ecdysone 20‐hydroxylation in M. sexta midgut. © 1996 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

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“…7,12 Research detailing the role of the cytochrome P-450 steroid hydroxylase E-20M has established its role in the NADPH-dependent conversion of the molting hormone, ecdysone, to its physiologically active form, 20-HE. 3,[16][17][18] Because this activity requires NADPH, and is predominantly associated with the mitochondria of the ecdysone target tissues such as midgut, fatbody, and Malphighian tubules 5,19 an association with the reversible transhydrogenase seems likely. In view of the coincidental peaks in ecdysone 20-monooxygenase activity and transhydrogenase activity, a developmental relationship between these mitochondrial proteins has been suggested.…”

Section: Discussionmentioning

confidence: 99%

“…5 Additionally, a marked preference for NADPH as the source of reducing equivalents is displayed for M. sexta mitochondrial E20-M's. 3 Recent molecular studies established that E20-M is indeed a P450-dependent steroid hydroxylase which is highly regulated and encoded by the gene shade. 6 Further evidence demonstrating the physiological importance of E20-M-mediated ecdysone conversion is evident in the identification of several different ecdysteroid cellular receptors which show over 1000 fold greater affinity for 20-HE then ecdysone.…”

Section: Introductionmentioning

confidence: 99%

Sequence and Structure of the Tobacco Hornworm,Manduca sexta, Transhydrogenase Gene

et al. 2012

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Abstract:The reversible mitochondrial transhydrogenase in Manduca sexta has been characterized and is involved with endocrinemediated post-embryonic larval development in this model insect. While biochemical in vivo and in vitro studies have been accomplished, robust molecular studies of the transhydrogenase have not been possible due to deficient genomic data. In the present study, using a combination of degenerate oligonucleotide primers and raw genomic data, we have determined the structure and sequence of the transhydrogenase gene from the model insect Manduca sexta. The encoded protein is highly similar to other transhydrogenase proteins and this sequence is the first lepidopteran sequence reported to date.

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Ecdysone 20-monooxygenase: Characterization of an insect cytochrome P-450 dependent steroid hydroxylase

Cited by 92 publications

References 26 publications

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

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

Ecdysone 20-hydroxylation inManduca sexta midgut: Kinetic parameters of mitochondrial and microsomal ecdysone 20-monooxygenases

Sequence and Structure of the Tobacco Hornworm,Manduca sexta, Transhydrogenase Gene

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