Ecdysterone Biosynthesis: A Microsomal Cytochrome-P-450-Linked Ecdysone 20-Monooxygenase from Tissues of the African Migratory Locust

Durst, Francis

doi:10.1111/j.1432-1033.1978.tb12420.x

Cited by 128 publications

(34 citation statements)

References 52 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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.

410

373

View full text Add to dashboard Cite

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.

show abstract

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.

410

373

View full text Add to dashboard Cite

show abstract

“…For example, the electron transfer from NADPH through the flavoprotein NADPH:ferrihemoprotein oxidoreductase (EC 1.6.2.4) to multiple forms of P-450 proteins appears to be identical (5,6). Insect P-450 monooxygenases are involved in the metabolism of insect hormones (7,8), secondary plant chemicals (9), and synthetic insecticides (6). Insecticide resistance in insect pests of agricultural or medical importance has been attributed in many cases to a modified cytochrome P-450 system (6,10).…”

mentioning

confidence: 99%

Isolation and sequence of cDNA encoding a cytochrome P-450 from an insecticide-resistant strain of the house fly, Musca domestica.

Feyereisen

Koener

Farnsworth

et al. 1989

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

126

View full text Add to dashboard Cite

A cDNA expression library from phenobarbital-treated house fly (Musca domestica) was screened with rabbit antisera directed against partially purified house fly cytochrome P450. Two overlapping clones with insert lengths of 1.3 and 1.5 kilobases were isolated. The sequence of a 1629-base-pair (bp) cDNA was obtained, with an open reading frame (nucleotides ) encoding a P450 protein of 509 residues (Mr = 58,738). The insect P-450 protein contains a hydrophobic NH2 terminus and a 22-residue cysteine-containing fragment near the COOH terminus that is known to bind the heme; both of these features have been found in the more than five dozen vertebrate P-450 proteins of which the sequences are presently known. Interestingly, the termination codon UAA may be contained in a putative polyadenylylation signal (AAUAAA) of the mRNA. This P-450 protein exhibits the most similarity (27% amino acid positional identity) with mammalian proteins of the P450111 family but qualifies as a member of another family, which we propose to designate the P450VI gene family.This cDNA and deduced protein sequence should provide important information in the study of evolution of the P450 gene superfamily, as well as provide an important probe for studying the regulation of insect P450 and understanding the molecular genetics of insecticide resistance.Cytochrome P-450 monooxygenases are ubiquitous enzymes, involved in the metabolism of endogenous compounds and of xenobiotics, such as pesticides, drugs, and chemical carcinogens (1). Thirteen P450 gene families comprised of more than five dozen members have been described to date, chiefly from mammals, yeast, and bacteria (2-4).Microsomal P-450 enzymes from insects have many common features with those from other organisms (5,6). For example, the electron transfer from NADPH through the flavoprotein NADPH:ferrihemoprotein oxidoreductase (EC 1.6.2.4) to multiple forms of P-450 proteins appears to be identical (5,6). Insect P-450 monooxygenases are involved in the metabolism of insect hormones (7,8), secondary plant chemicals (9), and synthetic insecticides (6). Insecticide resistance in insect pests of agricultural or medical importance has been attributed in many cases to a modified cytochrome P-450 system (6, 10). However, little is known about the molecular basis ofP-450-linked resistance, because of the difficulty in purifying and characterizing P-450 from insect sources. Higher P-450 levels, as well as differences in P-450 catalytic and spectral characteristics, have been observed in insecticide-resistant strains of Musca domestica (11) and other insects (6). Such "qualitative" changes have also been observed in phenobarbital-treated house flies (12) and may result either from the increased expression of specific P-450 genes or from the selection of flies carrying a mutant P-450 gene that would confer a selective advantage during insecticide exposure.In this paper we report the isolation and sequence of a P-450 cDNA from the house fly, Musca domestica. Our strategy was to use an insec...

show abstract

“…20-Hydroxy-'Hlecdysoiie was produced from ['Hlecdysone by incubation with microsoines prepared from malpighian tubules of the migratory locust, Locusta nzigratoria (Feyereisen and Durst, 1978). Microsomes from malpighian tubules were prepared in 20 mM potassium phosphate, 160 mM potassium chloride, pH 7.5, following a standard protocol, and purified by centrifugation through 2.5% (by mass) sucrose and sedimentation onto a 40% (by mass) sucrose cushion at 1500OOg for 1 h (Beckman L8-70M with rotor 70.1 Ti).…”

Section: Methodsmentioning

confidence: 99%

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

Kayser¹,

Wtnkler²,

Spindler-Barth³

1997

European Journal of Biochemistry

View full text Add to dashboard Cite

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.

show abstract

Ecdysterone Biosynthesis: A Microsomal Cytochrome-P-450-Linked Ecdysone 20-Monooxygenase from Tissues of the African Migratory Locust

Cited by 128 publications

References 52 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

Isolation and sequence of cDNA encoding a cytochrome P-450 from an insecticide-resistant strain of the house fly, Musca domestica.

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

Contact Info

Product

Resources

About