Molecular cloning and expression of CYP6B8: a xanthotoxin-inducible cytochrome P450 cDNA from Helicoverpa zea

Abstract

Xanthotoxin, a plant allelochemical, induces α-cypermethrin insecticide tolerance in Helicoverpa zea (corn earworm); inhibition of tolerance by piperonyl butoxide implicates cytochrome P450 monooxygenases (P450s) in the detoxification of this insecticide. To characterize the xanthotoxin-inducible P450 that might mediate α-cypermethrin tolerance in this species, a cDNA library prepared from xanthotoxin-induced H. zea fifth instar larvae was screened with cDNAs encoding furanocoumarin-metabolizing P450s from Papilio polyxenes ( CYP6B1v2) and P. glaucus ( CYP6B4v2) as well as a sequence-related P450 from Helicoverpa armigera ( CYP6B2). One full-length cDNA isolated in this screening shares 51–99% amino acid identity with the CYP6B subfamily of P450s isolated from Papilio and Helicoverpa species and, thus, has been designated CYP6B8. All of these CYP6B subfamily members share a number of highly conserved domains, including substrate recognition site 1 (SRS1) that is critical for xanthotoxin metabolism by CYP6B1v2 from Papilio polyxenes and coumarin metabolism by CYP2a5 from Mus musculus. Northern and RT-PCR analyses indicate that CYP6B8 expression is strongly induced by xanthotoxin and phenobarbital and negligibly induced by α-cypermethrin.