Characterization of a hostplant-inducible cytochrome P450 from the black swallowtail caterpillar, Papilio polyxenes (Lepidoptera: Papilionidae)

Cytochrome P450 monooxygenases (P450s) function in the oxidation of countless xenobiotic and endogenous substrates, and are among the most important enzymes utilized by herbivores in detoxifying plant defensive compounds. This thesis describes the identification and molecular cloning of a P450 isozyme associated with the detoxification of one such compound, xanthotoxin, by larvae of the black swallowtail, Papilio polyxenes (Lepidoptera:Papilionidae).When added to the diet of final instar larvae, xanthotoxin can induce by up to seven-fold the P450 activity in midgut microsomes by which it is detoxified. This induction involves only a subset of P450 isozymes in the midgut, as determined by a comparison of the induction of enzyme activity and total P450 content, and by Coomassie-blue staining and immunological screening of microsomal proteins.A xanthotoxin-inducible microsomal protein of approximately 55 kD was partially sequenced to generate a probe for screening a cDNA library constructed from xanthotoxin-induced midgut poly(A)$\sp+$ RNA. Two full-length cDNA clones, 1.6 kb in length, were isolated; conserved sequences indicate that they encode P450s. The clones are 98.2% similar in deduced amino acid sequence and probably represent two alleles of a single locus. On Northern blots, the clones cross-react with mRNAs highly inducible by xanthotoxin and by parsnip, a black swallowtail hostplant with high levels of xanthotoxin and other furanocoumarins. The xanthotoxin-inducible protein is most similar in amino acid sequence (32.1%) to P450VIA1 from the house fly and has been designated P450VIB1.P450-mediated metabolism was inducible by xanthotoxin in three additional swallowtail species, P. brevicauda, P. cresphontes, and P. glaucus, but not in P. troilus, Battus philenor, or Eurytides marcellus. Northern analysis of midgut poly(A)$\sp+$ RNA from these species and the parsnip webworm revealed a xanthotoxin-inducible P450 orthologous to P450VIB1 in P. brevicauda, which like P. polyxenes is a member of the P. machon species group, but not in the other species. Thus, P450VIB1 may have evolved in an ancestral species of the machaon group in association with its colonization of hostplants in the family Umbelliferae.U of I OnlyETDs are only available to UIUC Users without author permissio