Diversification of furanocoumarin-metabolizing cytochrome P450 monooxygenases in two papilionids: Specificity and substrate encounter rate

Abstract

Diversification of cytochrome P450 monooxygenases (P450s) is thought to result from antagonistic interactions between plants and their herbivorous enemies. However, little direct evidence demonstrates the relationship between selection by plant toxins and adaptive changes in herbivore P450s. Here we show that the furanocoumarin-metabolic activity of CYP6B proteins in two species of swallowtail caterpillars is associated with the probability of encountering host plant furanocoumarins. Catalytic activity was compared in two closely related CYP6B4 and CYP6B17 groups in the polyphagous congeners Papilio glaucus and Papilio canadensis. Generally, P450s from P. glaucus, which feeds occasionally on furanocoumarin-containing host plants, display higher activities against furanocoumarins than those from P. canadensis, which normally does not encounter furanocoumarins. These P450s in turn catalyze a larger range of furanocoumarins at lower efficiency than CYP6B1, a P450 from Papilio polyxenes, which feeds exclusively on furanocoumarin-containing host plants. Reconstruction of the ancestral CYP6B sequences using maximum likelihood predictions and comparisons of the sequence and geometry of their active sites to those of contemporary CYP6B proteins indicate that host plant diversity is directly related to P450 activity and inversely related to substrate specificity. These predictions suggest that, along the lineage leading to Papilio P450s, the ancestral, highly versatile CYP6B protein presumed to exist in a polyphagous species evolved through time into a more efficient and specialized CYP6B1-like protein in Papilio species with continual exposure to furanocoumarins. Further diversification of Papilio CYP6Bs has likely involved interspersed events of positive selection in oligophagous species and relaxation of functional constraints in polyphagous species.

Fig. 1.

Reduced representation of the most parsimonious phylogeny of insect and mammalian P450s. The CYP subfamily was highlighted with gray background. The non-CYP6B sequences were retrieved from GenBank. The phylogenetic tree has been drawn proportional to branch lengths calculated by using maximum likelihood method. The numbers below internal branches are bootstrap values. The species origin of each CYP6B protein is indicated in parentheses after each sequence name.

Fig. 2.

Relative distance of ancestral P450s to CYP6Bs. Grouped bars represent the ratio of average amino acid distance between ancestral sequence and the upper branch to that of the lower branch. The distance ratio was computed either based on active site residues (filled bars) or on the complete protein sequence (open bars). The ratios indicated as >1 are significantly different from unity (P < 0.05).

Fig. 3.

A multiple sequence alignment of the amino acid residues in the active sites of CYP6B and their ancestral P450s. Three ancestral sequences are shown, the ancestral CYP6B sequence (Anc1), the ancestral Papilio CYP6B (Anc2) and the ancestral P. glaucus/P. canadensis CYP6B (Anc3). Residue positions are labeled according to their position in CYP6B4 and CYP6B1. The residues inside the black boxes represent SRS domains (35). Amino acids identical to Anc1 were highlighted in yellow; amino acids identical to Anc2 but not Anc1 are shown in blue; amino acids identical to Anc3, but neither Anc1 nor Anc2, are shown in red. Amino acids that were proposed to be critical to P450 substrate specificity (Fig. 4) are indicated in bold.

Fig. 4.

Superposition of the active sites of ancestral CYP6B proteins with CYP6B1 and CYP6B4. (a) Anc1. (b) Anc2. The amino acid residues of CYP6B1 and CYP6B4 and the ancestral sequences are shown with the residues in the CYP6B1 sequence or progenitor to it labeled in red and amino acids in the CYP6B4 sequence or progenitor to it labeled in yellow. Residues involved in the aromatic interactive network in the CYP6B1 model are displayed as balls and sticks (33).