One challenge in the heterologous expression of microsomal cytochrome P450 monooxygenases (P450s) is fulfilling their obligatory requirement for electrons transferred from NADPH P450 reductase. We have established co-expression parameters for Papilio polyxenes CYP6B1 and house fly P450 reductase in baculovirus-infected Sf9 cells that allow for efficient expression of both components and significantly enhance metabolic turnover of this insect P450's substrates. These expression conditions have allowed us to reexamine the turnover capacities of CYP6B1 toward linear and angular furanocoumarins present in the host plants for the specialist caterpillar P. polyxenes. Coexpression of CYP6B1 and P450 reductase at equivalent viral concentrations [MOI (multiplicity of infection) ratio of 1] results in turnover rates for the linear furanocoumarins xanthotoxin and psoralen, which are increased 32-33 fold over the turnover rates obtained with CYP6B1 expressed alone. The turnover rate for the angular furanocoumarin angelicin is also significantly increased to 4.76 nmol/min/nmol P450 compared to its barely detectable level obtained with CYP6B1 expressed alone. Substrate binding analyses indicate that all three of these compounds elicit typical type I binding spectra but with varying magnitudes and affinities that are indicative of each substrate's effectiveness at coordinating with the heme iron. The relative proportions of high spin state generated with these substrates are consistent with CYP6B1 metabolizing these furanocoumarins in the rank order xanthotoxin>psoralen>angelicin. These differential activities for CYP6B1 suggest that it may have been an ancient participant in the coevolutionary arms race between papilionid butterflies and their apiaceous host plants. Due to its ability to handle a range of furanocoumarin structures, CYP6B1 may have contributed to P. polyxenes' early colonization of linear furanocoumarin-containing plants and to its subsequent colonization of angular furanocoumarin-containing plants.
- Baculovirus expression
- Furanocoumarin metabolism
- NADPH-dependent P450 reductase
- P450s (cytochrome P450 monooxygenases)
ASJC Scopus subject areas
- Molecular Biology
- Insect Science