Functional characterization and evolutionary analysis of lepidopteran cytochrome P450 genes involved in plant-insect interaction.

摘要

Herbivorous animals rely on very sophisticated detoxification systems, such as the cytochrome P450 monooxygenases (P450s), to overcome the chemical defense systems of plants. Understanding the driving force behind the evolution of these systems that is promoted by gene duplication will help us elucidate the mechanisms underlying the reciprocal selection between plants and their herbivores and provide useful information on the changing function of these enzymes. Here we demonstrate, using evolutionary, functional and structural methodologies to investigate the significance of positive selection for diversification of the lepidopteran CYP6B subfamily, that positive selection is the dominant force driving diversification of newly diverged P450s. Analysis applying maximum likelihood models that account for various selection pressure among codon sites has identified that 4.1% of the amino acids are under positive selection. Homology modeling and structural replacements have demonstrated that these sites are located mainly in three regions, corresponding to the extended I-H loop that is specific to this CYP6B subfamily, the C-terminal domain that protrudes into the catalytic pocket; and the entrance of the substrate access channel. Chimeric combinations of closely related CYP6B proteins have confirmed that the identity of residue 129, which is predicted to be under positive selection and interact with the extended I-H loop, defines the functional differences between the CYP6B4 and CYP6B25 proteins. These studies also indicate that variations in the ability of these proteins to metabolize angelicin, an angular furanocoumarin, can be attributed to adaptive substitutions in the C-terminal domain. Our findings demonstrate that positive selection is important for selection-associated functional divergence of P450 genes. Combined applications of evolutionary, functional and structural analyses can be very powerful tools for studying evolutionary process, as well as protein functionality and protein engineering.