CYP6B1 and CYP6B3 of the Black Swallowtail (Papilio polyxenes): Adaptive Evolution through Subfunctionalization

Abstract

Gene duplication provides essential material for functional divergence of proteins and hence allows organisms to adapt to changing environments. Following duplication events, redundant paralogs may undergo different evolutionary paths via processes known as nonfunctionalization, neofunctionalization, or subfunctionalization. Studies of adaptive evolution at the molecular level have progressed rapidly by computationally analyzing nucleotide substitution patterns but such studies are limited by the absence of information relating to alterations of function of the encoded enzymes. In this respect, evolution of the Papilio polyxenes cytochrome P450 monooxygenases (P450s) responsible for the adaptation of this insect to furanocoumarin-containing host plants provides an excellent model for elucidating the evolutionary fate of duplicated genes. Evidence from sequence and functional analysis in combination with molecular modeling indicates that the paralogous CYP6B1 and CYP6B3 genes in P. polyxenes have probably evolved via subfunctionalization after the duplication event by which they arose. Both enzymes have been under independent purifying selection as evidenced by the low dN/dS ratio in both the coding region and substrate recognition sites. Both enzymes have maintained their ability to metabolize linear and angular furanocoumarins albeit at different efficiencies. Comparisons of molecular models developed for the CYP6B3 and CYP6B1 proteins highlight differences in their binding modes that account for their different activities toward linear and angular furanocoumarins. That P. polyxenes maintains these 2 furanocoumarin-metabolizing loci with somewhat different activities and expression patterns provides this species with the potential to acquire P450s with novel functions while maintaining those most critical to its exclusive feeding on its current range of host plants.

Comparative molecular modeling of Anopheles gambiae CYP6Z1, a mosquito P450 capable of metabolizing DDT

“…Among plant P450s, spearmint CYP71D15 and peppermint CYP71D18 have different regiospecificities for limonene hydroxylation because of a single amino acid difference within the catalytic site (F363I in SRS5) (44,45). The closely related lepidopteran CYP6B1 and CYP6B3 also differ in their metabolism of plant allelochemicals (46). These metabolic variations highlight the fact that, even with high global sequence identity, only a small number of residues in each binding site define P450 substrate binding preferences.…”

Allelic Variation in the Depressaria pastinacella CYP6AB3 Protein Enhances Metabolism of Plant Allelochemicals by Altering a Proximal Surface Residue and Potential Interactions with Cytochrome P450 Reductase

“…A molecular model of CYP6AB3v1 contains three-dimensional elements similar to those predicted in the catalytic sites of the furanocoumarin-metabolizing CYP6B proteins in P. polyxenes, P. glaucus, and H. zea (25)(26)(27)31) including residues in the B-helix and BЈ-C loop of SRS1, the I-helix of SRS4, and the ␤-turn in ␤-sheet 4 of SRS6 (30). Amino acids are also conserved in SRS1, SRS4, and SRS5 that contribute to form the catalytic site of CYP6B1 from the specialist P. polyxenes, CYP6B4 from the generalist P. (25,31,32).…”

P450s in Plants, Insects, and Their Fungal Pathogens

“…While these examples highlight the breadth of compounds metabolized by insect P450s, comparisons of the predicted catalytic sites have provided more significant information on the amino acid variations between nonselective and selective P450s Recent reviews [291,305] provide numerous examples of the variations affecting particular P450 activities with most examples drawn from comparisons of closely related subfamily members and not from natural variations in individual insect P450s Not surprisingly, therefore, most of the highlighted variations map to residues in catalytic sites, substrate access channels, and proximal surfaces Highlighting a few of the important differences between closely related subfamily members, instances of catalytic site differences include A. gambiae CYP6Z2, where protrusions of Arg210 (SRS2), Ile298 and Glu302 (both in SRS4) are predicted to restrict its substrate range compared to CYP6Z1 [297], A. mellifera CYP9Q2, where protrusion of Arg246 (SRS3) into its catalytic site is predicted to prevent quercetin metabolism compared to CYP9Q1 [275] and the A. mellifera CYP6AS subfamily, where side chains on residues 107 (SRS1) and 217 (SRS2) and the carbonyl backbone between residues 302 and 303 (SRS4) moderate quercetin metabolism [306] Many other examples of catalytic site variations affecting activity exist in the Papilio and Helicoverpa CYP6B subfamily, where furanocoumarin metabolism rates are defined by the presence or absence of aromatic side chains in SRS1, SRS5, and SRS6 and other types of side chains in all six SRS regions [291] Instances of substrate access channel differences affecting activity include A. minimus CYP6P8, where Arg114 (SRS1) and Arg216 (SRS2) are predicted to extend into the CYP6P8 substrate access channel and prevent pyrethroid access compared to the closely related CYP6P7 that metabolizes this insecticide quite efficiently [307] Characteristic of the small number of natural and site-directed variants actually analyzed, some natural P. polyxenes CYP6B3 variants [263] and site-directed P. polyxenes CYP6B1 mutants [308][309][310] have identified particular SRS residues important in P450 folding, substrate turnover, and/or product exit Adding to this collection of important residues, natural D. pastinacella CYP6AB3 variants have identified proximal surface residues affecting catalytic efficiency with a single Val92Ala (B-helix on the proximal surface) switch substantially enhancing electron transfer from P450 reductase [265] And, several site-directed P. multicaudatus CYP6B33 mutants have identified residue 32 (in the linker preceding the proline-rich hinge) as important for folding of this P450 in insect cell expression systems [267] Not yet tested in site-directed mutants, other examples of potentially important residues likely exist in CYP6CM1 variants, where two changes in imidacloprid-resistant B. tabaci (His341Asn, Asn367Thr (numbered as in resistant compared to susceptible biotypes)) map to the proximal surface [282], CYP6A2 variants, where two changes...…”