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Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates.

ABSTRACT: The cytochrome P450 family 1 enzymes (CYP1s) are a diverse family of hemoprotein monooxygenases, which metabolize many xenobiotics including numerous environmental carcinogens. However, their historical function and evolution remain largely unstudied. Here we investigate CYP1 evolution via the reconstruction and characterization of the vertebrate CYP1 ancestors. Younger ancestors and extant forms generally demonstrated higher activity toward typical CYP1 xenobiotic and steroid substrates than older ancestors, suggesting significant diversification away from the original CYP1 function. Caffeine metabolism appears to be a recently evolved trait of the CYP1A subfamily, observed in the mammalian CYP1A lineage, and may parallel the recent evolution of caffeine synthesis in multiple separate plant species. Likewise, the aryl hydrocarbon receptor agonist, 6-formylindolo[3,2-b]carbazole (FICZ) was metabolized to a greater extent by certain younger ancestors and extant forms, suggesting that activity toward FICZ increased in specific CYP1 evolutionary branches, a process that may have occurred in parallel to the exploitation of land where UV-exposure was higher than in aquatic environments. As observed with previous reconstructions of P450 enzymes, thermostability correlated with evolutionary age; the oldest ancestor was up to 35 °C more thermostable than the extant forms, with a 10T50 (temperature at which 50% of the hemoprotein remains intact after 10 min) of 71 °C. This robustness may have facilitated evolutionary diversification of the CYP1s by buffering the destabilizing effects of mutations that conferred novel functions, a phenomenon which may also be useful in exploiting the catalytic versatility of these ancestral enzymes for commercial application as biocatalysts.

SUBMITTER: Harris KL

PROVIDER: S-EPMC9185370 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Genes vary greatly in their long-term phylogenetic stability and there exists no general explanation for these differences. The cytochrome P450 (CYP450) gene superfamily is well suited to investigating this problem because it is large and well studied, and it includes both stable and unstable genes. CYP450 genes encode oxidase enzymes that function in metabolism of endogenous small molecules and in detoxification of xenobiotic compounds. Both types of enzymes have been intensively studied. My analysis of ten nearly complete vertebrate genomes indicates that each genome contains 50-80 CYP450 genes, which are about evenly divided between phylogenetically stable and unstable genes. The stable genes are characterized by few or no gene duplications or losses in species ranging from bony fish to mammals, whereas unstable genes are characterized by frequent gene duplications and losses (birth-death evolution) even among closely related species. All of the CYP450 genes that encode enzymes with known endogenous substrates are phylogenetically stable. In contrast, most of the unstable genes encode enzymes that function as xenobiotic detoxifiers. Nearly all unstable CYP450 genes in the mouse and human genomes reside in a few dense gene clusters, forming unstable gene islands that arose by recurrent local gene duplication. Evidence for positive selection in amino acid sequence is restricted to these unstable CYP450 genes, and sites of selection are associated with substrate-binding regions in the protein structure. These results can be explained by a general model in which phylogenetically stable genes have core functions in development and physiology, whereas unstable genes have accessory functions associated with unstable environmental interactions such as toxin and pathogen exposure. Unstable gene islands in vertebrates share some functional properties with bacterial genomic islands, though they arise by local gene duplication rather than horizontal gene transfer.

Project description:BackgroundSpinosad is important in pest management strategies of multiple insect pests. However, spinosad resistance is emerging in various pest species. Resistance has in some species been associated with alterations of the target-site receptor, but in others P450s seems to be involved. We test the possible importance of nine cytochrome P450 genes in the spinosad-resistant housefly strain 791spin and investigate the influence of spinosad on P450 expression in four other housefly strains.ResultsSignificant differences in P450 expression of the nine P450 genes in the four strains after spinosad treatment were identified in 40% of cases, most of these as induction. The highly expressed CYP4G2 was induced 6.6-fold in the insecticide susceptible WHO-SRS females, but decreased 2-fold in resistant 791spin males. CYP6G4 was constitutively higher expressed in the resistant strain compared to the susceptible strain. Furthermore, CYP6G4 gene expression was increased in susceptible WHO-SRS flies by spinosad while the expression level did not alter significantly in resistant fly strains. Expression of CYP6A1 and male CYP6D3 was constitutively higher in the resistant strain compared to the susceptible. However, in both cases male expression was higher than female expression.ConclusionCYP4G2, CYP6A1, CYP6D3 and CYP6G4 have expressions patterns approaching the expectations of a hypothesized sex specific spinosad resistance gene. CYP4G2 fit requirements of a spinosad resistance gene best, making it the most likely candidate. The overall high expression level of CYP4G2 throughout the strains also indicates importance of this gene. However, the data on 791spin are not conclusive concerning spinosad resistance and small contributions from multiple P450s with different enzymatic capabilities could be speculated to do the job in 791spin. Differential expression of P450s between sexes is more a rule than an exception. Noteworthy differences between spinosad influenced expression of P450 genes between a field population and established laboratory strains were shown.

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Ancestral Sequence Reconstruction of a Cytochrome P450 Family Involved in Chemical Defense Reveals the Functional Evolution of a Promiscuous, Xenobiotic-Metabolizing Enzyme in Vertebrates.

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