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.

Keywords: CYP1A2; CYP1B1; ancestral sequence reconstruction; cytochrome P450; drug metabolism; thermostability.

Fig. 1.

CYP1 ancestral nodes reconstructed and correlation of thermostability with evolutionary age. Main figure: The phylogenetic relationship between sequences used to reconstruct CYP1 family ancestors from the CYP1A, CYP1B, CYP1C, and CYP1D subfamilies. Sequences were collected from the NCBI and UniProt databases by BLAST searching for proteins with at least 40% sequence similarity to characterized forms, and aligned with selected outgroup sequences (human CYP2A6 [NP_000753.3] and mouse CYP2E1 [NP_067257]) using MAFFT. A maximum-likelihood tree was generated using the JTT evolutionary model, and ancestors were inferred using a joint reconstruction approach in GRASP (Foley 2022). Individual nodes selected for reconstruction are shown as circles, with names based on the node number and sequences from which they are derived. “CYP1-like” sequences are those from the basal chordate Ciona intestinalis determined by the NCBI sequence annotation algorithm to likely be CYP1 genes. Inset: The variation in the ⁶⁰T₅₀ values for CYP1 ancestors and extant forms with branch length (a measure of evolutionary age). Each subfamily is colored separately (CYP1A: red, CYP1B: orange, CYP1C: green, CYP1D: blue) and lines connect points on the same evolutionary branch. Data represent the mean ± SD (n = 2–3 ⁶⁰T₅₀ determinations). A general trend was observed toward increased thermostability correlating with greater evolutionary age.

Fig. 2.

Relative activity of CYP1 ancestors toward 7-alkoxyresorufin and luciferin derivatives as marker substrates of extant human CYP1 forms. Plots illustrate the relative activity of ancestral and extant forms toward the dealkylation of six CYP1 probe substrates: 7-ethoxyresorufin, 7-methoxyresorufin, 7-benzyloxyresorufin, 7-pentoxyresorufin, luciferin-CEE, and luciferin-ME, normalized to the activity shown toward that substrate by the most active form. The background signal as indicated by the negative controls (hCPR-only in the case of the 7-alkoxyresorufin assay; supplementary fig. S3, Supplementary Material online) or without NADPH in the case of the luciferin assays (supplementary fig. S4, Supplementary Material online) was subtracted before calculating relative activity. Only data that are significantly different (two-sided Student’s t-test; P < 0.05) to negative controls are shown. Plots are superimposed on the evolutionary tree of the CYP1 family to show evolutionary relationships; however branch lengths are not to scale. All reactions were carried out at 37 °C. Alkoxyresorufin O-dealkylations assays were performed using membranes normalized to a P450 concentration of 5 nM, and a substrate concentration of 5 µM in a total volume of 100 µl. Luciferin-based assays were performed with bacterial membranes, using a P450 concentration of 10 nM and a substrate concentration of 100 µM for luciferin-ME and 30 µM for luciferin-CEE in a volume of 50 µl. Data represent means of triplicates.

Fig. 3.

Relative activity of CYP1 ancestors toward drug substrates of extant human CYP1 forms. Plots illustrate the relative activity of ancestral and extant forms toward the metabolism of the pharmaceutical compounds, clozapine, tacrine, imipramine, and aminopyrine normalized to the activity shown toward the substrate and metabolite in question by the most active form. The background signal as indicated by the hCPR-only negative controls (supplementary figs. S5–S8, Supplementary Material online) was subtracted before calculating relative activity. Only data that are significantly different (two-sided Student’s t-test; P < 0.05) to negative controls are shown. Plots are superimposed on the evolutionary tree of the CYP1 family to show evolutionary relationships; however, branch lengths are not to scale. All reactions were carried out at 37 °C in a total volume of 250 µl, with a P450 concentration of 0.1 µM and substrate concentrations of 50 µM. Data represent means of triplicates. Numbers in the legend reference panel numbers in supplementary figs. S5–S8, Supplementary Material online.

Fig. 4.

Metabolism of E2 and testosterone by ancestral and extant CYP1 forms. Plots illustrate the relative activity of ancestral and extant forms toward the metabolism of the steroids, E2 and testosterone, normalized to the activity shown toward the substrate and metabolite in question by the most active form. The background signal as indicated by the hCPR-only negative controls (supplementary figs. S10 and S12, Supplementary Material online) was subtracted before calculating relative activity. Only data that are significantly different (two-sided Student’s t-test; P < 0.05) to negative controls are shown. Plots are superimposed on the evolutionary tree of the CYP1 family to show evolutionary relationships; however, branch lengths are not to scale. E2 metabolism was assessed in reactions carried out for 60 min at 37 °C using 300 μM E2 and 0.5 μM P450 added in bacterial membranes. All metabolites were quantified using a standard curve prepared using the authentic metabolite, with the exception of the unidentified metabolite, which was quantified using the standard curve for 2-OH-E2. Testosterone metabolism was assessed in reactions carried out for 60 min at 37 °C with microsomal membranes in 250 μl total volume using 100 µM testosterone and 0.1 µM P450 added in bacterial membranes. Testosterone was metabolized to five primary metabolites and one unidentified metabolite by the ancestors and extant forms. Metabolites were quantified using a standard curve prepared with authentic standards. Data represent means of triplicates.

Fig. 5.

Caffeine metabolism by ancestral and extant CYP1 forms. The metabolism of caffeine to four metabolites (paraxanthine, theobromine, theophylline, and TMU) by the ancestors and extant forms is shown. Plots illustrate the relative activity of ancestral and extant forms normalized to the activity shown toward that substrate and product by the most active form, and are superimposed on the evolutionary tree of the CYP1 family to show evolutionary relationships; however branch lengths are not to scale. The background signal inferred from an hCPR-only standard curve (supplementary fig. S13, Supplementary Material online) were subtracted before calculating relative activity. Only data that are significantly different (two-sided Student’s t-test; P < 0.05) to negative controls are shown. Reactions were carried out for 60 min at 37 °C using 2 mM caffeine and 0.5 μM P450 added in bacterial membranes. All metabolites were quantified using a standard curve prepared using the authentic metabolite. Data represent means of triplicates.

Fig. 6.

FICZ metabolism by ancestral and extant CYP1 forms. The metabolism of FICZ to five previously characterized and three unknown metabolites by the ancestors and extant forms is shown. Plots illustrate the relative activity of ancestral and extant forms normalized to the maximal activity shown toward that substrate by any form, and are superimposed on the evolutionary tree of the CYP1 family to show evolutionary relationships; however, branch lengths are not to scale. The background signal as indicated by reactions without added NADPH or quenched at 0 min (supplementary fig. S16, Supplementary Material online) was subtracted before calculating relative activity. Only data that are significantly different (two-sided Student's t-test; P < 0.05) to negative controls are shown. Reactions were prepared and carried out in the dark for 60 min at 37 °C using 25 µM FICZ and 0.5 μM P450 added in bacterial membranes. Data represent means of triplicates.