A cytochrome P450 CYP87A4 imparts sterol side-chain cleavage in digoxin biosynthesis

Abstract

Digoxin extracted from the foxglove plant is a widely prescribed natural product for treating heart failure. It is listed as an essential medicine by the World Health Organization. However, how the foxglove plant synthesizes digoxin is mostly unknown, especially the cytochrome P450 sterol side chain cleaving enzyme (P450_scc), which catalyzes the first and rate-limiting step. Here we identify the long-speculated foxglove P450_scc through differential transcriptomic analysis. This enzyme converts cholesterol and campesterol to pregnenolone, suggesting that digoxin biosynthesis starts from both sterols, unlike previously reported. Phylogenetic analysis indicates that this enzyme arises from a duplicated cytochrome P450 CYP87A gene and is distinct from the well-characterized mammalian P450_scc. Protein structural analysis reveals two amino acids in the active site critical for the foxglove P450_scc's sterol cleavage ability. Identifying the foxglove P450_scc is a crucial step toward completely elucidating digoxin biosynthesis and expanding the therapeutic applications of digoxin analogs in future work.

Fig. 1. Quantifying the relative transcript abundance of genes in the sterol and digoxin biosynthetic pathways.

a Total lanatosides, including lanatosides A, B, C, and E, in D. lanata leaves and roots relative to the deuterated digoxin-d3 internal standard. The data are normalized by dry weight and represent the average ± SD of three biological replicates. b Sterol and digoxin biosynthetic pathway genes that are differentially expressed in leaf and root tissues. Genes that do not have corresponding heat maps are not differentially expressed. SQE squalene epoxidase, CAS cycloartenol synthase, SMO C-4 sterol methyl oxidase, SMT sterol C-24 methyl transferase, SSR1 sterol side-chain reductase 1, C14-R sterol C-14 reductase, 8,7 SI sterol 8,7 isomerase, P450_scc cytochrome P450 sterol side-chain cleaving, 3βHSD 3β-hydroxysteroid dehydrogenase, P5βR progesterone-5β-reductase.

Fig. 2. Identifying and characterizing candidate D. lanata P450_sccs in tobacco.

a Quantifying the relative transcript abundance from candidate genes in root and leaf tissues of D. lanata. b qRT-PCR quantified relative transcript abundance of top candidate genes, DlCYP87A4 and DlCYP90A1. Data represent the average ± SD of three biological replicates. c LC/MS data from tobacco leaves transiently expressing candidate genes in various combinations. The theoretical m/z values of parent ion adducts are given. Detected m/z values of peaks are within 5 ppm of the theoretical value. Set 1 contained full-length DlCYP87A4, DlCYP90A1, 3βHSD, and P5βR. Note the minor peak is not pregnenolone because of the different retention time compared to the pregnenolone standard. Set 2 contained DlCYP90A1, 3βHSD, and P5βR. Set 3 contained DlCYP87A4, 3βHSD, and P5βR. Set 4 contained DlCYP87A4 only. Set 5 contained DlCYP90A1 only. GFP control: negative control expressing a green fluorescent protein (GFP) in tobacco. The bottom panel shows the authentic standards of the four expected pathway intermediates.

Fig. 3. Characterizing the D. lanata CYP87A4 in S. cerevisiae strains producing various sterols.

a Sterols the yeast strains produce. b Extracted ion chromatograms from yeast expressing the full-length DlCYP87A4 and ATR2 or the human P450_scc with its redox partners, adrenodoxin (ADX) and adrenodoxin reductase (ADR). The theoretical m/z values of parent ion adducts are given. Detected m/z values of peaks are within 5 ppm of the theoretical value. The bottom panel shows authentic standards of pregnenolone and pregnenolone acetate.

Fig. 4. Maximum-likelihood phylogenetic tree of the CYP87A family members from eudicot plants.

Protein sequences are retrieved from 1000 plant transcriptome project and are at least 50% identical to DlCYP87A4. Bootstrap values from 1000 replicates are shown at each node. D. lanata CYP87As are bolded. Asterisk denotes the characterized DlCYP87A4 in this study. The scale bar represents the mean number of substitutions per amino acid.

Fig. 5. Protein modeling identified critical amino acids for DlCYP87A4’s sterol cleaving activity.

Docking of campesterol a and cholesterol b into the active site of the DlCYP87A4 protein model. Side chains of key amino acids S123, A355, and L357 are highlighted in pink. The protein model is truncated of a predicted N-terminal signal peptide of 31 amino acids. c Alignment of CYP87A subfamily enzymes to D. lanata and other CYP87A enzymes. Three locations that are different between DlCYP87A4 and the canonical DlCYP87A1 are indicated as follows; m1 (S123), m2 (A355), and m3 (L357). d Extracted ion chromatograms from campesterol-producing yeast expressing DlCYP87A4 mutants, including S123A, A355V, and L357A, along with ATR2. The theoretical m/z values of parent ion adducts are given. Detected m/z values of peaks are within 5 ppm of the theoretical value. e Campesterol-producing yeast expressing the wildtype DlCYP87A4 or the canonical DlCYP87A1 along with ATR2. Authentic standard for pregnenolone is shown in the bottom panel.

Fig. 6. Docking cholesterol and phytosterols to the active center of the DlP450_scc.

Docking simulations of DlCPY87A4 with a campesterol, b cholesterol, c β-sitosterol, and d stigmasterol. Dashed lines show the distances of C20 and C22 to the heme center. Docking conformations shown are of the lowest energy in each simulation.

Fig. 7. Substrate binding sites of the DlP450_scc and the human P450_scc.

Comparison of substrate recognition sites of Digitalis lanata CYP87A4 a and human CYP11A1 b. Cholesterol is docked to the active sites. Amino acids at near-identical positions of DlCYP87A4 and CYP11A1 are shown. c List of active-site amino acids within 4.5 Å to cholesterol in DlCYP87A4 and human CYP11A1. Green: similar amino acids in the two active sites; tan: hydrophobic amino acids in both active sites; gray: distinct amino acids.