The enzyme that cleaves the sterol side chain to form pregnenolone — the first committed, rate-limiting step of cardiac glycoside (cardenolide) biosynthesis in plants — was identified only in 2023, in Digitalis foxglove (CYP87A4/DpCYP87A106) and, in parallel, in Calotropis procera and Erysimum; RNA-interference silencing in Digitalis purpurea has since confirmed the in-planta role of the CYP87A-type enzyme. The objective is to complete the biochemical and evolutionary account of this gateway step: (1) the catalytic mechanism by which the plant enzyme converts cholesterol and phytosterols to pregnenolone is supported only computationally (docking and enzyme-engineering studies suggest a mammalian-like three-step sequential hydroxylation-cleavage) and has never been experimentally verified, including whether substrate stereochemistry matters; (2) the biochemical function of the canonical, constitutively expressed CYP87A paralogs (DlCYP87A1/A2), which lack pregnenolone-forming activity, remains unknown; and (3) the sterol-cleaving enzyme is still unidentified and unvalidated in remaining cardenolide producers, most prominently the milkweeds (Asclepias), where a CYP87A candidate is only transcriptionally predicted and where the discovery of non-CYP87A plant P450scc enzymes in Marsdenia (CYP108/CYP150) shows the activity can reside in other P450 families.

Background

Cardenolides (cardiac glycosides) are steroidal plant defense metabolites that inhibit the animal Na+/K+-ATPase; the most famous, digoxin from foxglove (Digitalis lanata and D. purpurea), is a frontline drug for heart failure and atrial fibrillation. The pathway begins when a sterol side chain is cleaved to yield pregnenolone, a C21 steroid — the first committed and rate-limiting step. In mammals this reaction is catalyzed by the mitochondrial cytochrome P450 CYP11A1, termed P450scc (sterol side-chain cleaving enzyme): a well-studied three-step sequence of two stereoselective hydroxylations (at C22, then C20) followed by oxidative cleavage of the C20–C22 bond of 20,22-dihydroxycholesterol. Cytochromes P450 are heme-thiolate monooxygenases named CYP by family/subfamily; in plants they sit on the endoplasmic reticulum (ER) and receive electrons from a plant-type NADPH-cytochrome P450 reductase (CPR), unlike the mitochondrial, adrenodoxin-fed CYP11A1. A plant P450scc activity was described in Digitalis as early as 1972, and decades of isotope-feeding work left the sterol precursor (cholesterol versus the major plant phytosterols campesterol and β-sitosterol, 24-alkyl sterols) ambiguous. The enzyme was finally identified in 2023 by two groups independently: Carroll et al. showed that D. lanata CYP87A4 converts both cholesterol and campesterol to pregnenolone (assayed by transient expression in Nicotiana benthamiana leaves and in yeast), and Kunert et al. identified the D. purpurea ortholog DpCYP87A106 plus the Calotropis procera enzyme CpCYP87A103, which also accepts β-sitosterol, and the Erysimum cheiranthoides enzyme EcCYP87A126. Kunert et al. further provided the key in-planta evidence: silencing DpCYP87A106 by RNA interference (RNAi, post-transcriptional gene knockdown) in D. purpurea leaves eliminated detectable pregnenolone and almost all cardenolides. The CYP87A family is conserved across seed plants, but before these discoveries no CYP87A subfamily member had been functionally characterized. In D. lanata the family comprises the cardenolide-associated DlCYP87A4; DlCYP87A3, a 97.4%-identical duplicate that nonetheless does not cleave campesterol in yeast; and the canonical, constitutively expressed DlCYP87A1 and DlCYP87A2 (about 72–74% identity to DlCYP87A4), of unknown function. The source paper posed the resulting open questions; subsequent work has moved the field downstream (e.g., plastid 2-oxoglutarate-dependent dioxygenases — 2OGDs, soluble Fe(II)/2-oxoglutarate oxygenases — catalyzing stereoinverted C14β-hydroxylation, and metabolic-network reconstruction from pregnenolone to the cardenolide digitoxigenin) and into biotechnology (engineered yeast producing pregnenolone at gram-per-liter scale), while the chemistry of the side-chain-cleavage step itself, the role of the canonical paralogs, and the enzyme's identity in several cardenolide-producing lineages have remained without answers.

Problem Statement

Complete the biochemical characterization of the sterol side-chain-cleavage step (the P450scc step) that initiates cardenolide biosynthesis in plants. The enzyme is identified in Digitalis (CYP87A4/DpCYP87A106, confirmed in planta by RNAi silencing in D. purpurea) and in Calotropis procera and Erysimum cheiranthoides; the following source-faithful questions remain open: (1) Determine experimentally the catalytic mechanism of the CYP87A-type plant P450scc — does its conversion of cholesterol and the phytosterols campesterol (and β-sitosterol) to pregnenolone proceed, as mammalian CYP11A1 does, through sequential 22-hydroxylation, 20-hydroxylation, and oxidative C20–C22 cleavage of a 20,22-dihydroxy intermediate, or by an alternative chemical route, and does the stereochemistry of the campesterol 24-methyl group (24R versus 24S epimers) affect the reaction? (2) Determine the biochemical function of the canonical CYP87A enzymes — DlCYP87A1 and DlCYP87A2 in Digitalis lanata, which are constitutively expressed, lack pregnenolone-forming activity, and have no identified substrate: what reactions do they catalyze, and do they possess any sterol-modifying activity? (3) Identify and functionally validate the sterol side-chain-cleaving enzyme in the cardiac-glycoside-producing plants where it remains unknown; the required instance is the milkweeds (Asclepias species), where a CYP87A candidate is transcriptionally predicted but unvalidated, and where the Marsdenia tenacissima precedent (two P450scc enzymes from families CYP108 and CYP150) shows the activity may reside in a different cytochrome P450 family, whereas functional validation of the enzyme in the other remaining cardenolide-producing lineages would extend the account but is not required to resolve this question.

References

  1. Carroll E, Ravi Gopal B, Raghavan I, Mukherjee M, Wang ZQ (2023). A cytochrome P450 CYP87A4 imparts sterol side-chain cleavage in digoxin biosynthesis. Nature Communications 14:4042. DOI: 10.1038/s41467-023-39719-4. https://doi.org/10.1038/s41467-023-39719-4
  2. Carroll E, Ravi Gopal B, Raghavan I, Wang ZQ (2022). The P450 Sterol Side Chain Cleaving Enzyme (P450scc) for Digoxin Biosynthesis in the Foxglove Plant Belongs to the CYP87A Family. bioRxiv preprint 2022.12.29.522212. DOI: 10.1101/2022.12.29.522212. https://doi.org/10.1101/2022.12.29.522212
  3. Kunert M, Langley C, Lucier R, Ploss K, Rodríguez López CE, Serna Guerrero DA, Rothe E, O'Connor SE, Sonawane PD (2023). Promiscuous CYP87A enzyme activity initiates cardenolide biosynthesis in plants. Nature Plants 9(10):1607–1617. DOI: 10.1038/s41477-023-01515-9. https://doi.org/10.1038/s41477-023-01515-9
  4. Xu M, Kastner DW, Luo W, Li F-S, Müller P, Sun Y, Huang W, Glinkerman CM, Guempel M, Kulik HJ, Weng J-K (2026). Mammalian-like steroidogenesis in plants gives rise to endocrine-mimetic cardenolides. Science Advances 12(16):eaeb5460. DOI: 10.1126/sciadv.aeb5460. https://doi.org/10.1126/sciadv.aeb5460
  5. Li R, Guo S, Wang D, Yang T, Li W, Wang J, Huang L, Zhang X, Dai Z (2025). Elucidation of the plant progesterone biosynthetic pathway and its application in a yeast cell factory. Metabolic Engineering 90:197–208. DOI: 10.1016/j.ymben.2025.03.016. https://doi.org/10.1016/j.ymben.2025.03.016
  6. Feng H, Zhang J, Powell AF, Buttelmann GL, Yang L, Yan E, Wang F, Broyles SB, Jander G, Strickler SR (2025). Genome and tissue-specific transcriptome of the tropical milkweed (Asclepias curassavica). Plant Direct 9(3):e70031. DOI: 10.1002/pld3.70031. https://doi.org/10.1002/pld3.70031
  7. Tian W, Wei G, Duan B, Peng H, Liu H, Zheng M, Lin Z, Deng Z, Zhang Z, Qu X (2025). Unveiling cytochrome P450 enzymes that catalyze steroid side-chain cleavage in bacteria. Nature Communications 17:581. DOI: 10.1038/s41467-025-67278-3. https://doi.org/10.1038/s41467-025-67278-3
  8. Chao Z, Chen Q, Zhao W, He J, Li Q, Gao T, Wei W, Liu S, Zhou J, Zeng W, Xu S (2026). Engineering of plant-derived P450scc for de novo biosynthesis of pregnenolone. Synthetic and Systems Biotechnology 12:411–421. DOI: 10.1016/j.synbio.2026.01.027. https://doi.org/10.1016/j.synbio.2026.01.027
  9. Wang J, Zhou X, Ji W, Bian J, Zhang C, Zhou H, Shan C, An N, Li R, Li Q, Hou S-H, Liu Z (2026). Plastid 2-oxoglutarate-dependent dioxygenases mediate stereoselective C14β-hydroxylation in cardenolide biosynthesis. Proceedings of the National Academy of Sciences 123(8):e2525003123. DOI: 10.1073/pnas.2525003123. https://doi.org/10.1073/pnas.2525003123
  10. Zeng V, Carroll E, Burnside D, Wang ZQ (2026). Subcellular localization of enzymes involved in the biosynthesis of digoxin in Digitalis lanata. Frontiers in Plant Science 17:1703671. DOI: 10.3389/fpls.2026.1703671. https://doi.org/10.3389/fpls.2026.1703671
  11. Rubiano-Buitrago P, Hastings AP, Uematsu M, Baskin JM, Agrawal AA, Duplais C (2026). Structural innovation in the evolution of plant chemical defense. Proceedings of the National Academy of Sciences 123(11):e2535853123. DOI: 10.1073/pnas.2535853123. https://doi.org/10.1073/pnas.2535853123
  12. Jiao D, Zhang Y, Guo W, Liu S, Su P, Huang L (2026). Cardiac glycosides: structural diversity, chemical ecology, bioactivity, and artificial synthesis. Natural Product Reports 43(1):167–199. DOI: 10.1039/d5np00050e. https://doi.org/10.1039/d5np00050e