Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3′-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids

• Published in: Plant physiology and biochemistry 2024-05, Vol.210 (C), p.108612-108612, Article 108612
• Main Authors: Karimzadegan, Vahid, Koirala, Manoj, Sobhanverdi, Sajjad, Merindol, Natacha, Majhi, Bharat Bhusan, Gélinas, Sarah-Eve, Timokhin, Vitaliy I., Ralph, John, Dastmalchi, Mehran, Desgagné-Penix, Isabel
• Format: Article
• Language: English
• Subjects: Ascorbate peroxidase/4-coumarate 3-hydroxylase; Coumaric acid; Endoplasmic reticulum membrane localization; Metabolic profile; Phenylpropanoids; Specialized metabolism
• ISSN: 0981-9428; 1873-2690
• DOI: 10.1016/j.plaphy.2024.108612

Biosynthesis of Amaryllidaceae alkaloids (AA) starts with the condensation of tyramine with 3,4-dihydroxybenzaldehyde. The latter derives from the phenylpropanoid pathway that involves modifications of trans-cinnamic acid, p-coumaric acid, caffeic acid, and possibly 4-hydroxybenzaldehyde, all potentially catalyzed by hydroxylase enzymes. Leveraging bioinformatics, molecular biology techniques, and cell biology tools, this research identifies and characterizes key enzymes from the phenylpropanoid pathway in Leucojum aestivum. Notably, we focused our work on trans-cinnamate 4-hydroxylase (LaeC4H) and p-coumaroyl shikimate/quinate 3ʹ-hydroxylase (LaeC3′H), two key cytochrome P450 enzymes, and on the ascorbate peroxidase/4-coumarate 3-hydroxylase (LaeAPX/C3H). Although LaeAPX/C3H consumed p-coumaric acid, it did not result in the production of caffeic acid. Yeasts expressing LaeC4H converted trans-cinnamate to p-coumaric acid, whereas LaeC3′H catalyzed specifically the 3-hydroxylation of p-coumaroyl shikimate, rather than of free p-coumaric acid or 4-hydroxybenzaldehyde. In vivo assays conducted in planta in this study provided further evidence for the contribution of these enzymes to the phenylpropanoid pathway. Both enzymes demonstrated typical endoplasmic reticulum membrane localization in Nicotiana benthamiana adding spatial context to their functions. Tissue-specific gene expression analysis revealed roots as hotspots for phenylpropanoid-related transcripts and bulbs as hubs for AA biosynthetic genes, aligning with the highest AAs concentration. This investigation adds valuable insights into the phenylpropanoid pathway within Amaryllidaceae, laying the foundation for the development of sustainable production platforms for AAs and other bioactive compounds with diverse applications. [Display omitted] •Study of cinnamate 4-hydroxylase (C4H) &p-coumaroyl 3′-hydroxylase (C3′H) enzymes.•Characterization of Leucojum aestivum C4H & C3′H unveils their substrate specificity.•Elucidate the roles of LaeC4H &LaeC3′H in Amaryllidaceae alkaloids (AA) biosynthesis.•Their ER-membrane localization provides insights into their cellular dynamics.•Tissue-specific expression analysis reveals bulbs as hotspots for AA-related transcripts.