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

Karimzadegan, V., Koirala, M., Sobhanverdi, S., Merindol, N., Majhi, B. B., Gélinas, S. E., Timokhin, V. I., Ralph, J., Dastmalchi, M. et Desgagné-Penix, I. (2024). Characterization of cinnamate 4-hydroxylase (CYP73A) and p-coumaroyl 3′-hydroxylase (CYP98A) from Leucojum aestivum, a source of Amaryllidaceae alkaloids. Plant Physiology and Biochemistry, 210 . Article 108612. ISSN 0981-9428 1873-2690 DOI 10.1016/j.plaphy.2024.108612

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Abstract

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.

Type de document:	Article
Mots-clés libres:	Phenylpropanoids Specialized metabolism Ascorbate peroxidase/4-coumarate 3-hydroxylase Endoplasmic reticulum membrane localization Coumaric acid Metabolic profile
Date de dépôt:	22 avr. 2025 14:43
Dernière modification:	22 avr. 2025 14:43
Version du document déposé:	Version officielle de l'éditeur
URI:	https://depot-e.uqtr.ca/id/eprint/11883

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