Kinetic and in silico structural characterization of norbelladine O-methyltransferase of Amaryllidaceae alkaloids biosynthesis

• Published in: The Journal of biological chemistry 2024-09, Vol.300 (9), p.107649, Article 107649
• Main Authors: Koirala, Manoj, Merindol, Natacha, Karimzadegan, Vahid, Gélinas, Sarah-Eve, Liyanage, Nuwan Sameera, Lamichhane, Basanta, Tobón, Maria Camila García, Lagüe, Patrick, Desgagné-Penix, Isabel
• Format: Article
• Language: English
• Subjects: Amaryllidaceae alkaloids; Amaryllidaceae Alkaloids - chemistry; Amaryllidaceae Alkaloids - metabolism; biosynthesis; catalytic potential; Kinetics; Methyltransferases - chemistry; Methyltransferases - genetics; Methyltransferases - metabolism; molecular docking; Molecular Docking Simulation; Narcissus - chemistry; Narcissus - enzymology; Narcissus - metabolism; Nicotiana - genetics; Nicotiana - metabolism; O-methyltransferase; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; regio-selectivity; Substrate Specificity
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2024.107649

Amaryllidaceae alkaloids are a diverse group of alkaloids exclusively reported from the Amaryllidaceae plant family. In planta, their biosynthesis is still not fully characterized; however, a labeling study established 4ˈ-O-methylnorbelladine as the key intermediate compound of the pathway. Previous reports have characterized O-methyltransferases from several Amaryllidaceae species. Nevertheless, the formation of the different O-methylnorbelladine derivatives (3ˈ-O-methylnorbelladine, 4ˈ-O-methylnorbelladine, and 3ˈ4ˈ-O-dimethylnorbelladine), the role, and the preferred substrates of O-methyltransferases are not clearly understood. In this study, we performed the biochemical characterization of an O-methyltransferase candidate from Narcissus papyraceus (NpOMT) in vitro and in vivo, following biotransformation of norbelladine in Nicotiana benthamiana having transient expression of NpOMT. Docking analysis was further used to investigate substrate preferences, as well as key interacting residues of NpOMT. Our study shows that NpOMT methylates norbelladine preferentially at the 4ˈ-OH position in vitro and in planta. Interestingly, NpOMT also catalyzed the synthesis of 3ˈ,4ˈ-O-dimethylnorbelladine from norbelladine and 4ˈ-O-methylnorbelladine during in vitro enzymatic assay. Furthermore, we show that NpOMT methylates 3,4-dihydroxybenzylaldehyde and caffeic acid in a nonregiospecific manner to produce meta/para monomethylated products. This study reveals a novel catalytic potential of an Amaryllidaceae O-methyltransferase and its ability to regioselectively methylate norbelladine in the heterologous host N. benthamiana.