Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, Canada.
Department of Biochemistry, Microbiology and Bioinformatics, Laval University, Québec, Canada.
J Biol Chem. 2024 Sep;300(9):107649. doi: 10.1016/j.jbc.2024.107649. Epub 2024 Aug 8.
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 4'-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 (3'-O-methylnorbelladine, 4'-O-methylnorbelladine, and 3'4'-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 4'-OH position in vitro and in planta. Interestingly, NpOMT also catalyzed the synthesis of 3',4'-O-dimethylnorbelladine from norbelladine and 4'-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.
石蒜科生物碱是一类独特的生物碱，仅在石蒜科植物中报道。在植物体内，其生物合成尚未完全阐明；然而，一项标记研究确定 4'-O-甲基去甲基石蒜碱是该途径的关键中间化合物。以前的报告已经从几种石蒜科物种中鉴定了 O-甲基转移酶。然而，不同的 4'-O-甲基去甲基石蒜碱衍生物（3'-O-甲基去甲基石蒜碱、4'-O-甲基去甲基石蒜碱和 3'4'-O-二甲基去甲基石蒜碱）的形成、O-甲基转移酶的作用和偏好的底物并不清楚。在这项研究中，我们在体外和体内对来自水仙的 O-甲基转移酶候选物（NpOMT）进行了生化表征，该候选物在瞬时表达 NpOMT 的黄花烟草中对石蒜碱进行生物转化。进一步使用对接分析来研究 NpOMT 的底物偏好和关键相互作用残基。我们的研究表明，NpOMT 在体外和体内优先在 4'-OH 位置甲基化石蒜碱。有趣的是，NpOMT 在体外酶促测定中还催化了石蒜碱和 4'-O-甲基去甲基石蒜碱合成 3'4'-O-二甲基去甲基石蒜碱。此外，我们表明 NpOMT 以非区域特异性方式甲基化 3,4-二羟基苯甲醛和咖啡酸，生成间/对位单甲基化产物。这项研究揭示了石蒜科 O-甲基转移酶的一种新的催化潜力及其在异源宿主 N. benthamiana 中对石蒜碱进行区域选择性甲基化的能力。
