Characterization of norbelladine synthase and noroxomaritidine/norcraugsodine reductase reveals a novel catalytic route for the biosynthesis of Amaryllidaceae alkaloids including the Alzheimer's drug galanthamine

Bharat Bhusan Majhi; Sarah-Eve Gélinas; Natacha Mérindol; Simon Ricard; Isabel Desgagné-Penix

doi:10.3389/fpls.2023.1231809

Characterization of norbelladine synthase and noroxomaritidine/norcraugsodine reductase reveals a novel catalytic route for the biosynthesis of Amaryllidaceae alkaloids including the Alzheimer's drug galanthamine

Front Plant Sci. 2023 Aug 30:14:1231809. doi: 10.3389/fpls.2023.1231809. eCollection 2023.

Authors

Bharat Bhusan Majhi¹, Sarah-Eve Gélinas¹, Natacha Mérindol¹, Simon Ricard¹, Isabel Desgagné-Penix^{1

2}

Affiliations

¹ Department of Chemistry, Biochemistry and Physics, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, QC, Canada.
² Plant Biology Research Group, Université du Québec à Trois-Rivières, Trois-Rivières, Québec, QC, Canada.

Abstract

Amaryllidaceae alkaloids (AAs) are a large group of plant specialized metabolites with diverse pharmacological properties. Norbelladine is the entry compound in AAs biosynthesis and is produced from the condensation of tyramine and 3,4-dihydroxybenzaldehyde (3,4-DHBA). There are two reported enzymes capable of catalyzing this reaction in-vitro, both with low yield. The first one, norbelladine synthase (NBS), was shown to condense tyramine and 3,4-DHBA, while noroxomaritidine/norcraugsodine reductase (NR), catalyzes a reduction reaction to produce norbelladine. To clarify the mechanisms involved in this controversial step, both NBS and NR homologs were identified from the transcriptome of Narcissus papyraceus and Leucojum aestivum, cloned and expressed in Escherichia coli. Enzymatic assays performed with tyramine and 3,4-DHBA with each enzyme separately or combined, suggested that NBS and NR function together for the condensation of tyramine and 3,4-DHBA into norcraugsodine and further reduction into norbelladine. Using molecular homology modeling and docking studies, we predicted models for the binding of tyramine and 3,4-DHBA to NBS, and of the intermediate norcraugsodine to NR. Moreover, we show that NBS and NR physically interact in yeast and in-planta, that both localize to the cytoplasm and nucleus and are expressed at high levels in bulbs, confirming their colocalization and co-expression thus their ability to work together in the same catalytic route. Finally, their co-expression in yeast led to the production of norbelladine. In all, our study establishes that both NBS and NR participate in the biosynthesis of norbelladine by catalyzing the first key steps associated in the biosynthesis of the Alzheimer's drug galanthamine.

Keywords: Amaryllidaceae alkaloids; Leucojum aestivum; Narcissus papyraceus; enzyme activity; norbelladine synthase; noroxomaritidine/norcraugsodine reductase.

Grants and funding

This work was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC) award number RGPIN-2021-03218 (Discovery) to ID-P This work was also supported by the Canada Research Chair on plant specialized metabolism Award No 950-232164 to ID-P Thanks are extended to the Canadian taxpayers and to the Canadian government for supporting the Canada Research Chairs Program.