Structure-Based Catalytic Mechanism of Amaryllidaceae O-Methyltransferases

Narcissus pseudonarcissus norbelladine 4 '-O-methyltransferase (NpN4OMT), Lycoris longituba OMT (LlOMT), Lycoris aurea OMT (LaOMT), and Lycoris radiata OMT (LrOMT) have been identified as the key enzymes that produce a common intermediate, 4 '-O-methylnorbelladine, from norbelladine in the biosynthesis of Amaryllidaceae alkaloids such as galanthamine, a therapeutic agent for Alzheimer's disease. Among them, only LrOMT was reported to produce not only 4 '-O-methylnorbelladine but also 3 '-O-methylnorbelladine from norbelladine as the plausible key intermediate in the biosynthesis of pseudolycorine-type Amaryllidaceae alkaloids. Here, in vitro functional analyses revealed that NpN4OMT, LlOMT, and LaOMT can also produce 3 '-O-methylnorbelladine with 7, 62, and 62% meta-O-methylation preferences, respectively. X-ray crystallographic studies of NpN4OMT, LlOMT, and LaOMT demonstrated that these enzymes possess an exceptionally large active site cavity including a 4-hydroxyphenyl moiety binding pocket, Region P, which has never been observed in previously reported homologous enzymes, catechol OMTs and caffeoyl coenzyme A OMTs. We also found that the native Amaryllidaceae OMTs regulate their para/meta-O-methylation preference for norbelladine by subtly balancing the polar contacts from the residues at positions 10 and 186 to the amine moiety of norbelladine in Region A. A structure-based mutagenesis study focusing on Asp230 led to the conversion of LlOMT to an unprecedented biocatalyst that specifically catalyzes the meta-O-methylation of norbelladine to produce 3 '-O-methylnorbelladine. The results provide an atomic-level understanding of the factors controlling the para/meta-O-methylation preferences of Amaryllidaceae OMTs for 4 '- and 3 '-O-methylnorbelladine syntheses.