Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities

Matthew B Kilgore,Cynthia K Holland,Joseph M Jez,Toni M Kutchan

doi:10.1074/jbc.m116.717827

Abstract

Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities. Several groups of Amaryllidaceae alkaloids including the hemanthamine- and crinine-type alkaloids show promise as anticancer agents. Two reduction reactions are required for the production of these compounds: the reduction of norcraugsodine to norbelladine and the reduction of noroxomaritidine to normaritidine, with the enantiomer of noroxomaritidine dictating whether the derivatives will be the crinine-type or hemanthamine-type. It is also possible for the carbon-carbon double bond of noroxomaritidine to be reduced, forming the precursor for maritinamine or elwesine depending on the enantiomer reduced to an oxomaritinamine product. In this study, a short chain alcohol dehydrogenase/reductase that co-expresses with the previously discovered norbelladine 4'-O-methyltransferase from Narcissus sp. and Galanthus spp. was cloned and expressed in Escherichia coli Biochemical analyses and x-ray crystallography indicates that this protein functions as a noroxomaritidine reductase that forms oxomaritinamine from noroxomaritidine through a carbon-carbon double bond reduction. The enzyme also reduces norcraugsodine to norbelladine with a 400-fold lower specific activity. These studies identify a missing step in the biosynthesis of this pharmacologically important class of plant natural products.

Highlights

Fighting potential including hemanthamine, crinine, and lycorine [2, 4, 5]
For the biosynthesis of all Amaryllidaceae alkaloids, norbelladine, which is made from the condensation of tyramine and 3,4-dihydroxybenzaldehdye followed by imine reduction, is a pathway intermediate (6 – 8). 3,4-Dihydroxybenzaldehyde is hypothesized to originate from a branch of the phenylpropanoid pathway with demonstrated intermediates trans-cinnamic acid, 4-hydroxycinnamic acid, and either 3,4-dihydroxycinnamic acid or 4-hydroxybenzaldehyde [9]
The Amaryllidaceae alkaloids are a large group of plant natural products with over 300 documented structures and diverse biological activities

Summary

Identification of Noroxomaritidine Reductase

Norbelladine undergoes methylation and phenol-coupling reactions to produce the vast diversity of Amaryllidaceae alkaloids, including galanthamine, lycorine, hemanthamine, and crinine (Fig. 1) [1, 2]. Recent studies have identified enzymes catalyzing steps in the core Amaryllidaceae alkaloid biosynthesis pathway, including norbelladine 4Ј-O-methyltransferase (N4OMT) and the cytochrome P450 CYP96T1 [44]. The enzymes that reduce noroxomaritidine to normaritidine (a precursor of the common hemanthamine-type alkaloids) or oxomaritinamine (a precursor of less prolific alkaloids including maritinamine and elwesine) remain to be identified. Pseudonarcissus, Galanthus sp., and Galanthus elwesii transcriptomes successfully identified N4OMT and CYP96T1 in the core Amaryllidaceae alkaloid biosynthetic pathway [17, 18]. For several missing steps in the hemanthamine, crinine, maritinamine, and elwesine pathways, we hypothesized that members of either aldo-keto reductase or SDR enzyme families may catalyze norbelladine synthesis and/or reduction of the noroxomaritidine enantiomers. Pseudonarcissus SDR reduces the carbon-carbon double bond of the Amaryllidaceae alkaloid noroxomaritidine to form one of the enantiomeric forms of oxomaritinamine. Given the high specific activity for noroxomaritidine reduction relative to norcraugsodine reduction, we designate this gene/protein as noroxomaritidine reductase (NR)

Results

Piperonal and tyramine

Discussion

Experimental Procedures