Identification of a Noroxomaritidine Reductase with Amaryllidaceae Alkaloid Biosynthesis Related Activities

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-Omethyltransferase 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.

Exploiting the Catalytic Diversity of Short‐Chain Dehydrogenases/Reductases: Versatile Enzymes from Plants with Extended Imine Substrate Scope

“…NADPH‐dependent NR selectively reduces the C=C bond of the enone moiety of noroxomaritidine ( 1 ). In addition, this SDR exhibits an imine reductase activity with norcraugsodine ( 2 ), a precursor of 1 , albeit with 1/400th of its native activity (Scheme ) …”

“… Involvement of noroxomaritidine reductase (NR) in Amaryllidaceae alkaloid biosynthesis (modified from Kilgore et al …”

“…Examples of enzymatic reduction promiscuity have been reported for highly reactive iminium and activated keto substrates . NR, however, displays catalytic versatility with nonactivated substrates occurring in vivo . This makes the enzyme an interesting candidate for investigating its intrinsic versatility for the biocatalytic reduction of imines.…”

Extended Catalytic Scope of a Well‐Known Enzyme: Asymmetric Reduction of Iminium Substrates by Glucose Dehydrogenase

“…Our finding for GDH is in line with a recent example of promiscuous SDR activity: Kutchan and co‐workers discovered a C=C‐bond‐reducing SDR with an imine reductase side activity . Moreover, in Catharanthus roseus a medium‐chain alcohol dehydrogenase homologue that possesses iminium‐reducing activity has been found .…”

The Short‐chain Dehydrogenase/Reductase Engineering Database (SDRED): A classification and analysis system for a highly diverse enzyme family

“…Although this catalytic machinery is conserved in almost all SDRs, they catalyze a wide spectrum of reactions. While Classical and Extended SDRs mainly catalyze the dehydrogenation of alcohols and the reduction of ketones, enoates, or imines, Extended SDRs also include epimerases, dehydratases, lyases, and isomerases. Intermediate SDRs consist mainly of epimerases and dehydratases and contain one superfamily with ADHs from the genus Drosophila .…”