CYP719B1 Is Salutaridine Synthase, the C-C Phenol-coupling Enzyme of Morphine Biosynthesis in Opium Poppy

Abstract

Morphine is a powerful analgesic natural product produced by the opium poppy Papaver somniferum. Although formal syntheses of this alkaloid have been reported, the morphine molecule contains five stereocenters and a C-C phenol linkage that to date render a total synthesis of morphine commercially unfeasible. The C-C phenol-coupling reaction along the biosynthetic pathway to morphine in opium poppy is catalyzed by the cytochrome P450-dependent oxygenase salutaridine synthase. We report herein on the identification of salutaridine synthase as a member of the CYP719 family of cytochromes P450 during a screen of recombinant cytochromes P450 of opium poppy functionally expressed in Spodoptera frugiperda Sf9 cells. Recombinant CYP719B1 is a highly stereo-and regioselective enzyme; of forty-one compounds tested as potential substrates, only (R)-reticuline and (R)-norreticuline resulted in formation of a product (salutaridine and norsalutaridine, respectively). To date, CYP719s have been characterized catalyzing only the formation of a methylenedioxy bridge in berberine biosynthesis (canadine synthase, CYP719A1) and in benzo[c]phenanthridine biosynthesis (stylopine synthase, CYP719A14). Previously identified phenol-coupling enzymes of plant alkaloid biosynthesis belong only to the CYP80 family of cytochromes. CYP719B1 therefore is the prototype for a new family of plant cytochromes P450 that catalyze formation of a phenol-couple.The C-O or C-C phenol-couple is widely present in the plant kingdom in natural product biosynthetic processes such as alkaloid (1), lignan (2), lignin (3), and gallotannin (4) formation. Phenol-coupling reactions in nature were thought to be catalyzed by a variety of oxidative enzymes with broad substrate specificity such as peroxidases, polyphenol oxidases, and laccases. More recently, several enzymes discovered to be responsible for the formation of intermolecular C-O phenol and intramolecular C-C phenol-couples were found to be highly regio-and/or stereoselective catalysts. The first intermolecular C-O phenol-coupling enzyme identified was the cytochrome P450-dependent oxidase berbamunine synthase (CYP80A1) of bisbenzylisoquinoline alkaloid biosynthesis in Berberis cell cultures (5, 6) ( Fig. 1). This enzyme is regiospecific, but will accept either (R)-and (S)-N-methylcoclaurine to form R-R and R-S phenol-coupled products. Absolute regio-and stereospecificity is demonstrated in the formation of the lignan (ϩ)-pinoresinol from two molecules of coniferyl alcohol, a reaction guided by dirigent proteins that can be catalyzed by a range of oxidases or oxidants (7). The aporphine alkaloid intramolecular C-C phenol-couple is catalyzed in Coptis japonica cell cultures by the cytochrome P450-dependent oxidase CYP80G2; this enzyme accepts six tetrahydrobenzylisoquinoline alkaloids as substrate (8).Morphine has often been described as the king of alkaloids. Although formal syntheses of this powerful analgesic have been reported, yields are low (Ref. 9 and references therein); attempts in organic chemist...

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…(R)-Reticuline is converted by the CYP salutaridine synthase (CYP719B1) to salutaridine (Gesell et al 2009), which is then converted by the short-chain dehydrogenase/reductase salutaridine reductase (SalR) (Ziegler et al 2006). Subsequently, salutaridinol is O-acetylated by salutaridinol 7-O-acetyltransferase (SalAT) (Grothe et al 2001;Lenz and Zenk 1995).…”

Impacts of Diversification of Cytochrome P450 on Plant Metabolism

“…61) CYP719B1 of Papaver somniferum is the salutaridine synthase catalyzing C-C phenol coupling in morphine biosynthesis. 62) Thus, CYP80s and CYP719s are the P450 families characteristic of alkaloids-producing plants, and they are not found in the genomes of Arabidopsis, rice, poplar, and lower plants. These results suggest that the evolution of these P450 families is closely related to the diversification of BIAs.…”

Biosynthesis of the Amaryllidaceae alkaloids

“…In addition, the latter steps of AA biosynthesis involve multiple C-C and C-O bond creation including methyledioxy-bridge formation. In other alkaloid pathways such as the BIA, these reactions are catalyzed by CYP719s and CYP80s (Diaz Chavez, Rolf, Gesell, & Kutchan, 2011;Gesell et al, 2009;Ikezawa et al, 2008). Altogether, these studies suggest that the phenol coupling and the methyledioxy-bridge formation steps in AA biosynthesis are likely to involve CYP450 of the CYP80 family.…”

“…Another example is salutaridine synthase, a member of the CYP719B1, which catalyzes the para-ortho coupling of reticuline in the biosynthesis of morphinan alkaloids in P. somniferum. CYP719B1 is highly stereospecific as it catalyzes the C-C phenol coupling of only (R)-reticuline resulting in formation of salutaridine (Gesell et al, 2009). These examples demonstrate that specific CYP450s are able to catalyze intramolecular C-C phenol coupling reactions in alkaloid biosynthesis and that members of two CYP450 families, CYP80 and CYP719, have been identified.…”