Characterization of Three O-Methyltransferases Involved in Noscapine Biosynthesis in Opium Poppy  

Abstract

Noscapine is a benzylisoquinoline alkaloid produced in opium poppy (Papaver somniferum) and other members of the Papaveraceae. It has been used as a cough suppressant and more recently was shown to possess anticancer activity. However, the biosynthesis of noscapine in opium poppy has not been established. A proposed pathway leading from (S)-reticuline to noscapine includes (S)-scoulerine, (S)-canadine, and (S)-N-methylcanadine as intermediates. Stem cDNA libraries and latex extracts of eight opium poppy cultivars displaying different alkaloid profiles were subjected to massively parallel pyrosequencing and liquid chromatographytandem mass spectrometry, respectively. Comparative transcript and metabolite profiling revealed the occurrence of three cDNAs encoding O-methyltransferases designated as SOMT1, SOMT2, and SOMT3 that correlated with the accumulation of noscapine in the eight cultivars. SOMT transcripts were detected in all opium poppy organs but were most abundant in aerial organs, where noscapine primarily accumulates. SOMT2 and SOMT3 showed strict substrate specificity and regiospecificity as 9-Omethyltransferases targeting (S)-scoulerine. In contrast, SOMT1 was able to sequentially 9-and 2-O-methylate (S)-scoulerine, yielding (S)-tetrahydropalmatine. SOMT1 also sequentially 39-and 7-O-methylated both (S)-norreticuline and (S)-reticuline with relatively high substrate affinity, yielding (S)-tetrahydropapaverine and (S)-laudanosine, respectively. The metabolic functions of SOMT1, SOMT2, and SOMT3 were investigated in planta using virus-induced gene silencing. Reduction of SOMT1 or SOMT2 transcript levels resulted in a significant decrease in noscapine accumulation. Reduced SOMT1 transcript levels also caused a decrease in papaverine accumulation, confirming the selective roles for these enzymes in the biosynthesis of both alkaloids in opium poppy.

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…A collection of diverse O-methytransferases catalyze the regiospecific transfer of a methyl group from S-adenosylmethionine to a free hydroxyl moiety on a variety of BIA subgroups, but appear to primarily target 1-benzylisoquinolines and protoberberines (Dang and Facchini 2012). N-methyltransferases involved in BIA biosynthesis display substantial sequence similarity and catalyze the transfer of a methyl group to the nitrogen on the isoquinoline ring.…”

“…(S)-Scoulerine is converted via methylenedioxy bridge formation and O-methylation to a variety of protoberberines including (S)-canadine, (S)-stylopine and (S)-sinactine. (S)-Canadine, the precursor to the protopine allocryptopine and the phthalideisoquinoline noscapine, is formed sequentially from (S)-scoulerine by (1) scoulerine 9-O-methyltransferase (SOMT1) (Dang and Facchini 2012;Takeshita et al 1995) yielding tetrahydrocolumbamine and (2) the methylenedioxy bridge forming enzyme canadine synthase (CAS), which is a member of the CYP719A subfamily (Díaz Chávez et al 2011;Ikezawa et al 2003;winzer et al 2012). Similarly, (S)-sinactine is formed from (S)-scoulerine by (1) the addition of a methylenedioxy bridge by cheilanthifoline synthase, also a member of the CYP719A subfamily (Díaz Chávez et al 2011;Ikezawa et al 2009), followed by (2) 2-O-methylation of cheilanthifoline by an enzyme not yet characterized from opium poppy.…”

“…Noscapine also occurs in other members of the Papaveraceae and Menispermaceae. Owing to its cough-suppressing activity and potential as an anticancer drug (Mahmoudian and RahimiMoghaddam 2009), the biosynthesis of noscapine has been the subject of renewed interest (Dang and Facchini 2012;Facchini et al 2007;winzer et al 2012). The formation of noscapine has not been as extensively investigated as other alkaloids, such as morphine.…”

“…The scheme involves sequential hydroxylation of the isoquinoline ring, oxidative cleavage of the berberine bridge and two subsequent oxidations to form the characteristic phthalideisoquinoline lactone ring. Following the identification of relevant O-methyltransferases (Dang and Facchini 2012), a gene cluster proposed to encode all noscapine biosynthetic enzymes was identified in opium poppy. The functions for some gene cluster members were suggested using vIGS to show correlations between the suppression of a specific gene and the accumulation of compounds purported as pathway intermediates (winzer et al 2012).…”

Morphine Biosynthesis in Opium Poppy Involves Two Cell Types: Sieve Elements and Laticifers

“…Only the epimerization of reticuline has not been characterized at the molecular biochemical level (De-Eknamkul and Zenk, 1992). Additional cDNAs encoding several other BIA biosynthetic enzymes have been characterized from opium poppy (see Supplemental Figure 1 online), including reticuline 7-O-methyltransferase (7OMT) (Ounaroon et al, 2003), norreticuline 7-O-methyltransferase (N7OMT) (Pienkny et al, 2009), scoulerine O-methyltransferase (SOMT) (Dang and Facchini, 2012;Winzer et al, 2012), berberine bridge enzyme (Facchini et al, 1996), stylopine synthase (Díaz-Chávez et al, 2011), tetrahydroprotoberberine N-methyltransferase (TNMT) (Liscombe and Facchini, 2007), pavine N-methyltransferase (PavNMT) (Liscombe et al, 2009), and sanguinarine reductase (Vogel et al, 2010).…”