Norcoclaurine Synthase Is a Member of the Pathogenesis-Related 10/Bet v1 Protein Family

Abstract

Norcoclaurine synthase (NCS) catalyzes the first committed step in the biosynthesis of benzylisoquinoline alkaloids (BIAs). NCS from Thalictrum flavum (Tf NCS), Papaver somniferum (Ps NCS1 and Ps NCS2), and Coptis japonica (Cj PR10A) share substantial identity with pathogen-related 10 (PR10) and Bet v1 proteins, whose functions are not well understood. A distinct enzyme (Cj NCS1) with similarity to 2-oxoglutarate-dependent dioxygenases was suggested as the bona fide NCS in C. japonica. Here, we validate the exclusive role of PR10/Bet v1-type NCS enzymes in BIA metabolism. Immunolocalization of Ps NCS2 revealed its cell type-specific occurrence in phloem sieve elements, which contain all other known BIA biosynthetic enzymes. In opium poppy, NCS transcripts and proteins were abundant in root and stem, but at low levels in leaf and carpel. Silencing of NCS in opium poppy profoundly reduced alkaloid levels compared with controls. Immunoprecipitation of NCS from total protein extracts of T. flavum cells resulted in a nearly complete attenuation of NCS activity. A Ps NCS2-green fluorescent protein fusion introduced by microprojectile bombardment into opium poppy cells initially localized to the endoplasmic reticulum but subsequently sorted to the vacuole. In our hands, Cj NCS1 did not catalyze the formation of (S)-norcoclaurine from dopamine and 4-hydroxyphenylacetaldehyde.

Tyrosine Aminotransferase Contributes to Benzylisoquinoline Alkaloid Biosynthesis in Opium Poppy

“…In particular, transcripts encoding Tyr/ DOPA decarboxylase and NCS, the other known enzymes involved in the early step of the BIA pathway, were abundant in stems and roots and were found only at low levels in leaves and developing seed capsules (Facchini and De Luca, 1995;Lee and Facchini, 2010). The distribution of PsTyrAT transcripts is consistent with a preeminent role of stems and roots in the biosynthesis of BIAs in opium poppy (Fig.…”

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…Alternatively, l-tyrosine and DOPA are converted to tyramine and dopamine by tyrosine decarboxylase (TYDC) (Facchini and De Luca 1994). The stereoselective Pictet-Spengler condensation of (S)-norcoclaurine from 4HPAA and dopamine is catalyzed by norcoclaurine synthase (NCS), which is a member of the pathogenesis-related (PR)10/Bet v 1 protein family (Liscombe et al 2005;Samanani et al 2004;Lee and Facchini 2010). In addition to a PR10-type NCS, a second enzyme (CjNCS1) sharing similarity with ODDs was reported to catalyze the formation of (S)-norcoclaurine in Coptis japonica (Minami et al 2007).…”

“…The biosynthesis and storage of BIAs in opium poppy has been extensively investigated and shown to involve three distinct cell types (Facchini and De Luca 1995;Bird et al 2003;Samanani et al 2006;Lee and Facchini 2010). In situ hybridization and immunofluorescence labeling demonstrated the localization of biosynthetic gene transcripts and enzymes to companion cells and sieve elements, respectively.…”

“…The importance of transcriptional control in BIA metabolism is particularly evident from (1) the cell typespecific localization of biosynthetic gene transcripts (Bird et al 2003;Samanani et al 2006;Lee and Facchini 2010;Onoyovwe et al 2013) and (2) the coordinated induction of all expressed biosynthetic gene transcripts (Zulak et al 2007) and corresponding proteins (Zulak et al 2009;Desgagné-Penix et al 2010) in opium poppy cell cultures after the addition of a fungal-derived elicitor.…”

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

“…Among these, opium poppy (Papaver somniferum) remains a valuable source for several pharmaceuticals, including the narcotic analgesics morphine and codeine, the muscle relaxant papaverine, and the cough suppressant and potential anticancer drug noscapine. BIA biosynthesis begins with the formation of (S)-norcoclaurine via the condensation of the Tyr derivatives dopamine and 4-hydroxyphenylacetaldehyde (Samanani and Facchini, 2002;Minami et al, 2007;Lee and Facchini, 2010). (S)-Norcoclaurine is subsequently converted via several steps to the key branch point intermediate (S)-reticuline ( Fig.…”