Dioxygenases catalyze the O-demethylation steps of morphine biosynthesis in opium poppy

Abstract

Two previously undetected enzymes involved in morphine biosynthesis and unique among plants to opium poppy have been identified as non-heme dioxygenases, in contrast to the functionally analogous cytochrome P450s found in mammals. We used functional genomics to isolate thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM), the only known 2-oxoglutarate/Fe(II)-dependent dioxygenases that catalyze O-demethylation. Virus-induced gene silencing of T6ODM and CODM in opium poppy efficiently blocked metabolism at thebaine and codeine, respectively.

Benzylisoquinoline alkaloid biosynthesis in opium poppy

“…The application of trinexapac-ethyl, an inhibitor of ODDs involved in gibberellin (GA) biosynthesis, with or without methyl jasmonate results in the introduction of a putative catabolic bottleneck leading to an increase in the relative abundance of thebaine and codeine and a decrease in the accumulation of morphine (Cotterill 2010). Interestingly, trinexapac-ethyl and other acylcyclohexanediones do not inhibit T6ODM and CODM directly (Hagel and Facchini 2010). exogenous addition of GA 3 was reported to increase the yield of morphine (Khan et al 2007), suggesting a substantial role for phytohormone control in morphinan alkaloid metabolism.…”

“…Suppression of T6ODM transcript levels resulted in the accumulation of thebaine and oripavine and substantially reduced levels of codeine and morphine. Similarly, suppression of CODM transcript levels caused the accumulation of codeine at the expense of morphine (Hagel and Facchini 2010). The isolation of all biosynthetic genes facilitated a coordinated confirmation of the physiological role of each enzyme operating downstream of (R)-reticuline (wijekoon and Facchini 2012).…”

“…In contrast, FADOX homologs appear to only catalyze the formation of C-O bonds in the formation of non-alkaloid specialized metabolites in other plants (Custers et al 2004;Sirikantaramas et al 2004). Although ODDs are known to perform a variety of hydroxylations, epoxidations and desaturations in a variety of plant metabolic pathways (Prescott and John 1996), enzymes from this family have only been shown to catalyze the dealkylation of certain BIA subgroups (Hagel and Facchini 2010;Farrow and Facchini 2013).…”

“…The morphine pathway bifurcates at thebaine with the initial step in the major route catalyzed by thebaine 6-O-demethylase (T6ODM) yielding neopinone, which undergoes purportedly spontaneous rearrangement to codeinone (Hagel and Facchini 2010). Codeinone is then reduced by the aldo-keto reductase codeinone reductase (COR) (Unterlinner et al 1999) to codeine, which is converted to morphine by codeine O-demethylase (CODM).…”

“…The physiological roles of T6ODM and CODM were suggested in the opium poppy mutant top1 , which accumulates high levels of thebaine and oripavine at the expense of codeine and morphine. The absence of T6ODM transcripts in a highthebaine/oripavine, low-codeine/morphine opium poppy chemotype was compared to three high-codeine/morphine varieties to identify the corresponding gene using a microarray-based approach (Hagel and Facchini 2010). Suppression of T6ODM transcript levels resulted in the accumulation of thebaine and oripavine and substantially reduced levels of codeine and morphine.…”