The final two O-demethylation steps of morphine biosynthesis in opium poppy (Papaver somniferum) are catalyzed by thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM), which Hagel and Facchini (2010) identified as 2-oxoglutarate/Fe(II)-dependent dioxygenases — the first plant enzymes of this family shown to catalyze O-demethylation. That discovery report left five issues open: the physiological relevance of the enzymes' promiscuous activities (notably CODM-catalyzed 3-O-demethylation of the protoberberine alkaloid (S)-scoulerine), the indirect mechanism by which acylcyclohexanedione growth regulators block flux through the O-demethylation steps in whole plants without inhibiting either enzyme in vitro, the genetic lesion behind the specific loss of T6ODM in the top1 and T high-thebaine chemotypes, the structural basis of C3-versus-C6 regiospecificity, and the evolutionary origin of these unusual dioxygenase demethylases. Later literature resolved two facets only in part — T6ODM co-substrate-bound crystal structures with computational mechanism proposals (2018-2021) and the T-chemotype T6ODM-cluster deletion (2020) — while the remaining core is open: no substrate-bound or CODM structures, no identified top1 lesion, no mechanistic account of acylcyclohexanedione action, no in-planta demonstration for the scoulerine activity, and no functional reconstruction of the demethylases' evolutionary recruitment.

Background

Opium poppy (Papaver somniferum) is the sole commercial source of the analgesics codeine and morphine and of thebaine, the feedstock for semi-synthetic opioids such as oxycodone, buprenorphine, and naltrexone. These drugs belong to the benzylisoquinoline alkaloids (BIAs), a family of tyrosine-derived plant specialized metabolites. In the morphinan branch of BIA biosynthesis, the pathway intermediate thebaine is converted to morphine through a small network of intermediates (oripavine, neopinone, codeinone, codeine) in which two O-demethylation steps — enzymatic removal of methyl groups from oxygen atoms — alternate with reduction and isomerization: thebaine 6-O-demethylase (T6ODM) removes the 6-O-methyl group of thebaine (and of oripavine), and codeine O-demethylase (CODM, also called codeine-3-O-demethylase) removes the 3-O-methyl group of codeine (and of thebaine, yielding oripavine).

O-demethylation of small molecules is classically catalyzed by cytochrome P450 heme enzymes, as in mammalian metabolism of codeine and thebaine. Hagel and Facchini (2010) therefore surprised the field by identifying T6ODM and CODM as 2-oxoglutarate/Fe(II)-dependent dioxygenases (2OGDs, also called ODDs): non-heme iron enzymes that couple the oxidative decarboxylation of 2-oxoglutarate to succinate and carbon dioxide with the oxidation of a bound substrate. These enzymes perform O-demethylation by hydroxylating the methoxy group to a hemiacetal that fragments with release of formaldehyde. Virus-induced gene silencing (VIGS, a viral-vector-based post-transcriptional gene knockdown technique) of T6ODM or CODM in poppy blocked metabolism at thebaine or codeine, respectively, and phenocopied the industrial high-thebaine chemotypes: the mutant top1 ('thebaine oripavine poppy 1', commercialized as the cultivar Norman; Millgate et al. 2004) and the laboratory T chemotype accumulate thebaine and oripavine and lack codeine and morphine, which the discovery work traced to the absence of T6ODM transcripts.

The discovery work left five issues unresolved. (i) Both enzymes accept non-morphinan BIAs in vitro — notably CODM efficiently 3-O-demethylates the protoberberine alkaloid (S)-scoulerine (a precursor of antimicrobial protopine and benzo[c]phenanthridine alkaloids) — but whether such activities operate in the intact plant was not established. (ii) The acylcyclohexanedione plant growth regulators prohexadione-calcium and trinexapac-ethyl (inhibitors of 2OGD enzymes of gibberellin, i.e. plant hormone, biosynthesis) increase thebaine and codeine at the expense of morphine when applied to whole poppy plants, yet do not inhibit T6ODM or CODM in vitro, implying an unexplained indirect mode of action. (iii) The genetic and molecular basis of the specific T6ODM silencing in the top1 and T chemotypes was not determined. (iv) The evolutionary origin of these unusual dioxygenase O-demethylases — for example, whether they were recruited from ancestral ODDs serving protoberberine or protopine metabolism — remained conjectural. (v) Mechanistic and structural characterization (transient hydroxylated intermediates, active-site residues responsible for regiospecificity, high-resolution structures) was lacking. Later work settled parts of (iii) and (v): a crystal structure of T6ODM bound to its co-substrate analogues (Kluza et al. 2018), computational mechanism and regioselectivity models (Kachhap et al. 2020; Singh et al. 2021), and the finding that the entire tandemly duplicated T6ODM gene cluster is deleted in cultivar T (Li et al. 2020). Structural work has nevertheless not yielded a substrate- or product-bound complex of either enzyme, no structure of CODM has been reported, and residue-swap mutagenesis between the two enzymes has shown that C3/C6 selectivity can be altered without revealing the structural basis of the discrimination (Runguphan et al. 2012). Farrow and Facchini (2013) additionally discovered that T6ODM, CODM, and the paralog protopine O-dealkylase also catalyze O,O-demethylenation (cleavage of methylenedioxy bridges) and established by VIGS that these dioxygenases have physiological roles in protopine, benzo[c]phenanthridine, and rhoeadine metabolism in planta — without, however, establishing an in-planta role for the scoulerine 3-O-demethylation itself. The evolutionary recruitment of these demethylases has been discussed only as conjecture, which Farrow and Facchini (2013) held would require functional characterization of homologs in related Papaver species. The remaining facets of the five issues are the subject of this problem.

Problem Statement

Complete the biological characterization of the two 2-oxoglutarate/Fe(II)-dependent dioxygenase O-demethylases of morphine biosynthesis, T6ODM and CODM, in opium poppy beyond their in vitro enzymology. The 2010 discovery report left five issues open, of which later literature settled two parts — the co-substrate-bound crystal structure of T6ODM together with computational mechanism proposals, and the deletion of the tandem T6ODM gene cluster as the genetic basis of the T high-thebaine chemotype — leaving the following open core. Its facets jointly serve one objective, namely establishing how, and to what end, morphinan O-demethylation operates in the plant. Each of the following five facets states an independent part of the open core:

  1. In-planta relevance of the promiscuous O-demethylations: determine whether the activities of T6ODM and CODM on non-morphinan benzylisoquinoline alkaloids — in particular CODM-catalyzed 3-O-demethylation of (S)-scoulerine — contribute to metabolism in the intact plant, as opposed to being incidental in vitro activities, and identify the metabolic role and fate of their products.

  2. Indirect action of acylcyclohexanediones: identify the molecular mechanism by which prohexadione-calcium and trinexapac-ethyl reduce metabolic flux through the O-demethylation steps in whole poppy plants (elevating thebaine and codeine at the expense of morphine), given that these compounds do not inhibit T6ODM or CODM in vitro.

  3. top1 genetics: identify the causal genetic lesion producing the specific loss of T6ODM in the top1 (Norman) high-thebaine chemotype, whose molecular basis has not been reported, in contrast to the T chemotype's T6ODM-cluster deletion.

  4. Structural basis of regioselectivity: determine how T6ODM and CODM discriminate the 6-O-methyl versus the 3-O-methyl group of thebaine-like substrates.

  5. Evolutionary origin: establish by which route dioxygenase O-demethylase activity was recruited into morphinan biosynthesis — for example, whether T6ODM and CODM derive from ancestral 2-oxoglutarate/Fe(II)-dependent dioxygenases serving protopine or protoberberine metabolism.

References

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