Breaking bad buttons: mescaline biosynthesis in peyote

The small, globular cactus peyote (Lophophora williamsii) is known for its ability to produce mescaline, a phenethylamine protoalkaloid (Figure 1). Consuming the mescaline-containing dried tops or “buttons” of peyote has a psychedelic effect that has been used in religious ceremonies by Indigenous communities for over 5800 years (Cassels & Sáez-Briones, 2018). Nowadays, psychedelic drugs are used for treatment of mental disorders such as depression, anxiety, or post-traumatic stress disorder. Mescaline is currently being tested in clinical trials, as its effects are longer lasting than those of other psychedelic drugs and might therefore provide greater therapeutic benefits (Bender, 2022). Mescaline biosynthesis pathway in peyote. For mescaline and N-methylmescaline biosynthesis in peyote, L-tyrosine is 3-hydroxylated to L-DOPA (A), followed by decarboxylation to dopamine (B); dopamine undergoes 3-O-methylation to 3-methoxytyramine (C); then, an unknown 5-hydroxylase converts 3-methoxytyramine to 3-methoxy-4,5-dihydroxy-phenethylamine (PEA) (D); PEA is O-methylated to 3,5-dimethoxy-4-hydroxy-PEA, which is O-methylated yielding mescaline (E, F), or by N-methylation and 4-O-methylation into N-methylmescaline (G). The tetrahydroisoquinoline (THIQ) alkaloids are likely formed from the mescaline pathway intermediates 3-methoxytyramine and 3-methoxy-4,5-dihydroxy-PEA; modified from Watkins et al. (2023) background photograph of peyote courtesy of Peter Facchini. Besides the phenethylamine alkaloids, there are tetrahydroisoquinoline (THIQ) alkaloids in peyote that are thought to act in the absorption, metabolism, and excretion of mescaline and thereby contribute to the psychedelic experience (Chan et al., 2021). Our current understanding of mescaline and THIQ biosynthesis is based on early radioisotope feeding experiments, which measured the incorporation of putative radiolabelled precursors into the biosynthetic products (Battersby et al., 1968), but no mescaline or THIQ biosynthetic enzymes had been identified. Therefore, the authors of the highlighted publication set out to elucidate a near-complete biosynthetic pathway from L-tyrosine to mescaline in peyote, using both transcriptomics and a homology-guided gene discovery strategy (Watkins et al., 2023). The study is a collaboration between the research groups of Peter Faccini and Sam Yeaman at the University of Calgary. Jacinta Watkins, the first author, has a background in plant secondary metabolism and is interested in the diversity of plant compounds that are used as medicines, to flavour food or for nutrition. She joined Facchini's lab in late 2019 as a postdoc. Yeaman's research associate Qiushi Li conducted the bioinformatics analyses, particularly the genome and transcriptome assembly, molecular marker analysis, and gene expression analysis. Li is fascinated by medicinal plants, such as opium poppy, ephedra, ginseng, and peyote. Facchini has worked on the metabolic biochemistry of alkaloid biosynthesis in plants of medicinal, social, and economic importance for over 30 years, including elucidating several benzylisoquinoline alkaloids biosynthetic pathways in opium poppy (Papaver somniferum) and related plants (Ozber & Facchini, 2013). His group also identified genes involved in the biosynthesis of the amphetamines ephedrine and pseudoephedrine in Ephedra sinica (Morris et al., 2018), and they have recently made progress on elucidating the biosynthesis of N,N-dimethyltryptamine (DMT) and derivatives in the psychedelic cane toad (Rhinella marina). His answer to the question of how his laboratory became the Breaking Bad of plant biology research: “controlled plants and substances chose me” rather than the other way around. To analyse the biosynthesis of mescaline and THIQ in peyote, Watkins et al. dissected epidermal and chlorenchyma tissue as one fraction, as well as the vasculature and cortex in another fraction, for transcriptome sequencing and assembly, and used untargeted metabolomics to measure the alkaloid accumulation. Both fractions contained low levels of mescaline and N-methylmescaline, but large quantities of the putative di-O-methylated intermediate, suggesting that all enzymes for the biosynthesis pathway were present, and enzymes that led to accumulation of the N-methylated derivative. Conversion of L-tyrosine into mescaline requires hydroxylation of the benzene ring at the 3 and 5 positions before O-methylation. In sugar beet (Beta vulgaris), which like peyote belongs to the Caryophyllales, a member of the CYP76AD subfamily catalyses the 3-hydroxylation of L-tyrosine to L-DOPA (Hatlestad et al., 2012). Therefore, the authors looked for homologues of the sugar beet enzyme based on amino acid identity and found three members of the family in peyote. They transiently expressed the three candidates in yeast and found that LwCYP76AD94 catalysed the conversion of exogenous L-tyrosine to L-DOPA (Figure 1A). The conversion of L-tyrosine into mescaline requires aromatic amino acid decarboxylation (Figure 1B). The authors therefore searched for peyote homologues of the opium poppy tyrosine/DOPA decarboxylase. They expressed three candidate genes in Escherichia coli, purified the cognate recombinant enzymes and found that LwTyDC1 was able to decarboxylate both L-tyrosine and L-DOPA. In a similar approach, the authors searched for homologues of the opium poppy 4′-O-methyltransferase 2 (OMT). The 11 candidate genes were expressed in E. coli, and the recombinant enzymes were purified and biochemically characterised. The authors found that LwOMT10 catalysed the 4-O-methylation of dopamine, yielding 4-methoxytyramine (Figure 1C), as well as the 4-O-methylation of 3,5-dimethoxy-4-hydroxyphenethylamine (3,5- dimethoxy-4-hydroxy-PEA), yielding mescaline (Figure 1F), and N-methyl-3,5-dimethoxy-4-hydroxy-PEA, yielding N-methylmescaline (Figure 1G). LwOMT2 5-O-methylated the intermediate 3-methoxy-4,5-dihydroxyphenethylamine (3-methoxy-4,5-dihydroxy-PEA) (Figure 1E). Of the analysed enzymes, LwOMT3 had the largest substrate range. Because it had N-methyltransferase activity rather than O-methyltransferase activity, it was renamed LwNMT. It potentially produces N-methylated-mescaline (Figure 1G). N-methylation of dopamine or 3-methoxytyramine could be the first step in the production of the N-methylated THIQ alkaloids (Chan et al., 2021). The authors identified most mescaline biosynthetic enzymes and could thereby construct the biosynthesis pathway, although the enzyme that catalyses the 5-hydroxylation of the benzene ring remained elusive (Figure 1D). The strategy of a homology-guided gene discovery can limit the discovery of novel enzymes because it assumes amino acid homology on as a start point. This approach is more successful for some types of enzymes than for others. For example, virtually all O-methyltransferases involved in plant-specialised metabolism share substantial amino acid sequence identity, owing to the similarity of the reaction mechanism, regardless of the substrate or metabolic pathway. In contrast, N-methyltransferases include diverse protein types, likely because there are more mechanistic options capable of facilitating reactions with a similar outcome. The same is true for oxidative reactions, which can be catalysed by quite different enzymes, making it more difficult to find the enzyme catalysing the addition of the 5-hydroxyl moiety on mescaline. Other techniques, such as co-expression analysis or gene clustering, might be useful to identify the hydroxylase enzyme. Due to habitat loss and overharvesting, peyote is an endangered species (Ermakova et al., 2021). As an alternative to its extraction from peyote, mescaline can be synthesised chemically (Cassels & Sáez-Briones, 2018). The identification of the biosynthetic pathway will allow the development of synthetic biosystems for mescaline production on an industrial scale.

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