Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD

Misson, Laëtitia; Burn, Reto; Vit, A.; Hildesheim, Julia; Beliaeva, Mariia A.; Blankenfeldt, Wulf; Seebeck, Florian P.

doi:10.1021/acschembio.8b00127

Cited by 27 publications

(20 citation statements)

References 77 publications

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“…For heterologous production of S. cerevisiae TRP5, E. coli KRX transformed with pJF56 was used. Production of E. coli S ‐adenosylhomocysteine nucleosidase and adenine deaminase followed a described procedure . The above enzymes were purified by immobilized metal‐affinity chromatography on a His Trap FF Crude column (1 mL bed volume), fitted to an Äkta Pure 25 FPLC instrument (GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

Enzymatic Route toward 6‐Methylated Baeocystin and Psilocybin

et al. 2019

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Psilocybin and its direct precursor baeocystin are indole alkaloids of psychotropic Psilocybe mushrooms. The pharmaceutical interesti np silocybin as at reatment optiona gainst depression and anxiety is currently being investigated in advanced clinicalt rials. Here, we reportabiocatalytic route to synthesize 6-methylated psilocybin and baeocystin from 4-hydroxy-6methyl-l-tryptophan, which was decarboxylated and phosphorylated by the Psilocybec ubensis biosynthesis enzymes PsiD and PsiK. N-Methylation was catalyzed by PsiM. We further present an in silico structural model of PsiM that revealed aw ell-conserved SAM-binding core along with peripheraln onconserved elements that likely govern substrate preferences.The indole nucleus is ac ommon motif with bioactiven atural products that is, in numerous instances, modified duringb iosynthesis. Modifications include, for example, 7-chlorination in the case of rebeccamycin, prenyl transfer to various positions to make asterriquinones, or hydroxylation at C-5 and C-6 of serotonin and amanitins,r espectively.R eserpine, ibogaine, and harmaline are methoxy-substituted att hesec arbons. Dissimilar from the above positionsa nd substituents, psilocybin( 1, Scheme 1) features aunique 4-phosphoryloxy group.Compound 1 is the principal metabolite of Psilocybe carpophores ("magic mushrooms"), whereas baeocystin (2)a nd norbaeocystin (3), the immediate metabolic precursors, are present in minor quantities. [1] The dephosphorylated analogue of 1, psilocin (4), represents the pharmacologically active, that is, psychotropic compound as it is ap artial agonist at the human 5HT 2A receptor. [2] The pharmaceutical value of 1 in the treat-ment of end-of-life anxiety and therapy refractory depression has been documentedi nv ariousa dvanced clinicalt rials. [3] Syntheses of 1 congeners date back to the late 1950s. Following the discovery of 1 by Albert Hofmann and co-workers, various series of derivatives were synthesized,w hich also contained the 6-phosphoryloxy and 6-hydroxy isomerso f1 and 4,a long with a6 -methylated, side-chain-alkylated tryptamine. [4] Subsequent efforts were made to install bromine or af ormyl group at C-5-or C-7, vary the substituents at the w-N-atom of 4,o r add various substituentst ot he aromatic ring of N,N-dimethyltryptamine (5), to produce its 6-and 7-methyl analogues, among others. [5] Despite theser esultsa nd despite the synthesis of 2-methyl derivatives of 5 and N,N-diethyltryptamine, [6] none of the previouse fforts, using synthetic chemistry,h ad focused on introducing am ethyl substituent to the indole of 1 or 2.We report the in vitro synthesis of 6-methylpsilocybin (6, Scheme1)a nd, as main products,i ts immediate precursors 6methylnorbaeocystin (7)a nd 6-methylbaeocystin (8). We address the previous lack of ring-methylated 1 analogues and simultaneously provide am odel of the PsiM structure for insight into the origin of its methylating activity on 3 and 7.W ei dentified al oop structure as criticalf or methyl acceptors ubstrate selectivity.C oncu...

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Section: Methodsmentioning

confidence: 99%

Enzymatic Route toward 6‐Methylated Baeocystin and Psilocybin

et al. 2019

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“…In more recent work, it has been suggested that ERG was probably first biosynthesised by anaerobes using a slightly different route that converts hercynine directly to ERG (98)(99)(100) , and that was later repurposed. Three-dimensional structures are known for a number of the relevant enzymes, including mycobacterial EgtB (101) for example, PDB 4XBE, EgtC (102) for example, PDB 4ZFJ, EgtD (103)(104)(105) for example, PDB 4PIM, and Neurospora crassa early G1 transcript 2 (egt2) which is like egtE (106) for example, PDB 5UTS.…”

Section: Biosynthesis and Phylogenetic Distributionmentioning

confidence: 99%

The biology of ergothioneine, an antioxidant nutraceutical

et al. 2020

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Ergothioneine (ERG) is an unusual thio-histidine betaine amino acid that has potent antioxidant activities. It is synthesised by a variety of microbes, especially fungi (including in mushroom fruiting bodies) and actinobacteria, but is not synthesised by plants and animals who acquire it via the soil and their diet, respectively. Animals have evolved a highly selective transporter for it, known as solute carrier family 22, member 4 (SLC22A4) in humans, signifying its importance, and ERG may even have the status of a vitamin. ERG accumulates differentially in various tissues, according to their expression of SLC22A4, favouring those such as erythrocytes that may be subject to oxidative stress. Mushroom or ERG consumption seems to provide significant prevention against oxidative stress in a large variety of systems. ERG seems to have strong cytoprotective status, and its concentration is lowered in a number of chronic inflammatory diseases. It has been passed as safe by regulatory agencies, and may have value as a nutraceutical and antioxidant more generally.

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“…Of note, the residues of EgtD known to interact with the (methylated) α‐amino group of l ‐histidine, that is, Gly161 and Asn166, are conserved in TrpM (Gly192 and Asn197). Asn166 in EgtD is essential to make N , N ‐dimethyl‐ l ‐histidine a 100‐fold stronger ligand than l ‐histidine . Crystallographic work would be warranted to elucidate the respective roles of the active‐site residues for TrpM.…”

Section: Methodsmentioning

confidence: 99%

Iterative l‐Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication

et al. 2018

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Psilocybe mushrooms are best known for their l-tryptophan-derived psychotropic alkaloid psilocybin. Dimethylation of norbaeocystin, the precursor of psilocybin, by the enzyme PsiM is a critical step during the biosynthesis of psilocybin. However, the "magic" mushroom Psilocybe serbica also mono- and dimethylates l-tryptophan, which is incompatible with the specificity of PsiM. Here, a second methyltransferase, TrpM, was identified and functionally characterized. Mono- and dimethylation activity on l-tryptophan was reconstituted in vitro, whereas tryptamine was rejected as a substrate. Therefore, we describe a second l-tryptophan-dependent pathway in Psilocybe that is not part of the biosynthesis of psilocybin. TrpM is unrelated to PsiM but originates from a retained ancient duplication event of a portion of the egtDB gene that encodes an ergothioneine biosynthesis enzyme. During mushroom evolution, this duplicated gene was widely lost but re-evolved sporadically and independently in various genera. We propose a new secondary metabolism evolvability mechanism, in which weakly selected genes are retained through preservation in a widely distributed, conserved pathway.

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Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD

Cited by 27 publications

References 77 publications

Enzymatic Route toward 6‐Methylated Baeocystin and Psilocybin

Enzymatic Route toward 6‐Methylated Baeocystin and Psilocybin

The biology of ergothioneine, an antioxidant nutraceutical

Iterative l‐Tryptophan Methylation in Psilocybe Evolved by Subdomain Duplication

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