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Magic mushrooms....ohhhhhhh

continuing with our guest blogger Andre Jagger



In this study I describe some of the chemistry around Psilocybin. Almost everyone has heard of magic mushrooms, their magic deriving from perceived halucinogenic, precursor or actual, chemicals in the fungus carpophore (fruiting body). There may be more than one such chemical. Psilocin is not the only psilocybin derivative in magic mushrooms, there are others that include; aeruginascin, baeocystin, norbaeocystin, and norpsilocin. These are notably all amino derivatives of the aromatic heterocyclic compound indole. There are also different genera of magic mushrooms that contain psilocybin and psilocin, these being; Inocybe, Conocybe, Panaeolus, Gymnopilus, and Pluteus. Note very well that not all species in these genera have psilocin or psilocybin and further note that these fungal genera all belong to the

new suborder Agaricineae - proposed by B. Dentinger et al in 2016.


Psychedelic is apparently the preferred term for these potentially hallucinogenic chemicals, principally due to the fact that the hallucinogenic response is not necessarily "enjoyed" by all who partake in the ingestion of them. So it is that psilocybin and psilocin are thought of as psychedelic or psychotomimetic drugs.

In this case Psilocybin is the supposed candidate pschedelic and is found in the TRUE species Gymnopilus aeruginosus, for which the species name aeruginosus was given due to the greenish or bluish staining of the carpophore (basidiocarp in this case) under the action of aeration ('oxidation').


The first question that needs to be addressed is how does one know that this is THE G. aeruginosus and not something else? The first study, previous post, addresses the question of whether this is or is not a Gymnopilus species. It would definitely appear to be a Gymnopilus. That it is or is not G. aeruginosus takes the work of microscopy and also molecular phylogenetics to determine completely. Suffice to say, from the macroscopic point of view it looks pretty close to known specimens of G. aeruginosus and apparently G. aeruginosus has been found in both Korea and Japan. However, there is a gap in that I can't remember seeing it described in the "The Macrofungus Flora of China's Guangdong Province", Bi Zhishu, Zheng Guoyang, and Li Taihui (1993).[ I will endeavour to find time to check the Central Library which has this book in the reference collection. ]


The second question, given this is G. aeruginosus or a closely related Asian look alike, is whether this fungus contains Psilocybin or Psilocin. Rumours have it that this fungus, seen in Hong Kong by others, may stain greenish or bluish when bruised (severed) or through deterioration in age. Whilst I did not see the staining analogy to Psilocybe cubensis ( a role model for such staining - both via injury and natural aging ) I did see that the button mushrooms of this species ( small basidiocarps of 0.5 - 3 mm in size ) were a grey, green, and blue in hue. All of these colours would justify the possible presence of Psilocin, most likely formed from dephosphorylated Psilocybin. Whether or not the mature basidiocarps stain in old age or stain when cut remains a mystery as I witnessed neither! Currently, I suspect that Psilocybin may be present and in large quantities while the basidiocarp is embryonic to early developmental and only in small quantities later. The larger question of whether Gymnopilus species contain Psilocybin has been reviewed and some do and some don't! Those that do belong mainly to the Aeruginosus-Luteofolius clade in the Genus.


I've glossed over a significant point, since when did colour change Blue-Green indicate the presence of psilocybin? This will be discussed soon ...


There are several items to be addressed here in the chemistry. The first is how is psilocybin potentially halucinogenic? This question is more about psilocin than psilocybin. The acidic stomach environment gives rise to rapid dephosphorylation (phosphate removed) of psilocybin and enzymes like alkaline phosphatases and other non-specific esterases dephosphorylate psilocybin in the intestines, kidneys, and the blood, in human metabolism.


The phosphate group on psilocybin is highly polar, that is the unequal sharing of electrons in the bonds creates charge separation and a dipole moment. The amine group at the other side of the molecule exhibits a strong attraction for an additional hydrogen atom making this area positively charged. This polarity along with the positively charged amine group creates a zwitterion and so psilocybin is more soluble in water than psilocin. Without a phosphate group, psilocin is far more lipid soluble than psilocybin. This makes psilocin metabolically available in the body and more easily absorbed in the intestines. As such, psilocin is distributed all over the body via the circulatory system. Being lipid soluble allows psilocin to cross the blood-brain barrier. Thus psilocin is indeed the psychedlic active drug and psilocybin only the precursor drug.


I will not dwell on the discussion about how psilocin mimics serotonin, a monoamine neurotransmitter. This piece of the picture is filled in nicely from many sources found widely in Pharmaceutical and Toxicological Handbooks and is published on the internet in much greater detail. Needless to say this is where the anticipated psychedelic and emotional behaviour arises from, the expected "Magic". See my Facebook blog for some of these details.


The next question is how does Psilocybin form in this fungus and what does the fungus use it for?


While L-Tryptophan is an essential amino acid in humans it is not so for fungi. Fungi can manufacture L-Tryptophan. It is the action of four main enzymes on the various intermediates constructed from L-Tryptophan that produces Psilocybin. This process has been for the most part resolved in the fungus Psilocybe cubensis. However, there is key piece of information that permits an extrapolation to Gymnopilus aeruginosus, discussed soon.


The names given to the the four enzymes that create Psilocybin from L-Tryptophan were actually derived from the names of genes that code for them, in the fungal genome :


PsiD – L-tryptophan decarboxylase (EC 4.1.1.105)

PsiK – 4-hydroxytryptamine kinase (EC 2.7.1.222)

PsiM – Psilocybin synthase (EC 2.1.1.345)

PsiH – Tryptamine-4-monooxygenase {a cytochrome P450 class enzyme} (EC 1.14.99.59)


INTERNATIONAL UNION OF BIOCHEMISTRY AND MOLECULAR BIOLOGY (IUBMB) Enzyme Catalogue denoted as (EC ...)

See "https://www.qmul.ac.uk/sbcs/iubmb/".


The interesting fact to note is that in many of the other genera of psilocybin producing fungi, like Gymnopilus, a similar locus in the genome can be found that has four genes with similar information that code for four enzymes that will produce psilocybin analogously to that of P. cubensis. That would seem bizarre to say the least ... However, if it were the case that this gene cluster could be transported from one species to another or across genera then you may anticipate something like Horizontal Gene Transfer. Indeed, some researchers are looking into horizontal gene cluster transfer as a means for other non-psilocybe genera having a similar mechanism to produce psilocybin. The question needing to be addressed is a mechanism that is plausible for the gene cluster transfer, since bacteria or viruses would seem to be the candidates of choice. Might the appropriate bacterium or virus be transported by a vector that could be a fly, noting that in the second study I have depicted fly larvae found in the decaying basidiocarp of the fungus? Later this crops up again with the discussion of the Blue - Green staining. So the mechanism in this picture that provides for psilocybin from L-tryptophan is likely very similar for G. aeruginosus. This mechanism was discovered over a few years by Janis Fricke, Felix Blei, and Dirk Hoffmeister, of Friedrich Schiller University, Jena, Germany. They identified and characterized the four enzymes that the Psilocybes use to make psilocybin. The team then developed the first enzymatic synthesis of the compound, setting the stage for its possible commercial production, in 2017.

What then does Psilocybin do for the fungus and why is it created? This question still remains a mystery. Some have hypothesised insecticidal advantages, however the adult mushrooms can still be consumed by fly larvae. So would this be the case for embryonic and juvenile basidiocarps? This is an open research question.


The other question I'll address now is how the colours blue and green are related to the presence of Psilocybin?


At the end of 2019 into early 2020 a paper appeared by a group of scientists, "Injury‐Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms"

C. Lenz, J. Wick, D. Braga, M. García‐Altares, G. Lackner, C. Hertweck, M. Gressler and D. Hoffmeister, working in Jena, Germany. This paper described another locus

in the genome of Psilocybe cubensis that had two genes that coded for two enzymes, one was a phosphatase and the other a multi-copper oxidase (laccase). It was also shown that Gymnopilus and other genera had similar loci with similar genes for their enzymes. In other words, this replicated what has just been seen in in the previous description for psilocybin production from L-Tryptophan. This set of genes would best be described as a means for psilocybin degradation, in a rather odd fashion!


To be brief, the phosphatase (PHAP - as seen in the lower left of the picture) takes the phosphate group off of psilocybin and creates psilocin. Now comes the interesting part. The multi-copper oxidase (laccase)(as seen bottom right in the picture) has three coordination domains for ligand and hydrogen bonding. At the Type I site the psilocin is oxidised and one electron is stripped leaving the psilocyl radical. (In fact, this is high through-put catalyis. Four electrons are needed to enable a reverse step to happen that will return the laccase back to its initial state.) Not one but four serial oxidations are done with four psilocyl radicals produced ... The copper ions move from the 2+ valence state to the + valence state. Oxygen is then used to combine with the copper ions in the Type II coordination domain and its reduction begins. Electrons move through the backbone of rings and bonds to switch copper domains. The copper ions move from the + valence state back to the 2+ valence state and the output is water from the oxygen reduction. The enzyme is now returned to its inital state. That is the simple picture of what the enzyme does. However, note that the Type I copper binding site may have psilocin look alikes bind and get oxidised. This becomes important for oligomerisation as more complex molecules consisting of monomers of psilocyl radicals possibly also undergo this laccase oxidation and form large radicals, a cascade style of reaction.


From the inital production of psilocyl radicals under various acidity conditions dimers form from two psilocyl radicals, by direct radical coupling. These dimers can be colourless or blue. As the process continues the quinoid dimers, trimers, and tetramers, turn out to be blue, whilst the hydroquinoid dimers, trimers, and tetramers, turn out to be colourless. The hybrid polymers vary - either colourless or blue. This reaction is the source of the injury / bruising colour change in the presence of aeration (oxygen) and the laccase.


Without the laccase, under ordinary oxidation the psilocyl radicals still form, albeit slowly. They undergo a slightly different mechanism, nucleophilic addition and direct radical coupling, become dimers and the Hydroquinoid dimer is brownish, the Oxoquinoid and Hydroxyquinoid dimers are greenish to brown. This reaction is the source of the natural bruising colour change in the presence of aeration (oxygen), the greenish tints. At the left-centre bottom of the picture underneath the text is one of the greenish hydroxyquinoid dimers.


In reality often a mix of natural oxidation and enzymatic oxidation occurs and so greens, blues, and browns, will appear with injury or aging in the basidiocarps.

    ©2018 by WildCreaturesHongKong.