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Internationale Ausgabe:DOI:10.1002/anie.201910175
Fungal Natural Products Very Important Paper Deutsche Ausgabe:DOI:10.1002/ange.201910175
Injury-Triggered Blueing Reactions of Psilocybe “Magic” Mushrooms
Claudius Lenz+,Jonas Wick+,Daniel Braga, Mar&aGarc&a-Altares,Gerald Lackner,
Christian Hertweck, Markus Gressler,and Dirk Hoffmeister*
Abstract: Upon injury,psychotropic psilocybin-producing
mushrooms instantly develop an intense blue color,the
chemical basis and mode of formation of which has remained
elusive.Wereport two enzymes from Psilocybe cubensis that
carry out atwo-step cascade to prepare psilocybin for oxidative
oligomerization that leads to blue products.The phosphatase
PsiP removes the 4-O-phosphate group to yield psilocin, while
PsiL oxidizes its 4-hydroxygroup.The PsiL reaction was
monitored by in situ 13CNMR spectroscopy, whichindicated
that oxidative coupling of psilocyl residues occurs primarily via
C-5. MS and IR spectroscopyindicated the formation of
aheterogeneous mixture of preferentially psilocyl 3- to 13-mers
and suggest multiple oligomerization routes,depending on
oxidative power and substrate concentration. The results also
imply that phosphate ester of psilocybin serves areversible
protective function.
Mushrooms from the genus Psilocybe accumulate psilocy-
bin (1,Scheme 1), which makes them strongly hallucino-
genic.[1] As arare reaction among natural product biosyn-
theses, 1assembly includes aphosphotransfer step,catalyzed
by the kinase PsiK.[2] Upon ingestion of the mushrooms,the
phosphate ester is cleaved to yield psilocin (2), the actual
psychotropic congener that profoundly alters human percep-
tion by binding agonistically to 5-hydroxytryptamine recep-
tors.[3] Ecologically,the presence of monomeric 1in the
fungus has been attributed to protection from mycophagous
insects by interfering with their behaviour.[4] Asecond very
prominent phenomenon is the blueing reaction of Psilocybe
mushrooms,reflected by species names such as P. cyanescens
or P. azurescens. Upon bruising or cutting, 1-containing
fruiting bodies instantaneously develop adark blue color at
the site of injury (Figure 1, Movie S1). Likewise, Psilocybe
specimens may turn blue as they age.Ablue coloration that
instantly develops upon injury is observed with other mush-
rooms as well. In the case of the dotted stem bolete[5] and the
cornflower mushroom, the blueing can be attributed to
pulvinic acid or diarylcyclopentenone derivatives.[6]
However,the structure of the chromophore and the
biochemical basis how this blue pigment is formed in
Psilocybe has remained elusive,although this conundrum
has attracted natural product chemistsQattention for de-
cades.[7,8] When oxidized chemically,for example,byferric
iron or base,[1c] or enzymatically, 2but not 1is converted to
ablue pigment.[7] It has been hypothesized that the chromo-
phore is most likely a 2-derived ortho-coupled biarylidene-
dione.[8] Furthermore,the existence of astable radical was
assumed.[7d,8] Precedence exists for enzymatic biaryl coupling
in fungal natural products,catalyzed by cytochrome P450 or
flavin-dependent monooxygenases,orlaccases whose selec-
tivity may be intrinsic or conferred by auxiliary proteins.[9]
Scheme 1. Chemical structures of psilocybin (1)and psilocin (2).
Figure 1. The blueing reaction of Psilocybe cubensis:intact (left) and
scalpel-injured mushroom (right).
[*] C. Lenz,[+] Dr.J.Wick,[+] Dr.M.Gressler,Prof. Dr.D.Hoffmeister
Department Pharmaceutical Microbiology,Hans-Knçll-Institute
Friedrich-Schiller-Universit-t
Beutenbergstr.11a, 07745 Jena (Germany)
E-mail:dirk.hoffmeister@leibniz-hki.de
Dr.D.Braga, Dr.G.Lackner
Synthetic Microbiology,Friedrich-Schiller-Universit-t, Leibniz Insti-
tute for Natural Product Research and Infection Biology –Hans-
Knçll-Institute, Winzerlaer Str.2,07745 Jena (Germany)
Dr.M.Garc&a-Altares, Prof. Dr.C.Hertweck
Department Biomolecular Chemistry, Leibniz
Institute for Natural Product Research and Infection Biology –
Hans-Knçll-Institute, Beutenbergstr.11a, 07745 Jena (Germany)
[++]These authors contributed equally to this work.
Supportinginformation and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201910175.
T2020 The Authors. Published by Wiley-VCH Verlag GmbH &Co.
KGaA. This is an open access article under the terms of the Creative
Commons AttributionNon-CommercialNoDerivs License, which
permitsuse and distribution in any medium, provided the original
work is properly cited, the use is non-commercial, and no
modifications or adaptations are made.
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Thepresence of reducing agents,such as sodium dithion-
ite,has been shown to discolor the blue matter,which is
reversible upon re-oxidation, thus supporting the notion of
aquinoid chromophore.[7c] In the presence of aphosphatase,
ablue color develops from 1as well.[10] Sequential 1degrada-
tion through dephosphorylation, oxidation, and subsequent
oligomerization would explain the rapid bluing reaction of
Psilocybe mushrooms.Inthis work, we address this long-
standing problem with acombined approach primarily based
on activity-guided chromatographic purification of aphos-
phatase (PsiP) and alaccase (PsiL) of P. cubensis that carry
out atwo-step degradative cascade to convert 1into 2,which
then undergoes oligomerization (Scheme 2). MALDI-MS
and LC-MS,IRspectroscopy,and in situ time-resolved
NMR spectroscopy provided insight in the oligomerization
modes.
We used P. cubensis carpophores to isolate native enzymes
by protein chromatography (Figure S1). After each chroma-
tographic step,invitro activity assays were performed.
Phosphatase activity was traced using 1as an authentic
substrate for the production of 2,and 4-nitrophenyl dihydro-
gen phosphate as achromogenic standard substrate.Todetect
oxidase activity, 2and syringaldazine were used in parallel as
substrates to yield photometrically detectable products.Cell
extracts were first subjected to anion-exchange chromatog-
raphy (AIEC). Then, positively assayed fractions were further
separated by hydrophobic interaction (HIC) and size-exclu-
sion chromatography (SEC).
Active SEC fractions were tryptically digested and sub-
jected to MS/MS-based peptide fingerprinting.Using the
deduced proteome of P. cubensis as asearch database,the
fraction containing the phosphatase activity revealed six
candidates (Table S1 in the Supporting Information). Con-
sistently,the highest-ranking protein (JGI ProteinID 89927)
was annotated as aphosphatase.Another candidate phos-
phatase (JGI ProteinID 74822) displayed amuch lower
intensity and alower identification score (Figure S2) and
could not be detected reproducibly.The first putative
phosphatase (PsiP) is amember of the histidine acid
phosphatase superfamily.The DNAsequence encoding PsiP
(552 aa, calculated mass of 59.5 kDa, Table S2) was localized
in the P. cubensis genome and was not associated with
1biosynthesis genes.Bioinformatic analysis pointed to
alysosomal localization of PsiP (Figure S3, Table S3). Of
note,other 1-producing mushrooms encode extremely similar
phosphatases (Table S4). LC-MS analyses and an authentic
standard demonstrated that PsiP dephosphorylates 1to 2.
Signals of identical mass,retention time (tR=2.15 min), and
UV/Vis spectra were observed (Figure 2). We therefore
describe PsiP as 1phosphatase.
Peptide mass fingerprinting of the fraction with oxidizing
activity identified seven candidate proteins (Table S5 and
Figure S4). One of the top scoring proteins was annotated as
amulticopper oxidase (JGI ProteinID 71514) according to
Pfam[11] and InterPro[12] domain analyses and was similar to
alaccase,that is,amulti-copper oxidases that catalyze
removal of one electron from the target substrate.[13] In
addition to displaying 10 high-intensity peptides with strong
MS/MS support, the putative laccase,now referred to as PsiL,
was the only identified protein with an annotation that
provides areasonable explanation for the expected radical
oxidation reaction. The psiL gene was identified but, like psiP,
it is not located in the 1biosynthesis gene cluster.The psiL
gene (Table S2) encodes a528 aa protein with acalculated
mass of 57.5 kDa that shows 63 %amino acid identity to
ayellow laccase of the mushroom Stropharia aeruginosa
(GenBank accession code:AFE48786.2).[14] PsiL is likely an
extracellular enzyme (Figure S3), and PsiL-like laccases (61–
71%identical aa) are encoded in all 1-producing fungi whose
genomes are available (Figure S5 and Table S6). Optimum
reaction parameters were determined both for PsiL and PsiP
(Figure S6).
Oxidizing activity on 2,but not on 1,was observed for
PsiL based on aphotometrically detected blue color
(Figure 2) which was absent in acontrol with heat-treated
enzyme.The activity of PsiL was comparable to Mycelioph-
thora thermophila laccase,yet three orders of magnitude
above a Trametes laccase (Figure S7, Table S7). We therefore
conclude that PsiP initiates and PsiL continues an enzymatic
pathway to convert 1into a 2-based polymeric colored
material.
PsiL-analogous model reactions for chemical (FeIII)or
enzymatic oxidation (M. thermophila laccase/O2or horse-
radish peroxidase (HRP)/H2O2)were set up to produce the
blue color and analyze 2oxidation. In both cases,LC-MS
traces showed decreased 2content and formation of the blue
color without noticeable qualitative or quantitative differ-
ences.When oxidized with FeIII,that is,inaprotein-free
environment, the blue compound was amenable to character-
ization by MALDI-MS and LC-MS.However,when oxidized
enzymatically,the majority of colored product was chromato-
graphically inseparable and adhered to proteins,aspreviously
reported.[7d] TheMALDI-TOF mass spectra revealed acom-
plex pattern of discrete signals representing multiples of m/z
202, that is,the mass-to-charge ratio of 2monomers less two
protons,thus indicating oligomerization up to 13-mers,
accompanied by aconsistent pattern of co-occurring ions
(Figure S8). Smaller oligomers were analyzed by LC-MS,
which verified dehydro- and primarily didehydro-coupling
Figure 2. Analysis of in vitro reactions. A) To ptrace (composite indi-
vidual chromatograms): authenticstandards of 1and 2.Trace a: PsiP
(SEC fraction)catalyzing 1dephosphorylation. Trace b: negative con-
trol with heat-denatured enzyme. B) Typical PsiL-dependent blueing
reaction with 2as the substrate, measured photometrically as absorb-
ance at l=618 nm. t=0reflects the first measurementofabsorption.
The time delay between substrate addition and first photometric
measurementisapprox. 30 s. The negative control was run with
aheat-treated HIC fraction containing laccase.
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products,and oxidized derivatives thereof (Figure S9). The
complexity of the product mixture was reduced by replacing
water with methanol as the solvent, and by diluting and
cooling. Then, aclear blue solution was obtained that
predominantly contained the didehydro dimer of 2(m/z
405), which shows absorptivity around 600 nm. Its leuco form,
acolorless 2dehydrodimer (m/z407), was reversibly gen-
erated by lowering the redox potential of the solution, for
example,byaddition of Na2S2O4or EDTA(Figure S10).
Concurrently,IRspectra of blue oligomeric and polymeric
product fractions were recorded (Figure S11). Thespectra
show similarities with those of eumelanin,[15] featuring strong
O@Hand N@Hstretching bands between 3,500 and
3,100 cm@1,and distinct signals in the range of conjugated
C=Oand C=Cbonds (1,610 and 1,641 cm@1).
While these features become less defined in the polymeric
fraction spectrum, absorptions of various C@H, C@O, C@C
and N@Hvibrations (3,100–2,700 cm@1and fingerprint region)
increased, probably due to both higher structural inhomoge-
neity and degree of polymerization, as shown for substrate
concentration variation in laccase-mediated coupling of 3-
methylcatechol.[16]
Thefinding of carbonyl functions and oligomeric,2H-
spaced mass clusters (e.g. m/z405–407 [M++H]+), combined
with reversible FeIII-mediated redox chemistry,suggests initial
formation of biindolidene quinonic products,asproposed
previously.[8] Subsequently,MSwas used to compare with the
enzymatic reaction. Adiluted laccase/O2oxidation of 2was
repeatedly and directly subjected to LC-MS (Figure 3, Fig-
ure S12). As the substrate 2was gradually consumed and the
blue color concurrently formed, dimeric 2(m/z405–407)
became less abundant over time while masses of higher
oligomers appeared.
We further characterized 2autoxidation by LC-MS,which
indicated oligomerization as well. However,inaqueous
buffers,the color ranged from greenish to taupe or brown
(Figure S10). Prominent product ions include m/z219 (2+
[O] @2[H]), 419 (dimeric 2+[O] @6[H]), and 621 (trimeric
2+[O] @8[H]). With higher concentrations of 2,increasing
amounts of ion m/z407, but not m/z405, were detected. For
all detected product species,the expected sum formulae were
confirmed by HRMS (Table S8). Themost prominent ones
were analyzed by MS/MS to verify the presence of intact
dimethylaminoethyl sidechains.The observed differences
between autoxidation and chemically boosted or enzyme-
mediated oxidation may be attributed to different mecha-
nisms and pathways (Figure S13), that both originate from
psilocylradicals.Boosted oxidations produce an excess of
radicals that enable direct radical coupling and subsequent
oxidation of hydroquinoid compounds,which would explain
the blue quinoid dimer (m/z405). Autoxidation yielded low
radical concentrations,where the average lifetime of the
psilocylradical may be insufficient to find asuitable radical
coupling partner.Inthis scenario,psilocylcations may be
formed,[17] which would favor reactions driven by nucleophilic
attacks to yield compounds such as m/z219, 419, and others.
At this stage,MShad provided valuable information
about general patterns of product formation. However,
product complexity,product binding to proteins,polymeri-
zation, and autoxidation prevented purification of the indi-
vidual products to homogeneity.Wethus resorted to in situ
NMR spectroscopy to monitor the progress of the enzymatic
reaction. Buffered 2solutions in D2O(5mgmL@1)were
oxidized enzymatically.For comparison, autoxidation of 2by
O2from an airstream supplied to an enzyme-free solution was
monitored as well. Forinsitu 1Hand 13CNMR spectroscopy,
reactions were run directly in the spectrometer,recording
1HNMR spectra every 2–3 minutes.Under autoxidation
conditions,the signal of H-5 decreased by about 70 %
within 4h.After 24 h, signal H-5 had virtually disappeared,
the overlapping signals of H-6 and H-7 had decreased by
about 30%, while all other protons were only marginally
affected (Figure S14). However, no substantial substrate
turnover within the same time was observed in LC-MS
control measurements.Thus,the selective signal decrease
must be based on an H/D-exchange with the solvent ortho
(and to alesser extent para) to the hydroxy functionality due
to tautomerism.
We therefore proceeded with inverse-gated decoupling
(igd) 13CNMR of 13C12-2to monitor the reaction. When HRP/
Figure 3. Chemical analysis of the blueing reaction. A) LC-MS-analysis
of laccase-mediated 2oxidation and concomitant formation and
decrease of reduced or quinoid psilocyl dimers (m/z405–407).
B) Carbon signal intensity during 2oligomerization, measured by in
situ 13CNMR spectroscopy.The positions are color-coded.“New”
refers to an emerging signal, likely C-6.
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H2O2was used for oxidation, ablue color developed instantly
after mixing. However,after approx. 30–60 min, the blue
color faded and gradually turned into alight brown (Fig-
ure S10). Signal changes in the igd-13CNMR spectra revealed
distinct differences between the individual carbons of 2
(Figures 3and Figure S15). Thesidechain carbons (Me,C-1’,
C-2’)were affected least while indole core carbon signals
markedly decreased. TheH-bound carbons of the six-
membered ring were most affected (C-6 >C-5 >C-7). Addi-
tionally,anew signal was observed (putative dd or tatd=
123.1 ppm). We conclude that the reaction primarily favors
couplings via C-5 and secondly at C-7. Since the newly closed
C@Cbonds via C-5 and C-7 affect the signal of C-6 as
expected, its strong decrease is in accordance with this
assumption. Additionally,the new signal is likely due to the
former 6-position with adjacently formed C@Cbonds at C-5
and C-7.[18]
With mass spectrometric evidence for oxidative coupling
of 2,IRspectra indicating C=Oand C=Cfunctionalities,and
NMR spectroscopy suggesting C-5 as apreferred coupling
position, our findings strongly support the hypothesis of 5-5’
coupled quinoid subunits being responsible for the blue color
of oxidation products of 2,[8] and, hence,wounded Psilocybe
mushrooms.The reported stable radical properties of the blue
product[7d] are consistent with our findings,since semiqui-
nones are the radical intermediates between quinones and
hydroquinones detected during our study.[19] Likewise,semi-
quinoids exist within eumelanins,abiological polymer that
shares,toadegree,structural features with polymeric 2.
However,nosingle blue compound exists in Psilocybe
mushrooms.Various oxidative pathways contribute,todiffer-
ent extents,tothe reaction. Each of them produces various
chemical (including isomeric) species.Similar phenomena are
known from the (aut)oxidation of phenolic and indolic
substances.[18,20, 21] Thesubstrate to oxidant ratio seems to
affect the mechanism and color (Scheme 2, Table 1).
Natural products,among them polyenes,cyanohydrins,
and numerous others,serve as defense compounds or
precursors that are formed after injury.[22] To this end,
nature has evolved strategies to remove aprotection group
from an accumulated stable precursor.This initiates forma-
tion of the ecologically relevant monomeric defensive com-
pound. Cyanide-releasing plant glycosides or mushroom
metabolites,such as amygdaline or aleurodisconitrile,[22a]
follow asimilar strategy to quickly provide awound-activated
chemical defense when needed. However, such reactions
require permanent physical separation of precursor and
enzyme(s) that catalyze the cascade to convert the precursor
into the actual defensive compound. Furthermore,they are
triggered only upon injury,but ensure rapid product forma-
tion. In the case of Psilocybe mushrooms,this requires spatial
separation of 1and PsiL/PsiP for self-protection and to
prevent polymerization and fatal protein crosslinking in intact
cells.The sequences of PsiL and PsiP suggest extracellular or
lysosomal localization, respectively (Table S3, Figure S3).
Even though 2is not the biosynthetic precursor of 1,the
kinase PsiK can rephosphorylate 2to 1.[2] Thus,PsiK adds
Scheme 2. The blueing reaction in P. cubensis,catalyzed by PsiP and
PsiL. Stabilized electron positions within the psilocyl radical are
highlighted in red. Numbering of compounds corresponds to Fig-
ure S13.
Table 1: Primary ion species detected by LC-MS, generated under various oxidation conditions(see also Table S8 and Figure S13). Structural isomers
may occur.
Type m/z
(tRin min)
Annotation Neutral sum formula Observedcolor Mechanism[a]
FeIII-orlaccase-
mediated oxidation[b]
405 (3.57) Quinoid dimer 6C24H28O2N4blue R
407 (3.38) Hydroquinoid dimer 5C24H30O2N4colorlessR
607 (4.09)[c] Quinoid trimer 8C36H42O3N6blue R
609 (3.80)[c] Hydroquinoid trimer 7C36H44O3N6colorlessR
807 (3.87)[c] Fully quinoid tetramer 11 C48H54O4N8blue R
809 (3.93)[c] Hybrid tetramer 10 C48H56O4N8blue/colorless R
811 (3.67)[c] Hydroquinoid tetramer 9C48H58O4N8colorlessR
Autoxidation
219 (3.53)[c] Quinoid psilocin 16 C12H14O2N2brownish N
407[d] (3.38) Hydroquinoid dimer 5C24H30O2N4brownish R
419[e] (3.89) Oxoquinoid dimer 19 C24H26O3N4greenish/brownish NorR+N
421[e] (3.28) Hydroxyquinoid dimer 18a,bC24H28O3N4greenish/brownish NorR+N
[a] Plausible mechanism:R=direct radical coupling (Figure S13 II), R +N=nucleophilic addition on former radical coupling products (Figure S13
IV), N =nucleophilic addition (Figure S13 V). [b] Excess of oxidant favors quinoid species, oxidant deficiency favors hydroquinoid species. [c] Isomers
detected, retention time indicates most intensivepeak. [d] Abundantwith higher 2concentrations (25 mm). [e] Abundantwith lower 2concentrations
(1 mm).
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another layer of self-protection through its capacity to
remove 2.Oligo- and polymeric 2consists of an extended
conjugated system and, in the reduced state,numerous
hydroxy groups that are oxidation-prone.Strikingly,these
structure elements are similar to those of anti-oxidative
compounds,such as flavonoids and polyphenolic tannins.[23] In
the basic and oxidative environment of insect guts,such
compounds act as producers of reactive oxygen species and
can thus create intestinal lesions,which is presumably their
main mode of toxic action.[24] Therefore,polymeric 2may
serve adefense function.
Our results shed light on the blueing reaction of Psilocybe
mushrooms.Weidentified alaccase and aphosphatase that
degrade 1and initiate blueing in P. cubensis. MALDI-MS and
in situ 13CNMR spectroscopy show that the blue color is due
to aheterogeneous mixture of quinoid psilocyl oligomers,
primarily coupled via C-5. Our results further suggest that
kinase-mediated phosphorylation during 1biosynthesis rever-
sibly deactivates areactive natural product, and stabilizes it
with an easily removable protection group that allows instant
on-demand formation of an oligomeric natural product.
Acknowledgements
We thank A. Pschibul, A. Perner, H. Heinecke and S.
Schieferdecker (Leibniz Institute for Natural Product
Research and Infection Biology,Hans-Knçll-Institute Jena)
for providing syringaldazine and M. thermophila laccase,and
for recording HRMS and NMR spectra, respectively.Wealso
thank J. Greßler (Friedrich-Schiller-University Jena) for
technical assistance.C.L. acknowledges adoctoral fellowship
by the International Leibniz Research School (ILRS) for
Microbial Interactions.G.L. and D. B. thank the Carl Zeiss
Foundation for funding.This work was supported by the
Deutsche Forschungsgemeinschaft (DFG,grant HO2515/7-
1). C.H. and D.H. are supported by the DFG Collaborative
Research Center 1127 (ChemBioSys).
Conflict of interest
Theauthors declare no conflict of interest.
Keywords: enzymes ·laccase ·natural products ·phosphatase ·
psilocybin
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Manuscript received:August 13, 2019
Acceptedmanuscript online: November 14, 2019
Version of record online: December 4, 2019
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