10.1002/chem.201801047
Chemistry - A European Journal
COMMUNICATION
Sigma-Aldrich, Roth, TCI, and VWR. Alkaloids 2, 11, and 16 were
purified from P. cubensis carpophores.
Contrasting the use of the trpB gene in fungal genetics as a
standard selection marker in 1-auxotrophic hosts, very few
Detailed experimental procedures for microbiological, and genetic
methods, as well as for purification of enzymes and compound 10 are
described in the Supporting Information.
fungal
tryptophan
synthases
were
investigated
biochemically.[9a,13] P. cubensis TrpB is the first biochemically
characterized tryptophan synthase of the basidiomycetes, which
represent a phylum of more than 30,000 species. The intrinsic
tolerance of TrpB to substituted indoles allowed for its
integration into a biocatalytic process that produces 2 congeners,
including 10 and 12, and that translates into a facile and more
cost-effective enzymatic synthesis of 2. The value of the
substrate 4, used in the previous procedure, is approximately
US$ 180/mmol, whereas the combined sales price for 1 mmol
(each) of 6 and 7 as starting material in our refined process is
about US$ 2.
Acknowledgments
We thank A. Perner and H. Heinecke (Hans-Knöll-Institute Jena)
for recording high-resolution mass and NMR spectra,
respectively. F. Ba. acknowledges a doctoral fellowship by the
Excellence Graduate School Jena School for Microbial
Communication (JSMC). This work was supported by the
Deutsche Forschungsgemeinschaft (grant HO2515/7-1). D.H.‘s
group is also supported by the DFG Collaborative Research
Center ChemBioSys 1127.
Directed in vitro evolution and structure-based engineering of
genes encoding biosynthesis enzymes proved instrumental to
optimize catalytic activity, introduce new activities, relax
specificities, to create libraries of natural product derivatives.[14]
Thus, future work on enzyme engineering, in particular on the
strictly specific methyltransferase PsiM, is warranted to access a
larger structural diversity of 2 derivatives using only a small set
of enzymes.
Keywords: biosynthesis • enzymes • psilocybin • alkaloid •
tryptophan synthase
[1]
[2]
[3]
a) A. Hofmann, R. Heim, A. Brack, H. Kobel, A. Frey, H. Ott, T.
Petrzilka, F. Troxler, Helv. Chim. Acta 1959, 42, 1557-1572; b) A.Y.
Leung, A.H. Smith, A.G. Paul, J. Pharm. Sci. 1965, 54, 1576-1579.
a) D.E. Nichols, Pharmacol. Ther. 2004, 101, 131-181; b) F. Hasler, U.
Grimberg, M.A. Benz, T. Huber, F.X. Vollenweider, Psychopharmacol.
2004, 172, 145-156.
Experimental Section
a) C.S. Grob, A.L. Danforth, G.S. Chopra, M. Hagerty, C.R. McKay, A.L.
Halberstadt, G.R. Greer, Arch. Gen. Psychiat. 2011, 68, 71-78; b) M.W.
Johnson, A. Garcia-Romeu, M.P. Cosimano, R.R. Griffiths, J.
Psychopharmacol. 2014, 28, 983-992; c) A. Mahapatra, R. Gupta, Ther.
Adv. Psychopharmacol. 2017, 7, 54-56.
Enzymatic reactions: All enzymatic in vitro reactions were carried out in
triplicate. The enzyme concentration was 200 nM. In the case of multi-
enzyme assays, each enzyme was present at this concentration. The
reactions were stopped after the indicated incubation times by freezing
and lyophilization. Subsequently, the residue was dissolved in methanol
(MeOH), centrifuged for 10 min at 20,000 g, and the supernatant was
collected. The solvent was removed under reduced pressure, and the
residue dissolved in H2O:acetonitrile (9:1, v/v), filtered, and used for
chromatography (see below). The in vitro characterization of TrpB was
[4]
a) A. Hofmann, A. Frey, H. Ott, T. Petrzilka, F. Troxler, Experientia
1958, 15, 397-399; b) F. Troxler, F. Seemann, A. Hofmann, Helv. Chim.
Acta 1959, 42, 2073-2103; c) S. Ametamey, F. X. Vollenweider, J. Patt,
D. Bourquin, F. Hasler, H.-F. Beer, P. A. Schubiger, J. Label. Compd.
Radiopharm. 1998, 41, 585-594; d) F. Yamada, M. Tamura, M. Somei,
Heterocycles 1998, 49, 451; e) H. Sakagami, K. Ogasawara,
Heterocycles 1999, 51, 1131-1135; f) D.E. Nichols, S. Frescas,
Synthesis 1999, 935-938; g) O. Shirota, W. Hakamata, Y. Goda, J. Nat.
Prod. 2003, 66, 885-887; h) N. Gathergood, P.J. Scammells, Org. Lett.
2003, 5, 921-923.
performed in a volume of 100 µL and 50 mM Tris-HCl buffer, pH=8.0 for 5
min at 30°C (varied between 16-42°C to determine the temperature
optimum, and between pH=6.0 through 9.5 for the pH optimum). The
substrates (6, and the respective indole substrate 5, 7, 8, 9, or 13) were
added at (each) 3 m
reactions including TrpB, PsiD, PsiK, and PsiM were set up in a volume
of 500 µL in 50 m Tris-HCl buffer, pH=8.0, and proceeded for 4 h at
25°C. The reactions yielding 2, 10, or 12 included 6 and indole substrates
7, 8, or 9 (3 m final concentration each) as well as SAM, ATP, and
MgCl2 (6 m each), the PLP concentration was 1 µ . Initially, all four
enzymes were added. For subsequent assays yielding 12, PsiK, PsiM,
ATP, and SAM was omitted. The yield of 2 (20.7%) was determined by
calculating the area under the curve of its chromatographic signal and
compared to a standard curve, recorded with authentic compound.
M, the PLP concentration was 1 µM. Multi-enzyme
[5]
[6]
J. Fricke, F. Blei, D. Hoffmeister, Angew. Chem. Intl. Ed. 2017, 56,
12352-12355.
M
a) E.W. Miles, H. Kawasaki, S.A. Ahmed, H. Morita, H. Morita, S.
Nagata, J. Biol. Chem. 1989, 264, 6280-6287; b) M.F. Dunn, D. Niks, H.
Ngo, T.R. Barends, I. Schlichting, Trends Biochem. Sci. 2008, 33, 254-
264.
M
M
M
[7]
a) R.J. Goss, P.L. Newill, Chem. Commun. 2006, 47, 4924-4925; b)
D.R. Smith, T. Willemse, D.S. Gkotsi, W. Schepens, B.U. Maes, S.
Ballet, R.J. Goss, Org. Lett. 2014, 16, 2622-2625; c) D. Francis, M.
Winn, J. Latham, M.F. Greaney, J. Micklefield, ChemBioChem 2017, 18,
382-386; d) M. Herger, P. van Roye, D.K. Romney, S. Brinkmann-Chen,
A.R. Buller, F.H. Arnold, J. Am. Chem. Soc. 2016, 138, 8388-8391; e)
A.R. Buller, S. Brinkmann-Chen, D.K. Romney, M. Herger, J. Murciano-
Calles, F.H. Arnold, Proc. Natl. Acad. Sci. USA 2015, 112, 14599-
14604.
Chemical analysis: LC-MS experiments were performed on an Agilent
1260 HPLC system equipped with a C18 column (Zorbax Eclipse XDB,
150 × 4.6 mm, 5 μm) and coupled to a 6130 Single Quadrupole mass
detector, high-resolution mass spectrometry was performed on a Thermo
Accela liquid chromatograph, following described parameters.[5] The 1D
and 2D NMR spectra of 10 and 2 were recorded at 300 K on a Bruker
Avance III spectrometer at 600 MHz for 1H and at 150 MHz for 13C
spectra. DMSO-d6 was used as solvent and internal standard. The
solvent signals were referenced to δH 2.50 ppm and δC 39.5 ppm.
Chemicals and solvents were purchased from Deutero, Key Organics,
[8]
[9]
D.K. Romney, J. Murciano-Calles, J.E. Wehrmüller, F.H. Arnold, J. Am.
Chem. Soc. 2017, 139, 10769-10776.
a) W.H. Matchett, J.A. DeMoss, J. Biol. Chem. 1975, 250, 2941-2946;
b) C.J. Bailey, P.D. Turner, Biochem. J. 1983, 209, 151-157.
This article is protected by copyright. All rights reserved.