Facile in Vitro Biocatalytic Production of Diverse Tryptamines

Article abstract of DOI:10.1002/cbic.201900069

Tryptamines are a medicinally important class of small molecules that serve as precursors to more complex, clinically used indole alkaloid natural products. Typically, tryptamine analogues are prepared from indoles through multistep synthetic routes. In the natural world, the desirable tryptamine synthon is produced in a single step by l-tryptophan decarboxylases (TDCs). However, no TDCs are known to combine high activity and substrate promiscuity, which might enable a practical biocatalytic route to tryptamine analogues. We have now identified the TDC from Ruminococcus gnavus as the first highly active and promiscuous member of this enzyme family. RgnTDC performs up to 96 000 turnovers and readily accommodates tryptophan analogues with substituents at the 4, 5, 6, and 7 positions, as well as alternative heterocycles, thus enabling the facile biocatalytic synthesis of >20 tryptamine analogues. We demonstrate the utility of this enzyme in a two-step biocatalytic sequence with an engineered tryptophan synthase to afford an efficient, cost-effective route to tryptamines from commercially available indole starting materials.

Full text of DOI:10.1002/cbic.201900069

Accepted Article
Title: Facile in vitro biocatalytic production of diverse tryptamines
Authors: Allwin McDonald, Lydia Perkins, and Andrew Richard Buller
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ChemBioChem
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COMMUNICATION
Facile in vitro biocatalytic production of diverse tryptamines
[
a]
[a]
[a]
Allwin D. McDonald , Lydia J. Perkins , Prof. Andrew R. Buller*
Tryptamines are a medicinally-important class of small molecules and
serve as precursors to more complex, clinically-used indole alkaloid
natural products. Typically, tryptamine analogs are prepared from
indoles through multi-step synthetic routes. In the natural world, the
desirable tryptamine synthon is produced in a single step by L-
tryptophan decarboxylases (TDCs). However, no TDCs are known to
combine high activity and substrate promiscuity, which might enable
a practical biocatalytic route to tryptamine analogs. Here, we identify
the TDC from Ruminococcus gnavus as the first highly active and
promiscuous member of this enzyme family. RgnTDC performs up to
96,000 turnovers and readily accommodates tryptophan analogs with
substituents at the 4, 5, 6, and 7 positions, as well as alternative
heterocycles, enabling the facile biocatalytic synthesis of > 20
tryptamine analogs. We demonstrate the utility of this enzyme in a
two-step biocatalytic sequence with an engineered tryptophan
synthase to afford an efficient, cost-effective route to tryptamines from
commercially-available indole starting materials.
Tryptamines are a prominent class of natural products that
include potent bioactive compounds such as the neurotransmitter
serotonin and psilocybin, precursor to the active component of the
hallucinogenic Psilocybe mushrooms (Figure 1).[1,2] A common
motif among alkaloid natural products is modification of the indole
ring, which can have a profound impact on the molecule’s
pharmacological properties. Similarly, several clinically-used
drugs are tryptamine derivatives, such as rizatriptan, and are
made through organic synthesis.[3] In Nature, formation of
tryptamine also serves as the starting point in the biosynthesis of
more sophisticated natural products, such as vincristine and
camptothecin.[3] Given the importance of tryptamines, this class of
Figure 1. a) Important tryptamine derivatives found in nature and in major
pharmaceuticals. b) Example of a reductive alkylation route to synthesize
tryptamines from indoles c) Nature’s route to tryptamine analogs imparts
chemical diversity on tryptophan with specialized tailoring enzymes, which are
subsequently decarboxylated d) The approach demonstrated here leverages
the abundant chemical diversity of commercially-available indoles and a highly
promiscuous tryptophan synthase and tryptophan decarboxylase.
compounds has been
a
frequent target for practical
demonstrations of synthetic methodology, many of which begin
with a substituted indole as the starting material. Classic synthetic
approaches include reductive alkylation[4,5] or, more recently, C-H
activation methodology.[6,7] While these traditional routes have
proven quite successful, it is also desirable to develop enzymatic
approaches, as they often better conform to the principles of
green chemistry and have the potential to be coupled with cellular
metabolism.[8,9] To date, biocatalytic methodology to produce
substituted tryptamines has lagged far behind traditional organic
synthesis.
Nature’s strategy to make modified tryptamines often starts
with L-tryptophan (Trp) and a tailoring enzyme that performs a
specific modification of the indole ring. The Trp derivatives are
subsequently decarboxylated and the resultant tryptamines
metabolized further into fully elaborated natural products.[ This
approach can be exploited synthetically to produce a few
tryptamine analogs, but the generation of further chemical
diversity quickly becomes cumbersome, as a separate enzyme is
required for each modification.[10] For example, the genetic
incorporation of Trp halogenases with distinct regiospecificities
into the medicinal plant Catharanthus roseus afforded access to
the halogenated alkaloids.[11] However these efforts were
hindered by the relatively poor efficiency of the Catharanthus
roseus TDC (CroTDC) with non-natural substrates. Indeed, most
enzymes that decarboxylate Trp are polyspecific for other,
smaller aromatic amino acids and are sluggish with non-standard
amino acids that are larger than Trp.[12] Moreover, there are many
desirable functional groups, such as fluoroindoles, that cannot be
made through any currently known enzymatic route.
1]
[
a]
Allwin D. McDonald, Lydia J. Perkins, Andrew R. Buller
Department of Chemistry
University of Wisconsin – Madison
Madison, Wisconsin 53705, USA
E-mail: arbuller@wisc.edu
Supporting information for this article is given via a link at the end of
the document.
An alternative biocatalytic approach to produce modified
tryptamines is to tap into the outstanding chemical diversity that
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is already commercially available for indoles. That is, rather than
relying on highly specific tailoring enzymes to introduce chemical
diversity, pre-modified indole building blocks can be converted
into their corresponding Trp analog through the promiscuous
action of a Trp synthase enzyme.[13–15] Natively, Trp synthase
uses the pyridoxal phosphate (PLP) cofactor to condense L-serine
We measured the progress of the native RgnTDC reaction in
vitro, which showed an impressive 20,000 turnovers in 4 h (Figure
2; see Figure S1 for a proposed mechanism). The total turnover
number (TTN) with Trp was improved 4-fold with 10 additional
equivalents of the PLP cofactor, up to ~80,000 turnovers in 4 h
(Figure S2). We hypothesize that over long time periods there is
some infrequent competing process that damages the cofactor.
This degradation process can be mitigated with the addition of
extra PLP, which swaps out the damaged cofactor and enables
catalysis to continue. In our hands, RgnTDC operated with a
catalytic efficiency (kcat/K ) of 6,900 M sec , ~7-fold lower than
M
reported previously, but with good agreement for the reported K
M
of 1.6 mM.[26] Serendipitously, this catalytic efficiency is also
similar to that observed for CroTDC, enabling direct comparison.
CroTDC has a much lower K with Trp (52 µM). In contrast, the
M
catalytic efficiency of RgnTDC is driven by kcat, which is ~130
times higher than that of CroTDC, accounting for the high TTN of
the R. gnavus enzyme.
Previously characterized TDC’s have had poor activity on
substrates that bear substitutions on the indole ring and are
therefore only of practical use with amino acids that are the size
of Trp or smaller.[27,28] The kinetic parameters of CroTDC with two
chlorotryptophans have been measured, which revealed that the
relatively poor efficiency of this reaction limits in vivo production
of chlorinated indole alkaloids in C. roseus (Table 1).[11,29]
(
Ser) and indole into Trp. Many substituted indoles and even other
amino acids can participate in this versatile reaction, affording
access to complex starting materials for downstream
modification.[16–19] Recently, these efforts have been made even
easier by evolving the β-subunit of tryptophan synthase (TrpB)
-1
-1
[
20–24]
(
EC 4.2.1.20) for efficient stand-alone function.
These
engineered TrpBs can operate at high temperatures, which is
helpful in solubilizing hydrophobic indoles, and provide access to
highly complex Trp analogs in good yield. Theoretically, these
diverse tryptophans could be converted to the analogous
tryptamines through the action of a promiscuous tryptophan
decarboxylase. Recently, the decarboxylase PsiD from the
psilocybin biosynthetic pathway was shown to act on both its
native substrate, 4-hydroxytryptophan, as well as two additional
regioisomers that were prepared using a TrpB enzyme.[25] These
enzymes were combined in a short and efficient cascade, but the
scalability and generality of this route for producing tryptamines
with alternative functionalities is not yet known.
Here, we report comprehensive characterization of the Trp
decarboxylase from the gut microbe Ruminococcus gnavus
(
RgnTDC) (EC 4.1.1.28) as the first broadly promiscuous member
Table 1. Comparison of kinetic parameters for Ruminococcus gnavus
tryptophan decarboxylase (RgnTDC) and Catharanthus roseus TDC (CroTDC)
on substituted tryptophans.
of its enzyme family. This enzyme was previously shown to be
specific for standard Trp over other aromatic amino acids and an
X-ray crystal structure with a substrate analog was determined,
revealing the indole binding pocket.[26] Structural analysis
suggested that this region of the active site could accommodate
substitution on the indole ring and therefore function on a range
of substrates.
To begin testing this hypothesis, we overexpressed RgnTDC in
E. coli BL21 cells and observed that cell cultures grew to
unusually low cell density in rich media, ~ 4 g per L culture. We
suspected RgnTDC was exerting a toxic effect by depleting cells
of Trp, and so exogenous indole (0.5 mM) was added at the time
of induction. This procedure significantly increased cell densities
to ~11 g per L culture, presumably by supplementing the native E.
coli Trp synthase enzyme with indole. As isolated, RgnTDC
copurified with covalently bound PLP and was reliably procured
in large quantities with yields of 200 mg purified enzyme per L
culture.
Fold-decrease
in activity
relative to Trp
k
cat
-1
K
(mM)
M
kcat/K
M
(M s )
Enzyme
Substrate
-1 -1
(s )
L-Trp
0.085
0.027
0.042
11
0.052
0.499
0.538
1.6
4560
55
-
CroTDC[a]
5-Cl-Trp
28
20
-
7
-Cl-Trp
L-Trp
77
6900
1700
4
-Cl-Trp
2.4
1.5
4.1
RgnTDC[b]
5-Cl-Trp
0.64
0.52
10
1.1
0.14
8.7
600
3600
1200
12
1.9
5.8
6
-Cl-Trp
-Cl-Trp
7
[
a] Values taken from Ref. 11. [b] Reactions conducted with 0.1 – 1.25 μM
RgnTDC in 50 mM potassium phosphate pH 8.0. Initial velocity data were
measured in at least duplicate and fit to the Michaelis-Menten equation.
Standard errors from a non-linear fit are reported in Table S1 and were typically
less than 25%.
To test the promiscuity of the RgnTDC, we sought to measure
its kinetic parameters with a complete set of 4-, 5-, 6-, and 7-
chlorotryptophans. The requisite enantiopure chlorotryptophans,
however, were not readily available. We therefore prepared each
Trp analog from their corresponding indole precursor through a
simple biocatalytic reaction using engineered TrpBs enzymes
from Pyrococcus furiosus with high stand-alone activity.[
Figure 2. Progress curve for the reaction of RgnTDC with tryptophan. Native
RgnTDC copurifies with its cofactor and 10 additional equivalents of PLP impart
a 4-fold boost in activity. Standard deviation is shown and, in many cases, is too
small to visualize.
20–24]
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Surprisingly, the kinetic parameters of RgnTDC with each
chlorotryptophan showed only modest decreases in catalytic
efficiency when compared to the native Trp substrate (Table 1,
histidine. In addition, no activity was observed with D-tryptophan.
Amino acids bearing indole isosteres were also prepared with
PfTrpB2B9 (Figure 3b). Indazole and indoline react with TrpB at N1
to yield 2-azaisotryptophan and 2,3-dihydro-isotryptophan,
respectively, and with which RgnTDC has modest activity. In
M
Figure S3). The K for the chlorotryptophans ranged from 5-fold
higher to 11-fold lower, whereas the kcat values were either similar
or lower compared to Trp. The overall effect is a smaller drop in
activity on substituted tryptophans when compared to CroTDC,
indicating that RgnTDC possesses relatively high native
promiscuous activity. More importantly, for synthetic purposes,
contrast, 7-aza-tryptophan is
a highly reactive substrate,
consistent with its steric profile being nearly identical to the native
Trp substrate.
M
we reason that high K values of RgnTDC are less indicative of
activity than kcat, since the enzyme can be kinetically saturated in
vitro simply by adding more substrate. Indeed, we measured
TTNs for each chlorotryptophan and observed an excellent
correlation between the kcat and the TTN for each substrate
(
Figure S4). In this light, RgnTDC is a particularly attractive
catalyst as it maintains high cat with each of the
a
k
chlorotryptophan substrates and performs thousands of turnovers
with four substrates that make distinct steric demands of the
active site.
We prepared a panel of substituted tryptophans using the
PfTrpB enzymes, relying heavily on the enzyme, PfTrpB2B9 (see
SI Methods for details).[21] This engineered TrpB variant is highly
promiscuous and thus acts as a ‘generalist’ catalyst for production
of tryptophan analogs.[21] Engineering and detailed study of this
engineered catalyst has been reported and the overall
engineering efforts for TrpB enzymes were recently reviewed.
[
21,30,31]
The collection of tryptophans encompasses a range of
sterically and electronically diverse substituents for each of the
−7 positions. Many of these Trp analogs have poor aqueous
4
solubility at high (0.1 M) concentrations, so reactions were carried
out with 10 mM substrate in aqueous phosphate buffer with 5%
methanol as a cosolvent. Generally, RgnTDC had the highest
activity with the 7-substituted tryptophans, consistent with the
enzyme’s high kcat with 7-chlorotryptophan (Table 1, Figure 3a).
The 5- and 6-substituted tryptophans were less well
accommodated by the enzyme. We analyzed the TTN with each
substrate as a function of the group van der Waals radius of the
substituent, which revealed the relative steric sensitivity of
RgnTDC to substitution at each position (Figure S5). RgnTDC
was most sensitive to steric perturbation at the 4-position (Figure
Figure 3. Total turnover numbers (log scale) for enzymatic decarboxylation of
tryptophan analogs by RgnTDC over 4 hours. a) Substrates with substituents at
indole ring positions 4–7. b) Activity on more structurally diverse substrates.
4). For the largest 4-substituent we tested, 4-cyanotryptophan,
only trace product formation (< 10 turnovers) was observed.
Satisfyingly, when we measured activity on a series of 4-
halotryptophans we noticed an order of magnitude increase in
activity as the size of the halide substituent decreases, up to
We sought to rationalize the structural basis for the reactivity
profile of RgnTDC using information from the previously-
determined structure with a suicide-inhibitor bound in the active
site (PDB ID: 4OBV).[ Using these data, we modelled the
covalent Trp-PLP external aldimine adduct that forms prior to
decarboxylation and whose geometry with the carboxylate
26]
~96,000 TTN with 4-fluorotryptophan (Figure 3a).
In contrast to substitution elsewhere on the ring, a methyl
substitution at the 2-position was poorly tolerated and RgnTDC
performed just 275 turnovers in 4 h (Figure 3b). When a methyl
group is added to the β-position of Trp, no reactivity was observed. through Dunathan’s stereoelectronic hypothesis.[ Addition of a
oriented perpendicular to the PLP ring can be readily inferred
32]
However, we assayed RgnTDC with a di-substituted Trp analog
and were pleased to observe formation of product corresponding
to 900 turnovers with 5,6-dichlorotryptophan as a substrate, which
chloro group to the 4-position of Trp clearly leads to a steric clash
with the carboxylate (Figure 4b). Given the steep drop in activity
with progressively larger substituents, the complex appears to
is lower than the activity on either of the mono-substituted analogs. distort away from an optimally reactive orientation to relieve this
Native TDC enzymes are often regarded as highly specific
catalysts and the broad promiscuity of RgnTDC was therefore
quite surprising. We sought to probe the substrate scope further,
testing for activity with substrates that are more structurally
distinct from Trp. In line with previous studies, RgnTDC shows
steric clash (Figure 4a). Bulky side chain residues in the RgnTDC
active site similarly appear to limit the size of the groups available
at the 5- and 6-positions and cause some structural
rearrangement of the active site to facilitate catalysis (Figure S5).
There is a noticeable sparsity of residues flanking the 7-position
of the indole ring, which is consistent with RgnTDC’s prodigious
ability to produce 7-substituted tryptamines.
[
26]
activity on L-tyrosine, but with 50-fold lower activity relative to
Trp. However, there is no observable decarboxylation of L-
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procedures detailed here do not require significant training in
organic synthesis or specialist equipment that is not already found
in most biochemistry labs. Hence, these enzymatic tools will
enable other biochemists to synthesize sophisticated analogs of
metabolic intermediates and spur new synthetic biology efforts to
engineer biosynthesis.
Figure 4. Effect of steric interactions on RgnTDC activity with 4-substituted
tryptophans. a) Plot showing the exponential correlation between RgnTDC’s
total turnover number and the substituent group van der Waals radius.[33] b)
Active site model of 4-chlorotryptophan depicts a steric clash when the
carboxylate is oriented in a reactive conformation.
This study of RgnTDC required synthesis of substituted Trp
substrates that were themselves produced in a biocatalytic
reaction. Naturally, we considered whether RgnTDC could be
combined with a Trp synthase, PfTrpB2B9, into an efficient two-
enzyme sequence to produce tryptamines from the corresponding
indole and serine. Although both enzymes have high activity on a
range of substrates, they operate at different optimal
Figure 5. One-pot sequential enzymatic reaction to produce tryptamine analogs
from L-serine (30 mM) and commercially available indole building blocks (10
mM). Enzyme concentrations were 0.4—4 μM for the engineered Trp synthase
o
temperatures (37 vs 75 C), which presents an inherent challenge.
b9
(
PfTrpB2 ) and 2—10 μM for the Trp decarboxylase (RgnTDC). One mmol
o
We first tested the enzymes in a cascade reaction at 37 C, which
scale reactions are shown with the total mg of product produced and the percent
isolated yield.
resulted in full conversion of indole to tryptamine (5000 turnovers,
Figure S6), demonstrating that the two enzymes can operate
efficiently under the same conditions. Although TrpB enzymes
have recently been engineered for higher activity at lower
Experimental Section
temperature,[
24]
the solubility of substituted indoles drops
precipitously at these temperatures, hindering preparative scale
reactions. Therefore, we adopted a sequential one-pot, two-
enzyme reaction setup. First, PfTrpB2B9 acts at 75 C on 0.01 M
substituted indole and three equivalents of Ser for 12 h. The
See Supporting Information for detailed experimental procedures.
o
Acknowledgements
o
reaction vessel was then cooled to 37 C and RgnTDC was added
along with 10 additional equivalents of PLP. Decarboxylation was
allowed to continue for several hours (times depend on the
substrate, see Supplemental Information for details) prior to
purification by a simple acid/base extraction procedure. We used
this two-enzyme sequence to produce six different tryptamines on
We thank David Romney and Christina Boville for samples of
substituted tryptophans. We acknowledge the invaluable support
and assistance from Jon Ellis, Prasanth Kumar, and other
members of the Buller Lab. Support for this research was
provided by the Office of the Vice Chancellor for Research and
Graduate Education at the University of Wisconsin – Madison with
funding from the Wisconsin Alumni Research Foundation. In
addition, we thank NIH Grant 1S10OD020022-1 for providing
funding for the Q Exactive Plus Orbitrap used for high resolution
mass spectrometry analysis of prepared compounds.
1.0 mmol scale with a range of yields (Figure 5). Isolated yield
tracked well with the efficiency of the analytical scale reactions.
While the one-pot reaction procedure was still sufficient for
substrates poorly tolerated by RgnTDC, these reactions suggest
that further improvements could be obtained through protein
engineering.
We have shown that the TDC previously isolated from R.
gnavus is the first broadly promiscuous enzyme for the in vitro
synthesis of tryptamines. Through kinetic experiments, we
determined that the catalytic efficiency of RgnTDC is driven by a
high kcat, which in turn governs reactivity of the catalyst in vitro.
We observed this enzyme performs up to 96,000 turnovers and
this high activity was leveraged to prepare > 20 different
tryptamine analogs. Hence, native RgnTDC breaks the mold of
both the generalist aromatic amino acid decarboxylases and the
specialist TDCs to provide access to valuable indole alkaloid
building blocks. Both the RgnTDC and the engineered TrpB
enzymes can be procured in high quantities (> 200 mg enzyme
per L culture) and combine to provide easy access to diverse
tryptamines from commercially abundant starting materials. In
contrast to previous methodology to produce tryptamines, the
Keywords: biocatalysis • tryptophan synthase • non-canonical
amino acids • indole alkaloid • decarboxylase
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ChemBioChem
10.1002/cbic.201900069
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Allwin D. McDonald, Lydia J. Perkins,
Andrew R. Buller*
Page No. – Page No.
Facile in vitro biocatalytic production
of diverse tryptamines
Diversifying the Tryptamine Product Line: Tryptamines are important synthons
in bioactive natural products, but general synthetic routes to tryptamine analogs are
limited. We present a highly active and promiscuous biosynthetic cascade using an
engineered tryptophan synthase (TrpB) and native tryptophan decarboxylase (TDC)
capable of producing a diverse set of tryptamines from readily accessible indole
starting materials.
For internal use, please do not delete. Submitted_Manuscript
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