Engineering the Active Site of an (S)-Selective Amine Transaminase for Acceptance of Doubly Bulky Primary Amines

Article abstract of DOI:10.1002/adsc.201901252

A protein engineering approach for expanding the substrate scope of the (S)-selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl- and benzyl-substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2-diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure (R)-1,2-diphenylethylamine with ee>99% under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20-30% (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)-selective amine transaminase showing activity towards benzyl-substituted primary amines. (Figure presented.).

Full text of DOI:10.1002/adsc.201901252

Title: Engineering the Active Site of an (S)-Selective Amine
Transaminase for Acceptance of Doubly Bulky Primary Amines
Authors: Henrik Land, Federica Ruggieri, Anna Szekrenyi, Wolf-Dieter
Fessner, and Per Berglund
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.201901252
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10.1002/adsc.201901252
Advanced Synthesis & Catalysis
Very Important Publication - VIP
FULL PAPER
DOI: 10.1002/adsc.201901252
Engineering the Active Site of an (S)-Selective Amine
Transaminase for Acceptance of Doubly Bulky Primary
Amines
Henrik Land,a, b Federica Ruggieri,a Anna Szekrenyi,c Wolf-Dieter Fessnerc and
Per Berglunda*
a
KTH Royal Institute of Technology, School of Engineering Sciences in Chemistry, Biotechnology and
Health (CBH), Department of Industrial Biotechnology, AlbaNova University Center, SE-106 91,
Stockholm, Sweden
Tel: +46 8 790 7037
E-mail: perbe@kth.se
b
Uppsala University, Department of Chemistry-Ångström Laboratory, Molecular Biomimetics, Box 523, SE-
751 20, Uppsala, Sweden
c
Technische Universität Darmstadt, Institut für Organische Chemie und Biochemie, Alarich-Weiss-Str. 4,
64287 Darmstadt, Germany
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/adsc.201######.
Abstract. A protein engineering approach for expanding the The variant L59A/F88A generated enantiomerically pure
substrate scope of the (S)-selective Chromobacterium (R)-1,2-diphenylethylamine with ee >99 % under all tested
violaceum amine transaminase is presented. Amino acid conditions. The variant also holds great promise for synthesis
residues in the small binding pocket of the active site were of hydrophobic compounds as it shows optimum activity
targeted in order to increase the pocket size for acceptance of when 20 % (v/v) DMSO is applied as cosolvent. The variant
primary amines bearing two bulky groups. A highly sensitive L59A/F88A provides a great addition to the available
fluorescence assay was then used to evaluate the generated catalyst toolbox for synthesis of chiral amines, as it is the
enzyme variants for their activity towards propyl- and benzyl- first published (S)-selective amine transaminase showing
substituted screening substrates. The best variant, activity towards benzyl-substituted primary amines.
L59A/F88A, was successfully applied in the kinetic
Keywords: Biocatalysis; Aminotransferase; Protein
resolution of 1,2-diphenylethylamine using different
engineering; Kinetic resolution; Enzymes
conditions and substrate loadings.
One important class of enzymes in the toolbox for
chiral amine synthesis are amine transaminases (ATA)
which has proven to be useful catalysts both for kinetic
resolution of racemic amines and asymmetric
synthesis from prochiral ketones. ATAs especially
show remarkable enantioselectivity compared to many
chemical catalysts and both (S)- as well as (R)-
selective enzymes are available.[1, 5] The ATA active
site consists of two binding pockets of different sizes,
one large (L) and one small (S) pocket next to the
pyridoxal-5´-phosphate (PLP) cofactor.[6] The L
pocket accepts larger substrate moieties such as
aromatic rings or aliphatic chains. There are even
ATAs, both wild-type (WT) and engineered variants,
that accept bulky biaryl substituents in the L pocket.[7]
Introduction
Chiral amines represent an important motif in many
groups of fine chemicals, including pharmaceuticals[1]
and agrochemicals[2]. To ensure high purity of these
compounds, synthetic methods need to be very
selective towards the desired enantiomer. Many
approaches have been developed towards this goal,
both chemo- and biocatalytic ones.[3] The field of
biocatalytic amine synthesis has been growing rapidly
over the last few decades, which has resulted in several
viable enzymatic strategies towards enantiomerically
pure amines.[4]
1
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10.1002/adsc.201901252
Advanced Synthesis & Catalysis
The S pocket is, however, generally limited to a methyl
group or similar small substituents (Figure 1). This
considerable spatial difference is the reason for ATA´s
excellent enantioselectivity, which is a major
advantage but it also significantly limits the allowable
substrate scope. Since many fine chemicals containing
chiral amine motifs present bulky units on both sides
of the amine functionality, enzymes that can accept
bulkier substrates while maintaining enantioselectivity
are of great interest. The possibility to expand the ATA
substrate scope by increasing the size of the S pocket
has previously been explored, but only to some extent.
The first example for engineering the size of the S
pocket was the directed evolution of an (R)-selective
Arthrobacter sp. ATA where the S pocket was
enlarged to accept a fluorinated benzyl substituent.[8]
The S pocket of the (S)-selective ATA from Vibrio
fluvialis has been engineered to accept
propyl/hydroxymethyl substituents[9], cyclohexyl/tert-
Figure 1. Active site of Cv-ATA WT (PDB ID: 4A6T). The
active site architecture consisting of a small (S) and a large
(L) binding pocket is illustrated with the binding of (S)-1-
phenylethylamine (shown in magenta). The two amino acid
residues targeted in this study, L59 and F88, are shown in
orange. Amino acids from the second subunit are marked
with an asterisk (*).
butyl substituents[10] or an isohexyl substituent[11]
.
Additionally, (S)-selective ATAs from Ruegeria sp.
and Ochrobactrum anthropi were recently engineered
to accept bulky aromatic substituents and
butyl/isopropyl substituents, respectively.[12]
In order to perform catalysis on any particular
substrate of choice, a toolbox of different catalysts
with individual substrate scope is needed. With this in
mind, we envisaged an (S)-selective ATA with the
ability to accept a benzyl substituent in the S pocket,
which would yield a catalyst complementary to the
previously mentioned (R)-selective Arthrobacter sp.
ATA[8]. When the active site of an enzyme is to be
engineered to accept substantially larger substrates, the
activity against the target substrate is initially expected
to be absent or very low. A highly sensitive assay for
detecting activity is therefore required. Previously, we
developed a fluorogenic assay for measuring ATA
activity based on the transamination of 1-(6-
methoxynaphth-2-yl)alkylamines (Scheme 1, 1). This
reaction produces an acetonaphthone product (2) that
can be measured with high sensitivity due to its strong
blue fluorescence.[13] A modular synthetic route
towards this group of assay substrates was also
developed, allowing the incorporation of structural
variations such as the desired benzyl moiety.
Initially, activity of Cv-ATA WT against screening
substrates 1a-c was determined using the previously
developed fluorescence assay[13] (Scheme 1). Not
surprisingly, activity was highest against 1a that bears
the small methyl substituent on the S pocket side. Low
but detectable activities of 0.33 % and 0.084 %
Here we present a rational design approach for
increasing the size of the S pocket of the (S)-selective
ATA from Chromobacterium violaceum (Cv-ATA) to
accept a bulky benzyl substituent, allowing for the
preparation of enantiomerically pure (S)-configured
doubly bulky primary amines.
Results and Discussion
Cv-ATA is one of the best known ATAs and
characteristics such as substrate scope,[14] reaction
kinetics,[13, 15] 3D-structure[16] and stability[16a, 17] have
all been studied in detail. The active site of Cv-ATA
has also been the subject of several engineering
studies.[15b, 15c, 18] With that in mind, Cv-ATA was
chosen as a starting point for rational design driven
expansion of the active site S pocket.
Scheme 1. Screening assay used for evaluation of ATA
variants. The ketone product generated upon reaction
between the amine substrate and pyruvate can be measured
using fluorescence (ex: 330 nm, em: 460 nm).
2
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10.1002/adsc.201901252
Advanced Synthesis & Catalysis
(leucine) and was therefore mutated into the only three
hydrophobic amino acids that are smaller (valine,
alanine and glycine). F88, one of the bulkiest
canonical amino acids, was mutated into an alanine.
The choice of mutating F88 into alanine instead of
glycine (the smallest amino acid) is rationalized by the
unpredictable nature of glycine mutations on the
secondary structure of the enzyme due to glycine´s
high rotational freedom[20]. Also, the mutation F88A
has previously been successful in enlarging the S
pocket of Cv-ATA.[15b]
The activity of variants L59V, L59A, L59G and
F88A against substrates 1a-c was subsequently
determined and all variants showed improved activity
against the bulky substrates 1b and 1c (Figure 2, Table
S1). Especially, variant L59A displayed an impressive
88-fold increased activity against 1b compared to WT
as well as a 8.8-fold improvement against 1c. All L59
variants were rather similar with regards to 1c while
F88A showed less improvement. In an effort to further
increase the size of the S pocket, all three L59 variants
were subsequently combined with the F88A mutation.
All three double-mutants showed decreased activity
against 1b and L59V/F88A had reduced activity
6
5
4
3
2
1
100
80
60
40
20
1a
1b
1c
0.35
0.30
0.25
0.20
0.15
6
5
4
3
2
0.05
0.04
0.03
0.02
0.01
0.00
0.8
0.6
0.4
0.2
0.0
A
100
WT
88A
F88A
L59V
L59A
L59G
L59V/F88LA59A/F88A
L59G/F
80
60
40
20
0
Figure 2. Activities of Cv-ATA WT and variants towards
screening substrates 1a-c. Left y-axis: Relative activities
where Cv-ATA is set as 100 %. Right y-axis: Specific
activities expressed as µmol formed 2a-c min-1 mg-1
enzyme. For tabulated specific activities, see Table S1.
compared to 1a were determined for 1b and 1c,
respectively (Figure 2, Table S1).
Next, the active site architecture of Cv-ATA WT
was investigated in order to identify amino acid
residues that could be mutated to increase the size of
the S pocket without compromising the catalytic
machinery. The X-ray crystal structure of Cv-ATA
WT (PDB ID: 4A6T)[16a] reveals that the L pocket in
the active site is mainly shaped by residues M56, W60,
F89, A231, C418 and V423 (Figure 1). These six
residues together form a large cavity where the bulky
substituent of the substrate fits nicely. Another critical
residue in the L pocket is R416, which previously has
been shown to flip in and out of the active site in order
to make dual substrate recognition between α-
carboxylic substrates and bulky uncharged substrates
possible.[19] In this crystal structure (4A6T) R416 is
adopting a stretched conformation, meaning that it is
pointing towards the active site. This residue has
therefore been removed from Figure 1 to simulate the
corresponding situation when the L pocket accepts a
bulky uncharged substituent. On the contrary, the S
pocket is effectively closed off by residues L59 and
F88, forming a small cavity opposite to the L pocket
where only small substituents such as a methyl group
fits well.
6.0
6.5
7.0
7.5
8.0
8.5
pH
B
100
50
0
0
10
20
30
40
50
DMSO (%)
Figure 3. Biochemical characterization of Cv-ATA
L59A/F88A with regards to (A) pH-optimum and (B)
influence of DMSO concentration. The pH-profile was
determined with the acetophenone assay at a range of
different pH-values. Citrate (pH 6.0 and 6.5) and HEPES
(pH 7.0, 7.5 and 8.2) buffers were used at a 50 mM
concentration. The DMSO-dependence was determined
with the acetophenone assay at a range of different DMSO
concentrations (0-50 %) in 50 mM HEPES buffer (pH 7.5).
Activities are reported as relative to the highest measured
activity within each dataset (pH 7.5 and 30 % DMSO,
respectively).
Amino acid residues L59 and F88 were therefore
targeted for site-directed mutagenesis in order to
increase the size of the S pocket. Position 59 already
hosts a medium-sized hydrophobic amino acid
3
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10.1002/adsc.201901252
Advanced Synthesis & Catalysis
against 1c. However, L59G/F88A caused 11-fold
increased activity against 1c compared to WT and
L59A/F88A even displayed a 32-fold improvement
(Figure 2, Table S1). This shows that the double-
mutant L59A/F88A is the best variant for
accommodation of a benzyl substituent in the S pocket.
Similar additive effects of mutations have previously
been shown when expanding the active site of ATAs.[9,
L59A/F88A variant (75°C) as compared to WT
(71°C).
After biochemical characterization, it has been
shown that the activity of variant L59A/F88A can be
greatly improved by optimizing reaction conditions.
As an example, the activity of L59A/F88A towards 1c
in the fluorescence assay was the highest among all
variants, but it was still rather low compared to the
activity of Cv-ATA WT towards 1a (2.7 %, Figure 2).
However, if the optimized conditions with regards to
pH (approximately two times improved activity,
Figure 3A) and DMSO concentration (approximately
four times improved activity, Figure 3B) were to be
applied it can be assumed that the activity of
L59A/F88A would be improved approximately eight
times, leading to 22 % activity towards 1c compared
to the activity of WT towards 1a.
12c]
Interestingly, all variants decreased their activity
against 1a. This can be explained by a decrease in
direct interactions between the methyl group and the S
pocket residues as the pocket enlarges. A similar
pattern can be seen with 1b where activity first
increases with an increasing pocket size but then
decreases as the pocket is further enlarged. (Figure 2).
To further examine the variant L59A/F88A, a
biochemical characterization was performed. By
measuring relative activities at different pH-values
using the acetophenone assay[21], a pH-optimum of 7.5
for this variant could be determined (Figure 3A). This
stands in contrast to the WT that has a pH-optimum of
8.3 (Figure S1).[15a] However, it is not surprising that
changes in the active site architecture can affect pH-
dependence as it was previously shown that the variant
Cv-ATA W60C has a significantly altered pH-
optimum of 7.0 (Figure S1).[15c]
Due to the hydrophobic nature of the target
compounds that variant L59A/F88A was designed to
act upon, an investigation on the influence of DMSO
on the enzymatic activity was performed. DMSO is a
commonly applied co-solvent in biocatalysis when the
solubility of hydrophobic substrates needs to be
increased and it is one of the best tolerated co-solvents
for various enzyme types, as recently demonstrated in
a systematic stability study involving eight common
water-miscible organic solvents.[22] Other examples of
co-solvents applied in ATA-catalyzed reactions
include 1,2-dimethoxyethane for solubilization of
steroids[23] and deep eutectic solvents for synthesis of
biaryl amines[24]. The influence of DMSO on the
transamination of (S)-1-phenylethylamine and
pyruvate was previously reported for Cv-ATA WT.[17a]
The enzyme displays maximum activity at 0 % (v/v)
DMSO and a steady activity decrease until it is almost
diminished at 50 % (v/v) DMSO. Interestingly, the
variant L59A/F88A shows a completely different
behavior towards DMSO as the activity first increases
and reaches a maximum around 20-30 % (v/v) DMSO
(Figure 3B). Activity then decreases and at 50 % (v/v)
DMSO, it attains a slightly higher level than for 0 %
(v/v) DMSO. The reason for this drastic change in
DMSO tolerance is yet unknown, but it holds great
promise for preparative applications with hydrophobic
substrates, both with respect to better substrate
solubility but also for increased stability of Cv-ATA
WT in the presence of elevated concentrations of
DMSO.[17a]
The utility of the engineered L59A/F88A catalyst
was evaluated next in the synthesis of 1,2-
diphenylethylamine (3, Scheme 2). This benzyl-
substituted primary amine motif is especially
interesting in view of its occurrence in pharmaceutical
compounds such as lefetamine,[26] diphenidine[27] and
diphenpipenol,[28] which have analgesic and
anaesthetic activities. The target compound holds a
phenyl-moiety on one side of the amine and a benzyl-
moiety on the other side. Even though both
substituents are similar in size, they display different
conformational rigidity and would point in different
directions when bound to the active site of Cv-ATA.
We hypothesized that the most successful design
found for the S pocket in the engineered L59A/F88A
variant would accommodate the benzyl substituent in
preference to the phenyl substituent, thereby resulting
in a certain degree of enantioselectivity towards 3.
An initial exploration of the variant´s ability to
catalyze the asymmetric synthesis of 3 starting from its
prochiral ketone precursor 4 was attempted using the
“smart” equilibrium shifting amino donor ortho-
xylylenediamine in equimolar (50 mM) amounts.[29]
However, it was not successful (Figure S2) and we
decided not to pursue this any further. The reason for
the low reactivity of 4 might be due to its low water
solubility or due to thermodynamic stabilization by
enol formation.
To
assess
the
principal
activity
and
enantioselectivity of L59A/F88A towards (R/S)-3, an
attempt for kinetic resolution (KR) was pursued
instead (Scheme 2) using 1 mM substrate. The results
The thermostability of variant L59A/F88A was
explored by measuring the melting point (Tm, the
temperature at which the folded and unfolded enzyme
species are at thermodynamic equilibrium) using
differential scanning fluorimetry.[25] The results of this
analysis show a slight increase in Tm for the
Scheme 2. Cv-ATA catalyzed kinetic resolution of 1,2-
diphenylethylamine (3). Pyruvate is used as amino acceptor,
resulting
in
the
generation
of
alanine,
2-
phenylacetophenone (4) and enantiomerically pure (R)-3.
4
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10.1002/adsc.201901252
Advanced Synthesis & Catalysis
100
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
A
B
80
60
40
20
0
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Time (h)
Time (h)
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
100
80
60
40
20
0
C
D
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Time (h)
Time (h)
Figure 4. Cv-ATA catalyzed kinetic resolution of 1,2-diphenylethylamine (3). (A) 5 mM 3, 7.5 mM pyruvate, 0 % (v/v)
DMSO, 0.5 mg Cv-ATA WT. (B) 5 mM 3, 7.5 mM pyruvate, 0 % (v/v) DMSO, 0.5 mg Cv-ATA L59A/F88A. (C) 20 mM
3, 30 mM pyruvate, 20 % (v/v) DMSO, 2 mg Cv-ATA L59A/F88A. (D) 100 mM 3, 200 mM pyruvate, 0 % (v/v) DMSO,
5.4 mg Cv-ATA L59A/F88A, magnetic stirring. All reactions were performed at room temperature in 3 ml reaction volumes
and under shaking unless otherwise specified. Conversion (●) and ee (■).
of this exploratory KR (Figure S3) revealed that
L59A/F88A completely consumed all (S)-3 while in
the WT-catalyzed reaction the remaining amine was
still racemic after 1.5 h. This confirms that
L59A/F88A is active towards 3 and that it shows high
enantioselectivity towards its (S)-enantiomer.
h, all of (S)-3 was consumed leaving enantiomerically
pure (R)-3 (eeS >99 %).
Conclusion
In order to explore the performance of the reaction
at higher substrate loadings, a KR of 3 catalyzed by the
variant L59A/F88A was performed using a slightly
elevated substrate concentration close to the observed
solubility limit (5 mM, Figure 4B). Substrate
conversion progressed steadily and after 4 h a high
enantiomeric excess of the substrate (eeS >99 %) was
accomplished. This stands in contrast to the low
activity of Cv-ATA WT under the same conditions
(Figure 4A).
The addition of 20 % (v/v) DMSO as co-solvent was
also investigated, thereby exploiting the increased
activity of the variant under those conditions (Figure
4C) and also enabling further increase of substrate
concentration (20 mM, close to the observed solubility
limit in 20 % (v/v) DMSO). Again, the reaction
proceeded steadily until an eeS >99 % was reached
already after 2 h.
We have demonstrated a rational design approach
towards the successful creation of a Cv-ATA variant,
L59A/F88A, that is able to kinetically resolve 1,2-
diphenylethylamine (3) efficiently by accommodating
its bulky benzyl substituent in the small S pocket of the
active site. It is also able to discriminate between a
benzyl substituent and a structurally similar phenyl
substituent, resulting in high enantioselectivity
towards (S)-3. The use of our previously published,
highly sensitive fluorescent screening method[13] with
specifically designed substrates (Scheme 1) enabled a
substrate walking approach where very low starting
activities could be detected and subsequently
improved by rational protein engineering. The variant
L59A/F88A also displayed interesting biochemical
properties as its alkaline pH optimum was displaced
by almost one unit towards an almost neutral pH. Its
behaviour towards the water-miscible co-solvent
DMSO also showed a drastic change. While the WT
enzyme has its maximum activity at 0 % (v/v) DMSO
with a steady decrease in activity as the concentration
increases, the variant shows an activity optimum at
20 % (v/v) DMSO. This shows great promise for
The applicability of the KR was further explored by
adding 3 at concentrations exceeding its solubility
limit in aqueous media (100 mM), leading to a non-
homogeneous mixture (Figure 4D). However,
L59A/F88A tolerated such conditions well and after 4
5
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10.1002/adsc.201901252
Advanced Synthesis & Catalysis
starting from 10 ml of overnight preculture. Expression was
induced at OD600 =0.8 with 0.3 mM IPTG and carried out at
15 °C in the dark over 24 h and 150 rpm agitation. Cells
were harvested by centrifugation (4 °C, 4000 xg, 30 min),
resuspended in 50 mM HEPES pH 7.5, 10 mM imidazole
and sonicated on ice at medium intensity with 50% pulses.
The lysate was clarified by centrifugation (4 °C, 30000 xg,
40 min) and filtration (0.45 μm) prior to loading on pre-
equilibrated 5 ml Ni-NTA cartridges (GE Healthcare). The
target protein was eluted in a linear gradient to 0.5 M
imidazole in 40 min at a flow rate of 2.5 ml/min. The
fractions containing the target were pooled together,
supplemented with PLP (1.2 mg) and stored overnight at 4
°C in the dark. The protein was then buffer-exchanged in 50
mM HEPES pH 7.5 using an Amicon Ultra Centrifugal
Filter (cut-off 30 kDa, Merck Millipore Ltd.). Protein
aliquots were flash frozen in liquid nitrogen and stored at -
80 °C until use.
future application as DMSO is commonly used to
increase solubility of hydrophobic substrates. Finally,
the generated variant L59A/F88A shows great promise
in the resolution of racemic 3 for generation of
enantiomerically pure (R)-3 with a highest tested
substrate loading of 100 mM (20 g/L).
Experimental Section
Materials
All chemicals were purchased from Sigma-Aldrich and used
without further purification, unless otherwise stated. The
HisPrep column used for enzyme purification and the PD10
desalting columns were purchased from GE Healthcare Life
Science (Sweden).
Screening substrate 1a-c synthesis
Site-directed mutagenesis
Screening substrates 1a-b were synthesized according to a
previously published protocol.[13] Screening substrate 1c
was synthesized as follows:
The Cv-ATA gene was previously inserted into pET-28a(+)
with a N-terminal His6-tag.[15a] Site-directed mutagenesis
was performed by the Quick Change method with
modifications according to Zheng et. al., using Phusion
High-Fidelity DNA Polymerase from Thermo Fisher. The
primers used are listed below, with the mutations indicated
in lower case:
Synthesis of 1-(6-methoxynaphth-2-yl)2-phenylethan-1-ol:
In
a
100 mL round bottom flask 6-Methoxy-2-
naphtaldehyde (2.5 g, 13.5 mmol, 1 eq.) was dissolved in
dry tetrahydrofuran (37 mL) under argon atmosphere, and a
solution of benzyl magnesium chloride (2.0 M in THF, 7.32
mL, 14,7 mmol, 1.1 eq.) was added slowly at 0 °C. Further
benzyl magnesium chloride (1.1 eq.) was added due to
incomplete conversion after 2 h, and the mixture was
allowed to stir at room temperature for an additional hour.
Then the mixture was quenched by the addition of saturated
aqueous NH4Cl (20 mL). After removal of THF under
reduced pressure, the aqueous phase was extracted with
diethyl ether (3 × 50 mL). The combined organic layers
were dried over Na2SO4 and the solvent was evaporated.
The crude product was recrystallized with n-
heptene/toluene to give light yellow crystals (2.07 g, 55%,
melting point: 133 °C.
Cv-ATA_L59G_f:
5´-GCAGGTggtTGGTGTGTTAATGTTG-3´
Cv-ATA_L59G_r:
5´-CACCAaccACCTGCCATACCATC-3´
Cv-ATA_L59A_f:
5´-CAGGTgcaTGGTGTGTTAATGTTG-3´
Cv-ATA_L59A_r:
5´-CACCAtgcACCTGCCATACC-3´
Cv-ATA_L59V_f:
5´-CAGGTgttTGGTGTGTTAATGTTG-3´
Cv-ATA_L59V_r:
5´-CACCAaacACCTGCCATACC-3´
Cv-ATA_F88A_f:
1H NMR (300 MHz, CDCl3): δ = 7.67-7.61 (m, 3H, 8’-, 4’-
, 1’-H), 7.38 (dd, J = 8.5 Hz, 1.8 Hz, 1H, 3’-H), 7.22-7.12
(m, 5H, Ph), 7.09 (d, J = 2.6 Hz, 1H, 7’-H), 7.05 (s, 1H, 5’-
H), 4.95 (dd, J = 8.1 Hz, 5.1 Hz, 1H, 1-H), 3.84 (s, 3H,
OCH3), 3.07-2.98 (m, 2H, 2-H); 13C NMR (CDCl3, 75
MHz): δ = 157.7 (C-6’), 139.0 (C-1”), 138.1 (C-2’), 134.1
(C-4a’), 129.5 (C-3” 2x), 129.4 (C-8’), 128.7 (C-3’), 128.5
(C-2” 2x), 127.0 (C-8a’), 126.6 (C-1’), 124.6 (C-4”), 124.5
(C-4’), 118.9 (C-7’), 105.7 (C-5’), 75.5 (C-1), 55.3 (OCH3),
46.0 (C-2).
5´-GTTTTATAATACCgccTTTAAAACCACCC-3´
Cv-ATA_F88A_r:
5´-GGTTTTAAAggcGGTATTATAAAACGGC-3´
Enzyme expression and purification
Expression of Cv-ATA WT and variants was performed by
inoculating 20 mL LB-medium (50 μg/mL kanamycin) with
Escherichia coli (E. coli) BL21 (DE3) cells containing the
Cv-ATA encoding plasmid. The culture was incubated at 37
°C and 210 rpm overnight. 180 mL LB-medium (50 μg/mL
Synthesis of 1-(6-methoxynaphth-2-yl)2-phenylethan-1-
one: In a 50 mL round bottom flask the alcohol (2.07 g, 7.33
mmol, 1 eq.) was dissolved in 14 mL acetone and cooled to
0 °C in an ice bath. A solution of CrO3 (6 mL, 12 mmol,
1.64 eq.) in 30% H2SO4 was added to the reaction mixture
slowly, so that the temperature remains below 5 °C. When
precipitation was observed, more acetone was added. After
the mixture was stirred for one hour, it was stopped by
quenching the excess oxidant with aqueous NaHSO3. The
product was precipitated by addition of water and isolated
by filtration, and dissolved in diethyl ether. The solution was
washed with 10% Na2CO3, water and brine. The organic
layer was dried over Na2SO4 and the solvent was evaporated
under reduced pressure. The crude product was
recrystallized from water/ethanol (1:3) to give yellow
crystals (1.26 g, 62%), melting point: 115 °C.
kanamycin
and
0.4
mM
isopropyl
β-D-1-
thiogalactopyranoside (IPTG)) was then inoculated with the
overnight culture and incubated at 25 °C and 140 rpm for 24
h. After expression, the cells were harvested by
centrifugation (8000 rpm for 15 min). The cell pellet was
dissolved in Immobilized Metal ion Affinity
Chromatography (IMAC) binding buffer (sodium
phosphate buffer (20 mM, pH 7.4) and NaCl (0.5 M)) and
cells were disrupted by sonication. Cell debris was removed
by centrifugation (20000 rpm for 15 min) and the
supernatant was collected and filtered (0.45 μm). The
enzyme was then purified by IMAC using a 5 mL HisPrep
column according to the manufacturers’ protocol.
Pyridoxal-5´-phosphate (PLP) was added in an excess (1
mM) and the solution was incubated in room temperature
for 30 minutes. Finally, a buffer exchange to HEPES buffer
(50 mM, pH 7.5 or 8.2) was performed by running the
solution through a PD-10 column according to the
manufacturers’ protocol. The enzyme solution was stored at
4 °C. Enzyme concentrations were determined according to
Pace et. al.[30]
1H NMR (300 MHz, CDCl3): δ = 8.41 (s, 1H, 1’-H), 7.98
(dd, J = 8.6 Hz, 1.8 Hz, 1H, 4’-H), 7.78 (d, J = 9.0 Hz, 1 H,
8’-H), 7.69 (d, J = 8.7 Hz, 1H, 3’-H), 7.26 (s, 5H, Ph), 7.21-
7.08 (m, 2H, 7’-H, 5’-H), 4.32 (s, 2H, 2-H), 3.88 (s, 3H,
OCH3); 13C NMR (CDCl3, 75 MHz): δ = 197.5 (C-1), 160.0
(C-6’), 137.4 (C-1”), 135.0 (C-2’), 132.2 (C-4a’), 131.3 (C-
8’), 130.4 (C-8a’), 128.8 (C-3” 2x), 129.6 (C-3’), 127.9 (C-
2” 2x), 127.3 (C-1’), 127.0 (C-4”), 125.2 (C-4’), 119.8 (C-
7’), 105.9 (C-5’), 55.5 (OCH3), 45.5 (C-2).
Larger scale Cv-ATA L59A/F88A expression and
purification was performed in
1
L
TB medium
supplemented with antibiotic (50 µg/mL kanamycin)
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.201901252
Advanced Synthesis & Catalysis
Synthesis
of
1-(6-methoxynaphthalen-2-yl)-2-
sodium pyruvate in different buffers at room temperature.
Acetophenone formation was monitored at 245 nm using a
Cary50 UV/Vis spectrophotometer.
phenylethanoneoxime: In a 50 mL round bottom flask the
ketone (1.26 g 4.56 mmol, 1 eq.) was dissolved in 33 mL
ethanol to this solution a mixture of hydroxylamine
hydrochloride (0.48 g, 6.85 mmol, 1.5 eq.) and 0.56 mL
pyridine (6.85 mmol, 1.5 eq.) was added. The reaction
mixture was stirred at 60°C for 16 h. A change in colour,
yellow to orange was observed. The solvent was evaporated
under reduced pressure, and the residue dissolved in 10 mL
dichloromethane. The dissolved product was washed with
water (2 × 10 mL) and brine (1 × 10 mL). The organic phase
was dried with Na2SO4 and the solvent removed under
reduced pressure. The obtained product was purified by
Biochemical characterization
pH-dependence: The pH-profile of Cv-ATA L59A/F88A
was determined with the acetophenone assay at a range of
different pH-values. Citrate (pH 6.0 and 6.5) and HEPES
(pH 7.0, 7.5 and 8.2) buffers were used at a 50 mM
concentration.
DMSO-dependence: The DMSO-dependence of Cv-ATA
L59A/F88A was determined with the acetophenone assay at
a range of different DMSO concentrations (0-50 %) in 50
mM HEPES buffer (pH 7.5). Every measurement was
performed in duplicate.
flash
column
chromatography
on
silica
gel
(cyclohexane/ethyl acetate 10-20%) to give orange crystals
(1.09 g, 82%).
1H NMR (300 MHz, CDCl3): δ = 8.50 (s, 1H, 1’-H), 8.07
(dd, J = 8.7 Hz, 1.8 Hz,1H, 4’-H), 7.90 (d, J = 8.9 Hz,1 H,
8’-H), 7.78 (d, J = 8.6 Hz, 1H, 3’-H), 7.39-7.31 (m, 5H, Ph),
7.29-7.16 (m, 2H, 7’-H, 5’-H), 4.41 (s, 2H, 2-H), 3.96 (s,
3H, OCH3); 13C NMR (CDCl3, 75 MHz): δ = 197.3 (C-1),
159.9 (C-6’), 137.3 (C-2’), 134.9 (C-4a’), 132.1 (C-8’),
131.2 (C-8a’), 130.3 (C-3’), 129.5 (C-3” 2x), 128.7 (C-2”
2x), 127.8 (C-1’), 127.2 (C-1”), 126.8 (C-4”), 125.0 (C-4’),
119.7 (C-7’), 105.8 (C-5’), 55.4 (OCH3), 45.4 (C-2).
Differential scanning fluorimetry: Melting temperature (Tm)
values were measured using differential scanning
fluorimetry (DSF).[25] The measurement was performed on
a CFX96 real-time PCR detection system and C1000
thermal cycler at 569 nm. The reaction sample (20 µL)
consisted of 1 mg/mL enzyme and 5x SYPRO Orange
protein gel stain (Sigma Aldrich, S5692) in HEPES buffer
(50 mM, pH 7.5). The temperature range was set to 25-95
°C, with an increase of 1 °C/min. Every measurement was
performed in duplicate.
Synthesis
of
1-(6-methoxynaphthalen-2-yl)-2-
phenylethanamine: The oxime (1.09 g, 3.74 mmol, 1eq.)
was dissolved in 50mL acetic acid and 3mL water. The
mixture was heated to 110°C and activated Zn (3.4 g, 52
mmol, 13.9 eq.) was added portion wise to the reaction over
the course of 25 minutes. After a total of 2 h reaction time
the mixture was cooled down and neutralized with a
saturated solution of NaHCO3. The product was extracted
with dichloromethane (3 × 50 mL) and the combined
organic layers were dried over Na2SO4. The solvent was
removed under reduced pressure to give pale yellow crystals
(892 mg, 86%).
Kinetic resolutions
Kinetic resolutions were performed in a 3 mL reaction
volume in 500 mM HEPES buffer (pH 7.5, 0 or 20 % (v/v)
DMSO). Substrate concentrations were 5-100 mM 3 and
7.5-200 mM sodium puruvate. 0.5, 2.0 or 5.4 mg enzyme
was used. See Figure 4 for specific conditions regarding
each reaction. Reactions were run for 6 h and samples were
collected regularly for analysis.
Conversion was analyzed using a Hewlett Packard 5890 gas
chromatograph (GC) equipped with an Agilent CP-Sil 5 CB
column (30m x 0.25 mm x 0.25 µm) and a flame ionization
detector (FID). Consumption of amine substrate was
determined with the following temperature gradient:
starting temperature 120 °C, 10 °C/min to 130 °C (hold 2
min), 5 °C/min to 180 °C, 25 °C/min to 300 °C (hold 2 min).
Retention times: 3 (12.1 min), 4 (12.7 min). Injector
temperature was 275 °C and detector temperature was 300
°C. Sample preparation for GC analysis was performed by
collecting 100 µL reaction sample. 10 µL NaOH (1 M) was
added and the amine was extracted into 200 µL hexane. The
organic phase was dried over anhydrous Na2SO4 and the
sample was then centrifuged and analyzed on GC.
Enantiomeric excess was analyzed with normal phase
HPLC on a Hewlett Packard 1100 liquid chromatography
system using a Chiralcel OD-H column. As mobile phase,
hexane:2-propanol:DEA (90:10:0.1) was used at a flow of 1
mL/min. Amine absorbance was detected at 215 nm.
Retention times: (R)-3 (8 min), (S)-3 (11 min). Sample
preparation for HPLC was performed by collecting 100 µL
reaction sample. 10 µL HCl (1 M) was added and the ketone
was removed by extraction to hexane. 20 µL NaOH (1 M)
was added to the aqueous phase and the amine was extracted
into 200 µL hexane. The organic phase was dried over
anhydrous Na2SO4 and the sample was then centrifuged and
evaporated. Amine was redissolved in 100 µL hexane:2-
propanol (90:10). 10 µL was then injected.
1H NMR (300 MHz, CDCl3): δ = 7.77-7.72 (m, 3H, 1’- ,3’-
, 4’-H), 7.51 (dd, J = 8.4 Hz, 1.8 Hz, 1H, 7’-H), 7.38-7.17
(m, 7H, Ph, 8’-H, 5’-H), 4.36 (dd, J = 8.8 Hz, 5.0 Hz, 1H,
1-H), 3.94 (s, 3H, OCH3), 3.16-2.90 (m, 2H, 2-H); 13C NMR
(CDCl3, 75 MHz): δ = 157.5 (C-6’), 140.8 (C-1”), 139.1 (C-
2’), 133.9 (C-4a’), 129.4 (C-3” 2x), 129.3 (C-8’), 128.9 (C-
8a’), 128.5 (C-2” 2x), 127.0 (C-3’), 126.4 (C-1’),125.6 (C-
4”), 124.7 (C-4’), 118.8 (C-7’), 105.7 (C-5’), 57.6 (C-1),
55.3 (OCH3), 46.4 (C-2).
Synthesis
of
1-(6-methoxynaphthalen-2-yl)-2-
phenylethanammonium chloride: In a 250 mL round bottom
flask the amine (892 mg, 3.2 mmol, 1eq.) was dissolved in
125 mL dry diethyl ether under argon atmosphere. HCl gas
was bubbled through the solution until salt precipitation
could be observed. The product was filtered and washed
with ether, and the residual solvent was removed under
reduced pressure to give white crystals (640 mg, 64%),
melting point: 275 °C (decomposition).
1H NMR (300 MHz, CDCl3): δ = 7.85-7.73 (m, 3H, 1’-, 3’-
, 4’-H), 7.51 (d, J = 8.6 Hz, 1H, 7’-H), 7.28-7.14 (m, 7H,
Ph, 5’-,8’-H, ), 4.67 (dd, J = 9.1 Hz, 6.1 Hz, 1H, 1-H), 3.89
(s, 3H, OCH3), 3.49-3.30 (m, 2H, 2-H); 13C NMR (CDCl3,
75 MHz): δ = 159.9 (C-6’), 137.0 (C-1”), 136.3 (C-2’),
132.6 (C-4a’), 130.6 (C-8’), 130.5 (C-3” 2x), 130.0 (C-8a’),
129.7 (C-2” 2x), 129.0 (C-4”), 128.2 (C-3’), 128.2 (C-1’),
125.9 (C-4’), 120.6 (C-7’), 106.7 (C-5’), 58.6 (C-1), 55.9
(OCH3), 41.9 (C-2).
All NMR spectra were recorded on a Bruker AX300
spectrometer
Acknowledgements
Enzyme activity assays
Dr Luuk van Langen, Viazym, Delft, NL, is gratefully
acknowledged for scientific advice. This project has received
funding through the German Federal Ministry of Education and
Research (BMBF) under Grant Agreement No 031B0595
(Tralaminol; to WDF) and the initiative ERA CoBioTech, which
has received funding from the European Union’s Horizon 2020
Research and Innovation Programme under grant agreement No
722361.
Fluorescence assay: The fluorescence assay was performed
as previously described. Substrate concentrations were set
to 1 mM 1a-c and 4.5 mM sodium pyruvate. Data was fitted
using the BMG CLARIOstar software. Every measurement
was performed in duplicate.
Acetophenone assay: The acetophenone assay[21] was
performed using 5 mM (S)-1-phenylethylamine and 5 mM
7
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10.1002/adsc.201901252
Advanced Synthesis & Catalysis
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10.1002/adsc.201901252
Advanced Synthesis & Catalysis
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10.1002/adsc.201901252
Advanced Synthesis & Catalysis
FULL PAPER
Engineering the Active Site of an (S)-Selective
Amine Transaminase for Acceptance of Doubly
Bulky Primary Amines
Adv. Synth. Catal. Year, Volume, Page – Page
Henrik Land,a, b Federica Ruggieri,a Anna
Szekrenyi,c Wolf-Dieter Fessnerc and
Per Berglunda*
10
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