Chromobacterium violaceum ω-transaminase variant Trp60Cys shows increased specificity for (S)-1-phenylethylamine and 4′-substituted acetophenones, and follows Swain-Lupton parameterisation

Article abstract of DOI:10.1039/c2ob25893e

For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have proven useful. Engineering of these enzymes has to some extent been accomplished by rational design, but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium violaceum ω-transaminase was found upon comparison with engineered variants from homologous enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and 4′-substituted acetophenones (～5-fold). To further study the effect of the mutation the reaction rates were Swain-Lupton parameterised. On comparison with the wild type, reactions of the variant showed increased resonance dependence; this observation together with changed pH optimum and cofactor dependence suggests an altered reaction mechanism.

Full text of DOI:10.1039/c2ob25893e

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PAPER
Chromobacterium violaceum ω-transaminase variant Trp60Cys shows
increased specificity for (S)-1-phenylethylamine and 4′-substituted
acetophenones, and follows Swain–Lupton parameterisation†
Karim Engelmark Cassimjee,a Maria Svedendahl Humble,‡a Henrik Land,a Vahak Abedib and Per Berglund*a
Received 11th February 2012, Accepted 31st May 2012
DOI: 10.1039/c2ob25893e
For biocatalytic production of pharmaceutically important chiral amines the ω-transaminase enzymes have
proven useful. Engineering of these enzymes has to some extent been accomplished by rational design,
but mostly by directed evolution. By use of a homology model a key point mutation in Chromobacterium
violaceum ω-transaminase was found upon comparison with engineered variants from homologous
enzymes. The variant Trp60Cys gave increased specificity for (S)-1-phenylethylamine (29-fold) and
4′-substituted acetophenones (∼5-fold). To further study the effect of the mutation the reaction rates were
Swain–Lupton parameterised. On comparison with the wild type, reactions of the variant showed
increased resonance dependence; this observation together with changed pH optimum and cofactor
dependence suggests an altered reaction mechanism.
synthesis of chiral amines of high enantiomeric excess,4–15 for
Introduction
use in e.g. pharmaceutical preparations.16 Successful reaction
For the synthesis of chiral amines, transaminase enzymes are an
attractive option. These enzymes interconvert amino and keto
groups, generally with high enantioselectivity, assisted by their
coenzyme pyridoxal-5′-phosphate (PLP).1 The transaminase sub-
group denoted ω-transaminases (ωTAs) can convert aliphatic
ketones and amines without a carboxylate group in the α-pos-
ition. The reaction mechanism, assumed to follow that of aspar-
tate transaminase,1,2 is composed of two half reactions. An
amine reacts with the holoenzyme to produce the corresponding
ketone and pyridoxamine-5′-phosphate (PMP), another ketone
can then react to form its corresponding amine and the holo-
enzyme is reformed. A simplified scheme is shown in Scheme 1;
see ESI Fig. S4† for a detailed mechanism. Generally, carboxy-
late-containing substrates, such as keto acids or amino acids, are
faster reacting. This also applies for the enzyme used in this
work, Chromobacterium violaceum ωTA (Cv-ωTA).3 There are
several examples of successful employment of ωTAs for
engineering approaches have dealt with the commonly unfavour-
able reaction equilibria.17–19
In general, the employment of an ωTA in a specific synthetic
reaction requires enzyme engineering.20 (Semi)rational
design,16,21–23 but mostly directed evolution24–27 has been the
method used for improvement of ωTA stability, activity and
enantioselectivity for a desired reaction. Advances in compu-
tational methods for finding suitable ωTAs have also been
made.28 Previously, we designed ωTA variants of both Cv-ωTA29
and of Arthrobacter citreus ωTA22 that showed improved
enantiospecificity or reversed enantiomeric preference. These
point mutations were guided by using homology models.
aKTH Royal Institute of Technology, Division of Biochemistry, School of
Biotechnology, AlbaNova University Centre, SE-106 91 Stockholm,
Sweden. E-mail: per.berglund@biotech.kth.se; Fax: +46 8 5537 8468;
Tel: +46 8 5537 8366
bAstraZeneca R&D, Pharmaceutical Development, SE-151 85
Södertälje, Sweden
†Electronic supplementary information (ESI) available: pH profiles,
active site quantification, rate equations and examples of plots, Swain–
Lupton parameterisation, reaction mechanism, materials and methods.
See DOI: 10.1039/c2ob25893e
‡Present address: Department of Organic Chemistry, Arrhenius Labora-
tory, Stockholm University, SE-106 91 Stockholm, Sweden.
Scheme 1 The ω-transaminase enzyme (Enz) catalysed reaction as half
transamination reactions, of isopropylamine/acetone (top) and aceto-
phenone/(S)-1-phenylethylamine (bottom).
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Another example is the tailoring of the ωTA from Vibrio fluvialis
(Vf-ωTA) by rational design to increase the activity for aliphatic
amines.21 In that work, a tryptophan residue in the active site
was replaced by a glycine (Vf-ωTA Trp57Gly) to create a larger
substrate binding pocket with space for larger hydrophobic
groups on substrates in the (S)-configuration. In another work, a
thermostable ωTA variant from Arthrobacter citreus denoted
CNB05-01, where a cysteine replaced the corresponding tyrosine
in the active site, was found by directed evolution.26
To measure and compare turnover numbers between different
enzyme variants or enzymes from different species, an active site
quantification method is required. Recently, we published such a
method for Cv-ωTA.30 This enzyme was now subject to protein
engineering, and the active site quantification method made
kinetic comparisons possible. Interestingly, the enzyme shows a
time dependent activation in solution,30 possibly due to struc-
tural rearranging upon cofactor binding.31 For this reason, the
enzymes used in this paper were after IMAC-purification incu-
bated with an excess of cofactor, desalted, and then the amount
of active enzyme was quantified.
In transaminase reactions with substituted acetophenones we
observed significant rate differences depending on the substi-
tuent, hypothetically due to electrochemical effects. In chemical
reactions with benzoic acid and analogues thereof, the Hammett
equation may be used to predict meta and para substituent
effects on reaction rate and reaction equilibrium.32 The coeffi-
cients for various substituents are derived by studying the pKa of
substituted benzoic acids. Swain and Lupton extended the work
of Hammett by deriving two coefficients for each substituent; by
assuming that field and resonance are independent of each other
these effects can be linearly combined with corresponding
coefficients.33–35
We studied transaminase reactions of two common amino
donors, and of chosen substituted acetophenones by their depen-
dence on field and resonance effects by employing the Swain–
Lupton equation, in the wild type and a variant derived from
comparisons with engineered homologous enzymes. To deter-
mine specificity constants (kcat/KM) we chose substrate concen-
trations near the value of KM (within the linear range), or derived
relative specificity constants by allowing several substrates to
compete for the active site. Then, we treated the specificity con-
stants as first-order rate constants.
the wild type was explored by measuring the absorbance spectra
of the holoenzymes, the pH optima, the effect of excess PLP and
the specificity constants for two amine substrates and several
selected 4′-substituted acetophenones. The values obtained for
the selected substituted acetophenones were used for Swain–
Lupton parameterisation to investigate the presence of putative
mechanistic differences in the variant compared to the wild type
enzyme.
The wild type holoenzyme has an absorbance maximum at
395 nm.30 Instead, Cv-ωTA Trp60Cys showed an absorbance
maximum at 407 nm, after incubation with PLP and desalting.
This is similar to free PLP and/or the hemimercaptal form of
PLP.36 This change of absorbance for the variant dictated the
modification and reestablishment of the active site quantification
method, since the method relies on the virtually irreversible
reaction of the wild type holoenzyme with an absorbance
maximum at 395 nm. The wild type was measured at 395 nm, as
previously described.30 The correlation between the Trp60Cys
putative holoenzyme amount of active sites and the absorbance
at 407 nm was measured, analogous to the said method, by
measuring the amount of consumed (S)-1-phenylethylamine
((S)-1-PEA) by HPLC, in a half transamination reaction, see
ESI† for details.
The pH rate dependence of the Cv-ωTA Trp60Cys variant was
measured in synthesis mode, i.e. for the amination of acetophen-
one by isopropylamine (IPA). Cv-ωTA wild-type has optimum
pH at 8.3,30,37 while the Trp60Cys variant showed an optimum
at pH 7.0, see ESI, Fig. S1.†
Cv-ωTA wild-type is inhibited by an excess of PLP,30 but the
Trp60Cys variant did not show this behaviour, see Fig. 1.
Instead, Cv-ωTA Trp60Cys required an excess of PLP to achieve
maximum rate; additionally, continuous measurements showed
that reactions without excess PLP stopped after ∼10 min.
The specificity constants for (S)-1-PEA and IPA were
measured for a transamination reaction with pyruvate catalysed
by Cv-ωTA wild type or variant Trp60Cys. The reaction of
(S)-1-PEA was followed spectrophotometrically at 245 nm.38
Initial rates for concentrations of (S)-1-PEA for transamination
Results
In this work, homology models of Vf-ωTA and CNB05-01 were
structurally aligned to the homologous Cv-ωTA to localize the
corresponding amino acid residue position from previous engin-
eering work by Cho et al.21 and by Cambrex North Brunswick
Inc.26 In Cv-ωTA, Trp60 was thereby identified and targeted for
site-directed mutagenesis. Previously, we showed that Cv-ωTA
Trp60Cys has increased enantiospecificity for 1-phenylethyl-
amine, 1-aminotetraline and 2-aminotetraline compared to the
wild-type enzyme.29 Additionally, Cv-ωTA Trp60Ala shows
reduced activity and enantiospecificity and gives poor cultivation
yields compared to the wild-type enzyme and was therefore not
further explored.29
Fig. 1 The effect of excess PLP on the initial rate of Cv-ωTAwild type
(blue dots) and variant Trp60Cys (red diamonds), obtained by following
the consumption of acetophenone at 285 nm, in HEPES buffer (50 mM,
pH 8.2 for the wild type and pH 7.0 for the variant) in a system with
IPA (50 mM for the wild type and 200 mM for the variant) as the amino
donor. As previously shown30 the wild type enzyme is inhibited. The
variant did not reach maximum rate below a two-fold excess of PLP.
The effect of the mutation Trp60Cys (a cysteine residue at the
corresponding position was found in CNB05-01) compared to
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Table 1 Kinetic constants for (S)-1-PEA and IPA for Cv-ωTA wild
Table 2 Specificity constants for chosen 4′-substituted acetophenones
for Cv-ωTAwild type and variant Trp60Cysa
type and variant Trp60Cysa
kcat/KM (s−1 mM−1
Cv-ωTAwild type
)
Cv-ωTATrp60Cys
kcat/KM
kcat/KM
KM
kcat
NO2–
Cl–
Br–
CN–
F–
H–
Me–
OH–
MeO–
0.030
0.029
0.028
0.026
0.020
0.0082b
0.0050
0.0024
n.d.c
0.16
(s–1 mM–1
)
(s−1 mM−1
)
(mM) (s−1
)
n.d.d
0.039
0.10
Cv-ωTAwild type
Cv-ωTA
Trp60Cys
0.68
20
0.20 0.004
0.024 0.002
73
190
15 0.3
4.6 0.4
n.d.c
0.034b
0.024
0.013
0.011
a Reactions performed in 50 mM HEPES buffer at pH 8.3 (wild type)
and pH 7.0 (Trp60Cys). Excess PLP (200 μM) was added to the
reactions with the variant, as dictated by its PLP-dependence curve
(Fig. 1).
a Reactions performed in 50 mM HEPES buffer pH 8.3 with 50 mM IPA
(wild type) or pH 7.0 with 200 mM IPA (Trp60Cys). Excess PLP
(200 μM) was added to the reactions with the variant, as dictated by its
PLP-dependence curve (Fig. 1). b Experimentally determined values
from which the remaining values were calculated from their relative
values. c Not determined, no observed reaction. d Not determined,
significant background reaction.
of pyruvate were collected. Rates which were linearly dependent
on the concentration were used to calculate the specificity con-
stant (kcat/KM). For an ordered ping-pong bi–bi reaction the true
specificity constants can be deduced in this manner, even though
pseudo-one-substrate kinetics are applied; see ESI† for the rate
equations and examples of the rate plots.
fixed values for field and resonance for each substituent.
The transamination reaction of IPA and pyruvate was
measured by, first, treating IPA as a competing substrate in the
above mentioned spectrophotometrically measurable reaction, by
measuring rates with and without IPA. From this the KI for IPA
was obtained. Then, since in this case KI = KM, an appropriate
concentration of IPA could be chosen for analysis of formed
alanine with ninhydrin, IPA does not give a colour change upon
incubation with ninhydrin.39 As shown in Table 1, variant
Trp60Cys showed a significantly increased specificity constant
for (S)-1-PEA; for IPA the specificity was reduced.
Further, the relative specificity constants of a variety of 4′-sub-
stituted acetophenones were investigated; 4′-fluoro-, chloro-,
bromo-, nitro-, cyano-, methyl-, hydroxyl-, methoxy-, and
unsubstituted acetophenone were used. By employing several
ketones simultaneously their relative reaction rates could be
obtained. Ketone consumption was followed by HPLC with IPA
as the amino donor. All ketones could not be used at once due to
peak overlap in the HPLC-measurement. Instead, four separate
reactions were prepared. Intercomparison was achieved by
adding common ketones in the reactions. Ketone concentration
was kept low (0.2 mM for each ketone, 0.6 mM total) to avoid
inhibition.
The division of two or more rate equations for a competing
substrate case, where the enzyme and substrate concentrations
are equal, will simplify to the division of the specificity con-
stants (kcat/KM), as described in ESI.† This applies irrespective
of the amount of competing substrates. Our experiment with sub-
stituted acetophenones can therefore be regarded as a comparison
of relative specificity constants. By experimentally determining
the specificity constant for acetophenone, by the method
described by Schätzle et al.,38 the remaining specificity constants
could be calculated from the relative values. Table 2 shows the
specificity constants for the chosen ketones in the wild type and
the mutant.
The equation can be written as
k
log ¼ fF þ rR
ð1Þ
k0
where k is the first order rate constant for a given substituted
species, k0 is the rate constant for the unsubstituted species,
F and R are the predetermined values for field and resonance for
a given substituent, and f and r are their coefficients which were
subject to least squares fitting to the given set of rate constants.
We treated the specificity constant (kcat/KM) as a first order
rate constant for this purpose. For the wild type enzyme, least
squares fitting revealed two points that were outliers. These
points were thereafter omitted from the fitting (but still shown in
the figure) to achieve a more accurate calculation of the coeffi-
cients for the remaining points which were in accordance with
the Swain–Lupton equation. Visually the plot appeared the same
with or without the inclusion of these points for the least squares
fitting. In the variant, in contrast, all points were included in the
least squares fitting and provided a good linear regression.
The redox state of the introduced cysteine residue was not
determined. Quantification of free cysteines, or disulfide bonds,
with Ellman’s reagent40,41 was attempted but the absorbance of
the holoenzyme together with the possibility of dissociation of
PLP with concomitant oxidation, yielding the carboxylic acid,
prevented accurate measurements; the absorbances of the said
species are in the same range as for the Ellman’s test.
Discussion
Cv-ωTA Trp60Cys shows an increased specificity constant for
(S)-1-PEA, which is consistent with the previously observed
increased enantiospecificity, compared to wild-type.29 The effect
is consistent with the hypothesis that a larger hydrophobic sub-
strate binding pocket favours the substrate phenyl group in the
(S)-configuration. However, this hypothesis cannot account for
The values in Table 2 were used to construct Swain–Lupton
plots,33–35 shown in Fig. 2. The Swain–Lupton equation has
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(S)-1-PEA will act as a competing substrate and virtually halt the
reaction. Continuous removal of the product and/or a large
excess of IPA are plausible solutions, if IPA remains the amino
donor of choice. Applying the appropriate amino acceptor–donor
pair has been shown to increase the yield.20
We observed a lower specificity for IPA in the variant com-
pared to the mutant. In the work by Cho et al. with Vf-ωTA21 the
corresponding tryptophan was mutated to a glycine which
yielded an enzyme with a two-fold increase of specific activity
for the resolution of 1-PEA, in agreement with our results, and a
19-fold increase for IPA, differing from our observations. Even
though we used a different enzyme similar results would be
expected. It is noteworthy that in the work by Cho et al. the
same pH was employed for both wild type and mutant. It is
possible that the apparent increase of activity for IPA is mainly
due to the conditions being more suitable for the mutant, and
that Vf-ωTA Trp57Gly has similar properties to Cv-ωTA
Trp60Cys in terms of pH optimum and PLP-dependence.
In our experiments with competing ketones the concentration
of IPA was chosen to be high enough for the ketone reaction to
be rate determining, but low enough for significant substrate
inhibition to be absent, if present. Complete two-substrate kine-
tics were not performed, but pseudo-one-substrate reactions with
acetophenone (1.0 mM) as the amino acceptor and varied con-
centrations of IPA as the amino donor were performed where no
substrate inhibition by IPA was detected within the chosen range
(5–100 mM for wild type and 5–250 mM for Trp60Cys). This
verifies that the 4′-substituted acetophenones are competing for
the enzyme : pyridoxamine-5′-phosphate-complex (E : PMP) in
our experiments and that the specificity constants in Table 2 are
valid (see ESI† for the reaction mechanism).
The Swain–Lupton parameterisation shows Cv-ωTA Trp60Cys
to be a more general catalyst for these substituted acetophenones
than the wild type since the agreement with the Swain–Lupton
equation was better over the substrate range. It can be assumed
that the enzyme contribution is approximately equal for all these
substrates in the Trp60Cys variant. The rate differences can be
attributed to the activation/deactivation by the substrate alone.
Whereas, in the wild type, the specificities for the 4′-nitro and
4′-cyano substituents were lower than the Swain–Lupton parame-
terisation would predict. The relative value of the Swain–Lupton
factors for the field and resonance effect is different for the
variant, compared to the wild type. The resonance effect contri-
bution has changed from 36% to 55%, in terms of the ratio of
the Swain–Lupton factors. This, together with the change of pH
optimum, indicates that the mechanism and/or the rate determi-
ning step in the amination reaction of the ketone have changed
upon mutation Trp60Cys. The increase of the resonance contri-
bution suggests that the increased space in the active site is not
the sole cause for the increased specificity. The putative role of
the introduced cysteine residue as a Lewis acid, or other electro-
chemical effects, can be regarded as a subject for further investi-
gations, based on these findings. The change of pH optimum to
7.0 in the variant may suggest that the introduced cysteine
residue actively partakes in the reaction. Possibly the cysteine
residue provides a field effect which lessens the importance of
field effects by the 4′-substituents; this is consistent with the
observed increased importance of resonance effects from the
substituents. The lack of inhibition of PLP in Trp60Cys, together
Fig. 2 Swain–Lupton plots for chosen 4′-substituted acetophenones
from the specificity constants listed in Table 2, for (A) Cv-ωTAwild type
and (B) variant Trp60Cys. The 4′-substitution of the acetophenones is
marked by the corresponding points on the graph. The values of the
coefficients f and r were calculated as 0.90 and 0.50 for the wild type,
and 0.28 and 0.34 for Trp60Cys. The percent resonance is 36% in the
wild type and 55% in the variant.
the successful mutation to a cysteine residue in contrast to an
alanine residue.
In a transamination reaction catalyzed by Cv-ωTA wild type
using IPA as the amino donor with any of the substituted aceto-
phenones, the second half reaction (the amination of the ketone)
can be regarded as rate determining. This is visible on compari-
son of the specificity constants; the values are the same for
a system with IPA as the amino donor for any of the aceto-
phenones. For the Cys variant, in contrast, the half reaction with
IPA is only rate determining for the two slowest reacting ketones
among the ones tested. One may also conclude that high yield
synthesis of (S)-1-PEA from acetophenone with IPA as the
amino donor is problematic since the enzymes favour the
product amine from IPA; as the reaction proceeds the formed
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9 K. O. Lidström, PharmaChem, 2007, 6, 5–7.
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of a difference of binding of the cofactor. Hypothetically, PLP
does not bind as a Schiff-base to the catalytic lysine, but is
bound in the PLP-binding pocket and may form a hemimercaptal
with the introduced cysteine residue. The impact of this cannot
be explained without a deeper investigation of the reaction mech-
anism and putative changes thereof and/or a crystal structure.
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Conclusions
We found a key point mutation in Cv-ωTA which increases the
specificity constant for the aliphatic amine (S)-1-PEA and a
range of 4′-substituted acetophenones. Plausibly, a more effective
enzyme for larger aliphatic substrates has been created. The
variant, Trp60Cys, has a more general nature than the wild type
for 4′-substituted acetophenones since their specificity constants
can be plotted in good agreement with the Swain–Lupton
equation, in contrast to the wild type enzyme. The point mutation
was found by structural alignments of homology models of other
engineered ωTAs and is therefore of interest for understanding
the mechanism and the development of rational design for this
enzyme group. The straightforward variant Trp60Ala was a poor
catalyst, the role of the cysteine residue in this position is not
trivial and a suitable subject for further investigation.
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Acknowledgements
The authors would like to acknowledge Prof. Wolfgang Kroutil
(Graz University) for supplying the gene for Cv-ωTA, Dr Linda
Fransson (KTH) for fruitful discussions and VINNOVA (The
Swedish Governmental Agency for Innovation Systems) for
financial support.
31 M. S. Humble, K. E. Cassimjee, M. Håkansson, Y. R. Kimbung,
B. Walse, V. Abedi, H.-J. Federsel, P. Berglund and D. T. Logan, FEBS
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