A colorimetric assay for screening transketolase activity

Article abstract of DOI:10.1016/j.bmc.2006.06.008

A tetrazolium red-based colorimetric assay has been devised to screen for transketolase activity with a range of aldehyde acceptors. The colorimetric TK assay is able to detect >8% bioconversion using non-α-hydroxylated aldehydes as acceptor substrates an

Full text of DOI:10.1016/j.bmc.2006.06.008

Bioorganic & Medicinal Chemistry 14 (2006) 7062–7065
A colorimetric assay for screening transketolase activity
Mark E. B. Smith,a Ursula Kaulmann,b John M. Wardb and Helen C. Hailesa,*
aDepartment of Chemistry, University College London, 20 Gordon Street, London WC1H OAJ, UK
bDepartment of Biochemistry and Molecular Biology, University College London, Gower Street, London WC1E 6BT, UK
Received 30 March 2006; accepted 6 June 2006
Available online 19 June 2006
Abstract—A tetrazolium red-based colorimetric assay has been devised to screen for transketolase activity with a range of aldehyde
acceptors. The colorimetric TK assay is able to detect >8% bioconversion using non-a-hydroxylated aldehydes as acceptor
substrates and is significantly faster and more convenient to use than chromatographic procedures.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
R
H
O
O
TK
Mg2+,TPP
+
OH
R
OH
O
LiO2C
OH
(S)-2
Transketolase (TK) (EC 2.2.1.1) is an important and
versatile enzyme that is used in stereospecific carbon–
carbon bond formation to give a,a0-dihydroxyke-
tones.1–12 In vivo TK catalyses the transfer of the
C1–C2 ketol unit from D-xylulose-5-phosphate to D-ri-
1
Scheme 1. Transketolase-catalysed reaction to generate a,a0-
dihydroxyketones.
bose-5-phosphate, in
a key step of the pentose
sensitive, easy to use and readily available assay is cru-
cial for the identification of active mutants.13 Indeed,
the lack of effective screening methods for high-through-
put usage has limited the number of variants that can be
screened. To date, TK activity has been monitored
using: a spectrophotometric method which measures
residual HPA using an NADH dependent enzyme as-
say;5,9 an HPLC assay for erythrulose product forma-
tion and HPA depletion,6 as well as preparative scale
product isolation. However, these methods suffer from
problems such as low sensitivities, or low throughput.
Recently an assay for TK was reported, able to detect
wild-type TK activity in vitro using fluorescence, but it
is limited to substrates containing the umbelliferone
moiety which may effect the bioconversion.14 It was de-
signed for the selection of mutants that produce D-threo
aldoses or L-erythro ketoses.14 In our studies, we sought
a general method for screening TKs with 2-non-hydrox-
ylated aldehyde acceptors as substrates. Herein we de-
scribe the use of tetrazolium red in a new assay for
TK activity.
phosphate pathway, and the enzyme requires magne-
sium(II) ions as well as thiamine pyrophosphate (TPP)
as cofactors.1 This stereospecific transfer is reversible,
however when b-hydroxypyruvic acid (HPA) is used as
a donor substrate for TK, the resulting loss of carbon
dioxide renders the reaction irreversible.12 The
TK-catalysed condensation of HPA (the free acid or
Li-salt 1) with a variety of 2-hydroxyaldehydes1–7 and
2-non-hydroxylated aldehydes8–11 has been performed
successfully in vitro to generate (S)-2 on a preparatively
useful scale (Scheme 1). Although the enzyme appears to
be highly specific for the donor substrate, more structur-
al variation in the aldehyde acceptor is tolerated; includ-
ing 2-non-hydroxylated aldehydes, although lower
relative rates of reaction are normally observed.8–11
Our current work is focusing on the evolution of TK to
generate variants with new or improved properties. In
particular, mutants are being generated with the aim
of increasing the rate of reaction with a range of accep-
tor aldehydes compared to wild-type. The use of a rapid,
2. Results and discussion
Keywords: Colorimetric assay; Transketolase; Directed evolution;
Tetrazolium red.
*
Our aim was to establish a colorimetric assay, particu-
larly for use as a first pass binary screen to rapidly iden-
Corresponding author. Tel.: +44 0 20 7679 4654; fax: +44 0 20 7679
7463; e-mail: h.c.hailes@ucl.ac.uk
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.06.008

M. E. B. Smith et al. / Bioorg. Med. Chem. 14 (2006) 7062–7065
7063
tify potential TK variants for further investigation.
Recently, a pyruvate decarboxylase assay has been
reported for the detection of aromatic acyloins, notably
phenylacetylcarbinol, using 2,3,5-triphenyltetrazolium
chloride (tetrazolium red).15 Tetrazolium red, which is
colourless, oxidized the 2-hydroxyketone to the dike-
tone, and the tetrazolium was reduced to the corre-
sponding formazane which has an intense red colour
(kmax ꢀ 485 nm). Although a 2-hydroxyketone motif is
present in the starting material Li-HPA (1) in the TK
reaction as well as in the expected product, we set out
to establish whether such a tetrazolium-based assay
could be used in assays with non-a-hydroxylated alde-
hyde acceptors and TK.
A
B
C
D
E
Figure 1. Calibration assay for the generation of 4, produced after the
addition of (S)-2a to 3. The wells (A–E) correspond to a bioconversion
that generates the following concentrations of 2a: A, 50 mM; B,
25 mM; C, 10 mM; D, 5 mM; E, 2.5 mM (OD485nm measured after
2 min).
As a model system propanal was used with Li-HPA (1)
and an Escherichia coli TK strain (XL10/pQR711),
capable of overexpressing wild-type E. coli TK (Scheme
2).6 Initial experiments were carried out using (3S)-l,3-
dihydroxypentan-2-one9 (2a) at a range of concentra-
tions, corresponding to typical concentrations to be used
in TK assays, in Gly-Gly buffer (pH 7.0) to determine
whether and at what concentration 2a could be detected
using tetrazolium red (3). A calibration assay protocol
was established to avoid signal saturation, but that
would be sufficiently sensitive at low concentrations of
bioconversion. Addition of an excess of tetrazolium
red and sodium hydroxide solution (3 M) led to a red
colouration due to the formation within 2 min of for-
mazane (4) and predominantly the diketone shown (or
a ketoaldehyde). The intensity displayed increased with
increasing concentrations of 2a (Fig. 1). Optical density
(OD) measurements were also carried out at 485 nm and
a calibration curve was constructed (Fig. 2).
Figure 2. Calibration curve for formazane 4, produced after the
addition of (S)-2a to 3 (r2 = 99.7%).
anal, since the effect of cofactors and other components of
the lysate was unknown. The TK reaction was performed
for 17 h. Control experiments readily established that
propanal, TPP, Mg(II) and TK lysate gave rise to no
red colouration (e.g., Fig. 3, well I). However the reaction
between Li-HPA (1) and tetrazolium red solution gave
rise to the distinctive red colouration of formazane due
to the presence of the hydroxyketone group in 1. Different
methods were therefore investigated to remove any
remaining Li-HPA at the end of the TK reaction which
could mask the assay. A protocol was initially envisaged
involving the addition of lactate dehydrogenase (LDH)
(and NADH as cofactor) at the end of the TK reaction
to reduce any remaining 1 to the corresponding dihydr-
oxyacid, which was shown to be unreactive with tetrazo-
lium red (3). Whilst effective in removing 1 in test
reactions with Li-HPA alone in buffer, when the same
conditions were used with TK present, a red colouration
still remained, even though it was less intense. It was pos-
sible that this was due to the non-specific binding of Li-
HPA to TK, leaving remains of 1 that are not oxidized
by the LDH, thus giving rise to a low level positive test
with 3.
In buffer only, 2a was detectable down to a correspond-
ing bioconversion of 2.5 mM (actual concentration after
dilution in the assay protocol 12.5 nmol; see Section 3).
An alternative substrate l,3-dihydroxy-3-phenylpropan-
2-one (Scheme 1, 2b R = Ph) was also tested in the assay
and comparable detection levels were determined.
With the sensitivity and rapid nature of the assay using 3
established, its use in a TK assay was investigated by the
addition of tetrazolium red after a TK reaction with prop-
O
R
H
O
TK
Mg2+, TPP
+
R
OH
OH
O
LiO2C
OH
1
(
S)-2a R = C2H5
2b R = Ph
N
Ph
Ph
Tetrazolium red-
colourless
N
+
N
N
3
Ph
–
Cl
NaOH
F
G
H
I
N
NHPh
Formazane-
Ph
4
red colouration
Figure 3. TK reaction with propanal to give 2a (Scheme 2) and
control experiments after addition of 3 and MP-carbonate resin: F
17 h reaction with TK/TPP/Mg2+, propanal, 1 (OD485nm 0.427); G
17 h reaction with TPP/Mg2+, propanal, 1 (OD485nm 0.0712); H 17 h
reaction with TK/TPP/Mg, 1 (OD485nm 0.1172); I 17 h reaction with
TK/TPP/Mg2+, propanal (OD485nm 0.0025).
N
N
Ph
O
R
OH
O
Scheme 2. Use of 3 in a colorimetric assay with (S)-2a and 2b.

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M. E. B. Smith et al. / Bioorg. Med. Chem. 14 (2006) 7062–7065
An alternative approach was used, to remove remain-
ing 1 by the addition of an ion exchange resin. The
use of secondary amine resins was avoided in case
any synthetic coupling reactions between Li-HPA (1)
and propanal were observed.16 The use of a quaterna-
ry amine functionalized MP-carbonate resin (Biotage)
again added prior to the addition of tetrazolium red
was found to be more effective than LDH, removing
all but trace quantities of the red colouration of 4
from solution. A representative experiment showing
the detection of 2a formed in a 50 mM TK reaction
and control reactions are shown in Figure 3. As be-
fore, the reactions were imaged after 2 min and
OD485nm measurements taken.
3.2. Calibration of the assay for (3S)-l,3-dihydroxypen-
tan-2-one
Stock solutions of (3S)-l,3-dihydroxypentan-2-one9 were
made up at 50, 25, 10, 5 and 2.5 mM concentrations in
Gly-Gly buffer (50 mM, pH 7.0). Each solution (5 lL)
was diluted with Gly-Gly buffer (95 lL, 50 mM, pH
7.0) prior to the addition of tetrazolium red solution
(20 lL, 0.2% 2,3,5-triphenyltetrazolium chloride in
methanol) and finally 3 M NaOH (aq) (10 lL) with
good mixing. The reactions were imaged after 2 min
and OD485nm measurements made (against buffer) using
a FLUOstar Optima plate reader (BMG Labtechnolo-
gies GmbH).
Well F contains all the components in a typical TK as-
say and the red colouration clearly indicated the forma-
tion of 2a. The OD485nm data indicated approximately
15 mM of 2a was formed which is consistent with previ-
ous work using propanal in the TK reaction using wild-
type E. coli TK.9 Well G had an almost undetectable
colouration and very low OD485nm value, most likely
due to traces of Li-HPA or the chemical decomposition
of 1 to give 2-hydroxycarbonyl products such as glycol-
aldehyde. Well H generated a colouration (OD485nm
0.1172) possibly again predominantly due to the non-
specific binding of Li-HPA (1) to TK, which is then
not fully removed from solution. Nevertheless, this ra-
pid, low cost assay will enable TK variants to be identi-
fied that are able to couple HPA and aldehyde acceptors
with a >8% bioconversion.
3.3. Assaying for TK-mediated synthesis of (3S)-l,3-
dihydroxypentan-2-one
A 90 mL reaction mixture containing propanal (50 mM),
lithium hydroxypyruvate (50 mM), TPP (2.4 mM),
MgCl2 (9 mmol) and cell-free lysate (50% total reaction
volume) in Gly-Gly (50 mM, pH 7.0) was incubated at
20 °C for 17 h. Ten microlitres of the reaction mixture
was then transferred to a microwell containing MP-car-
bonate resin (Biotage AB) (10 mg) and Gly-Gly buffer
(90 lL, 50 mM, pH 7.0) and the mixture was incubated
at 20 °C for 3 h. Fifty microlitres of this mixture (without
resin beads) was then diluted with further Gly-Gly buffer
(50 lL, 50 mM, pH 7.0), then tetrazolium red solution
(20 lL) and finally 3 M NaOH (aq) (10 lL) with good
mixing. Three control experiments lacking in TK, prop-
anal and lithium hydroxypyruvate, respectively, were al-
lowed to proceed in parallel and were worked up in an
identical fashion. All reactions were imaged after 2 min
and OD485nm measurements carried out.
In summary, a new low cost, rapid colorimetric TK as-
say has been identified, able to detect >8% bioconver-
sion using non-a-hydroxylated aldehydes as acceptor
substrates. This assay is significantly faster and more
convenient to use than HPLC and can be used with a
range of aliphatic and aromatic aldehydes. In addition,
analysis of the a,a0-dihydroxyketone produced in the
bioconversion can be quantified using this assay system
with high-throughput. Furthermore, this method has the
potential to be used to screen other chemical reactions
or bioconversions leading to the formation of products
possessing a 2-hydroxyketone motif.
Acknowledgments
The authors thank the UK Engineering and Physical
Sciences Research Council (EPSRC) for support of the
multidisciplinary Biocatalysis Integrated with Chemistry
and Engineering (BiCE) programme (GR/S62505/01)
(M.E.B.S. and U.K.). Financial support from the 13
industrial partners supporting the BiCE programme is
also acknowledged. We also thank Prof. N. J. Turner
for helpful discussions.
3. Experimental
3.1. TK biocatalyst preparation
References and notes
Escherichia coli transformant (XL10/pQR711),17 capa-
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obtained as previously reported. Inoculation in Luria–
Bertani medium containing ampicillin for 16–24 h at
37 °C gave a cell broth with an OD600nm ꢁ 5. The cell
broth was centrifuged (1 h, 10,500 rpm) and the super-
natant discarded. The wet cell pellet was resuspended
in cold sodium phosphate buffer (5 mM, pH 7.0) to a
dilution of 1 g wet cell pellet/10 mL buffer and sonicated
on ice to promote cell lysis. Following further centrifu-
gation (5 min, 10,500 rpm) the cell-free lysate was ob-
tained and used in experiments without further
purification.
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