Separating Thermodynamics from Kinetics—A New Understanding of the Transketolase Reaction

Article abstract of DOI:10.1002/cctc.201601649

Transketolase catalyzes asymmetric C?C bond formation of two highly polar compounds. Over the last 30 years, the reaction has unanimously been described in literature as irreversible because of the concomitant release of CO₂ if using lithium hydroxypyruvate (LiHPA) as a substrate. Following the reaction over a longer period of time however, we have now found it to be initially kinetically controlled. Contrary to previous suggestions, for the non-natural conversion of synthetically more interesting apolar substrates, the complete change of active-site polarity is therefore not necessary. From docking studies it was revealed that water and hydrogen-bond networks are essential for substrate binding, thus allowing aliphatic aldehydes to be converted in the charged active site of transketolase.

Full text of DOI:10.1002/cctc.201601649

Accepted Article
Title: Separating Thermodynamics from Kinetics - A New
Understanding of the Transketolase Reaction
Authors: Stefan R. Marsden, Lorina Gjonaj, Stephen J Eustace, and Ulf
Hanefeld
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemCatChem 10.1002/cctc.201601649
Link to VoR: http://dx.doi.org/10.1002/cctc.201601649
A Journal of
www.chemcatchem.org

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
Separating Thermodynamics from Kinetics – A New
Understanding of the Transketolase Reaction
[a]
[a]
[a]
[a]
Stefan R. Marsden , Lorina Gjonaj , Stephen J. Eustace and Ulf Hanefeld*
Transketolase catalyzes asymmetric C-C bond formation of two
highly polar compounds. Over the last 30 years, the reaction has
unanimously been described in literature as irreversible due to the
product. For this reason, the TK catalyzed reaction with
LiHPA and decarboxylation driven reactions in general are
[
2-10]
traditionally described as irreversible in literature.
concomitant release of CO
2
when using lithium hydroxypyruvate
Indeed, the first S. cerevisiae TK catalyzed synthesis of L-
(LiHPA) as substrate. When following the reaction over a longer
erythrulose was performed with LiHPA to ensure it to be
period of time however, it was now revealed to be initially kinetically
controlled. For the non-natural conversion of synthetically more
interesting apolar substrates, a complete change of active site
polarity is counterintuitively not necessary. Docking studies revealed
water and hydrogen bond networks to be essential in substrate
binding, thus allowing aliphatic aldehydes to be converted in the
charged active site of transketolase.
[11-13]
irreversible.
Yet in 2004, the coupling of two molecules
[14]
of glycolaldehyde to L-erythrulose was reported.
In
combination with the reversibility of the natural TK
catalyzed reactions, this renders an irreversible product
formation unlikely from a mechanistic point of view. In
recognition of the extensive use of decarboxylation in
contemporary C-C bond formation strategies, a correct
understanding of the actual impact of decarboxylation on
the overall reaction is thus of great importance. In particular
since this synthetically very powerful decarboxylation has
the disadvantage of a poor atom economy. Secondly,
although aliphatic substrates were successfully converted, it
remains yet to be fully understood how this is possible. With
phosphorylated polyols as typical substrates, TKs are
naturally not disposed towards aliphatic substrates.
Nevertheless, E. coli TK has successfully been engineered
Introduction
2
+
Transketolase (TK, E.C. 2.2.1.1) is a Mg and thiamine
diphosphate (ThDP) dependent enzyme which naturally
catalyzes the conversion of glycolysis derived metabolites
into carbohydrates utilized for nucleotide synthesis and the
production of essential aromatic amino acids via the
[
1]
Shikimate pathway. The overall reaction comprises of the
reversible transfer of a C -ketol group and an asymmetric
by single-point mutations to convert a variety of aromatic
2
[6,7]
and aliphatic aldehydes.
This is surprising, since the
C-C bond formation. This makes the reaction interesting for
synthetic applications. A multitude of enzymatic strategies
have been developed in order to address the substantial
importance of asymmetric C-C bond formation in organic
synthesis, of which many rely on decarboxylation as driving
mutations introduced in E. coli TK do not render the active
[6]
site highly lipophilic.
S. cerevisiae TK shares 47% sequence identity with E.
coli TK and the aligned crystal structures (1QGD and
1
TRK) have an RMSD of 0.81 indicating extensive
[
2-5]
2
force for the C -ketol transfer.
structural homology. Due to its facile heterologous
overexpression in E. coli, S. cerevisiae TK was chosen as
model enzyme to representatively investigate both the
actual impact of decarboxylation in asymmetric C-C bond
synthesis and the cause of enhanced activity towards
aliphatic aldehydes previously observed for single-point
Scheme 1: Natural TK reaction.
Scheme 2: Synthetic TK reaction.
[
6,7]
mutations.
In order to obtain an improved understanding of the TK
catalyzed reaction, two points will be addressed:
hydroxypyruvate (HPA) is currently utilized as the ketol
donor of choice, because the considerable change in Gibb’s
Results and Discussion
Free Energy, which follows from the liberation of CO
2
,
Figure 1: compounds overview.
results in an equilibrium constant entirely in favor of the
The E. coli TK mutants D469E and D469T described earlier
remarkably showed that highly polar or even charged amino
acids improved enzyme activity towards aliphatic
[a]
Stefan R. Marsden, Dr. Lorina Gjonaj, Dr. Stephen J. Eustace and
Prof. Ulf Hanefeld Biokatalyse, Afdeling Biotechnologie
Technische Universiteit Delft
van der Maasweg 9, 2629HZ Delft, The Netherlands
E-mail: u.hanefeld@tudelft.nl
[6]
aldehydes. This is in contrast to our results that non-
phosphorylated substrates are better converted by TK
mutants of reduced polarity (R528K, R528Q, R528K/S527T
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
[
15-16]
and R528Q/S527T).
Therefore, the equivalent
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
mutations D477E and D477T were created in S. cerevisiae
TK to allow for a direct comparison and an improved
understanding. Preparative reactions (Table 1) were in line
with those reported for E. coli TK. Again, mutant D477E
was surprisingly identified as the best catalyst for the
conversion of aliphatic aldehydes. While synthetically
relevant, these data however do not allow the evaluation of
the catalytic activity of the separate mutants.
Scheme 3: proposed mechanism for carbanion formation.
For the synthesis of L-erythrulose from glycolaldehyde and
LiHPA as substrates in aqueous solution under standard
0
r
conditions, the total change in Gibb’s Free Energy ∆ G
amounts to -264.5 kJ/mol (L-erythrulose, S18 ESI), largely
due to the contribution of decarboxylation. Overall, this
46
would correspond to an equilibrium constant of Keq = 10 in
favor of the product. In 2004, the one-substrate TK
catalyzed
reaction
coupling
two
molecules
of
Table 1: Isolated yields, enantiomeric excess.
[14]
glycolaldehyde to L-erythrulose was reported
and in
strong contrast to the decarboxylation driven reaction, an
Analysis of the Michaelis-Menten parameters confirmed
the results obtained preparatively. Mutant D477E was found
the most successful one in the conversion of aliphatic
aldehydes 1a and 2a showing an enhanced activity of 50 to
equilibrium constant of Keq = 5.0 was calculated from the
0
change in Gibb’s Free Energy, (∆
r
G
= 4.0 kJ/mol
L-erythrulose in aqueous solution under standard
conditions, S18 ESI). Supported by the reversibility of the
natural reactions, the one-substrate reaction should
therefore be a true equilibrium reaction. In the proposed
mechanism for TK catalyzed reactions with lithium
hydroxypyruvate, the thermodynamically irreversible
decarboxylation of LiHPA effects the direct formation of the
carbanion on the activated ketol. For the one-substrate
reaction however, the activated carbanion must be formed
by catalytic deprotonation from residue His481 as
alternative to decarboxylation, generating the activated
intermediate at a lower rate in comparison to its generation
by decarboxylation. At the stage of the activated ketol
bearing the carbanion, the enzyme can no longer
distinguish whether it was formed via a reaction pathway
involving decarboxylation, or via catalytic deprotonation.
The information about the thermodynamic driving force of
decarboxylation is therefore already lost prior to the actual
1
00 fold compared to the WT. While mutations at position
R528, which natively binds to the phosphate group in
[
15-16]
phosphorylated substrates
and the incorporation of a
[
17]
group mutation strategy did enhance enzyme activity, the
improvements were only minor compared to the effect of
mutation D477E.
Table 2: Michaelis-Menten parameters.
In silico docking studies
Figure 2: docked substrate in the model active site.
-
1
With an observed improvement of 50 to 100 fold in kcatK
M
for the conversion of substrates 1a and 2a with D477E by
only a single-point mutation, the mutation D477E was
introduced in silico into the corresponding crystal structure
[
18]
product
formation.
These
mechanistic
reflections
1
GPU in order to investigate the resulting changes in the
consequently suggest, that TK catalyzed synthesis
reactions are reversible via the mechanism of the one-
substrate reaction, splitting the product back into one
molecule of the respective acceptor aldehyde and one
active site. The obtained model was energy minimized
before docking of substrates 1a-4a into the active site using
[
19]
YASARA.
The model showed that extension of the
carbon chain by mutating aspartate to glutamate newly
enabled hydrogen bond interactions between the glutamate
carboxylate and the substrate carbonyl groups bridged by a
molecule of coordinated water at 1.7 Å each. In this
manner, the substrate is correctly aligned towards the
cofactor and the forming oxyanion is stabilized by charge
delocalization during nucleophilic attack. This interaction
was correctly predicted by the model for the converted
substrates 1a-3a and not predicted for the unconverted
substrate 4a (fig. 2 and fig. S5-S8 ESI). In combination with
preparative and kinetic data the docking studies illustrate,
that correct substrate orientation towards the activated
molecule
of
glycolaldehyde.
The
thermodynamic
contribution of decarboxylation therefore should not affect
the position of the overall equilibrium (compare scheme 3)
and argue against an irreversible product formation. In
conclusion, it should thus be possible to avoid the release
2
of CO and to improve the atom economy of the reaction.
Equilibrium analysis
Scheme 4: reaction schemes equilibrium analysis.
In order to experimentally confirm the reversibility of the TK
catalyzed product formation suggested by the mechanistic
reflections, L-erythrulose was synthesized both via the one-
cofactor (improving not only kcat, but potentially also K
of greater importance for catalysis than an increase based
solely on substrate affinity (improving only K ). This would
also explain why the introduction of an isoleucine into the
equivalent position in the TK of Geobacillus
stearothermophilus did not lead to such large rate
M
) is
substrate
reaction
coupling
two
molecules
of
M
glycolaldehyde and via the conversion of glycolaldehyde
with LiHPA to afford the product L-erythrulose in 100 mM
concentration for complete conversion using WT S.
cerevisiae TK. The reactions were performed in sealed
NMR tubes allowing for direct measurements of the product
[
20]
improvements.
[
21]
erythrulose
(fig. S40 ESI). The substrates were not
Mechanistic reflections
followed due to complete consumption within 30 minutes in
the case of LiHPA and due to the issue of oligomerization
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
and hydration of glycolaldehyde in aqueous solution.
[
22]
traditionally are misinterpreted in literature as irreversible,
Both reactions were followed over an extended period of
mechanistic
reflections
and
experimental
evidence
[
14]
unambiguously showed the reaction to initially be under kinetic
control. In the context of man-made climate change, we thus
have to extensively re-evaluate our choice of donor substrates
and the utilization of decarboxylation strategies in synthetic
applications.
time. In line with the results earlier published,
L-
erythrulose formation was observed. The one-substrate
reaction proceeds relatively rapidly (fig. 3A) but is limited to
less than 30% yield due to the thermodynamic equilibrium
of the reaction (fig. 3B).
Figure 3:
Experimental Section
When LiHPA was used as ketol donor, a fast and
[
3-10,15-16,20]
complete conversion was observed as expected
fig. 3A). If this reaction was thermodynamically controlled
by the release of CO it should stop here. However, in line
Materials
(
Chemicals and solvents were obtained as reagent grade from
Sigma-Aldrich. Aldehydes were freshly distilled and their purity
2
with a reversible reaction, a slow decline of L-erythrulose
concentration was subsequently observed ultimately
coinciding with the equilibrium concentration of the one-
substrate reaction at Keq=29.1±0.6 mM. The synthesis
reaction was thus shown to benefit from a kinetic effect
enabling high yields at the beginning of the reaction. The
reverse reaction causing thermodynamic equilibration to
occur over a time course of several weeks then shifted the
product distribution; in line with the outcome of the one-
substrate reaction (fig. 3B). In order to confirm that the
observed equilibration indeed was enzyme catalyzed,
another portion of LiHPA was added at the end. Retained
enzymatic activity was observed (fig. 3B, inset), while
control reactions without enzyme showed no conversion.
The representative formation of L-erythrulose from
glycolaldehyde and LiHPA was thus shown to be initially
kinetically controlled contrary to all earlier assumptions
1
confirmed by H NMR before usage. Petrolether (b.p. 40-60°C) was
freshly distilled before usage. Lithium hydroxypyruvate was
obtained both commercially and synthesized as previously
[
30]
described.
Methods
Reaction progress was monitored by TLC (TLC Silica gel 60 F254
,
Merck) using UV light and a potassium permanganate stain for
visualisation. NMR spectra were recorded using an Agilent 400
1
1
MHz ( H, 9.4 Tesla) spectrometer operating at 399.67 MHz for
at 298K and were subsequently interpreted using MNOVA. A
benzene-D NMR insert capillary (Sigma-Aldrich) was used for
H
6
external locking during water suppression experiments using the
PRESAT-PURGE pulse sequence in sealed Wilmad® screw-cap
NMR tubes (Sigma Aldrich). Spectra were recorded using a recycle
delay of
bioconversions were carried out in an Excella E24 Incubator Shaker
New Brunswick Scientific).
2 seconds and 64 repetitions. Preparative scale
2
about the thermodynamic driving force of CO release. The
(
proposed reaction mechanisms depicted in scheme 3
suggest these findings to generally hold true for all TK
catalyzed reactions with HPA. Following the example of the
pyruvate decarboxylase catalyzed synthesis of (R)-
phenylacetylcarbinol with acetaldehyde replacing the
Preparation of cell free extract. The cell pellet containing the
respective mutant TK was resuspended in sodium phosphate buffer
(5 mM, pH = 7.0, 10 mL/g cell pellet). A protease inhibitor (PMSF,
200 µL, 0.1M in EtOH) was added to each sample. Lysosyme was
added at 20 mg/g cell pellet and a spatula tip of DNAse was added
to each sample and incubated on ice for 30 minutes. The cells were
broken using a sonifier 250 (Branson) and the cell debris removed
by centrifugation.
[
23]
traditional donor substrate pyruvate,
the development of
novel strategies which do not rely on decarboxylation is of
commercial relevance. To do so, a correct understanding of
decarboxylation is of utmost importance. In syntheses
where aldehydes other than glycolaldehyde are used as
acceptors, formation of the desired product will be
competing with the one-substrate reaction. Active site
engineering as pioneered by Pohl for a range of ThDP
dependent enzymes could ensure that glycolaldehyde will
Enzyme purification. The cell pellet was resuspended in binding
buffer (5 mM sodium phosphate, pH = 7.4, 20 mM imidazole) and
incubated with PMSF, lysozyme and DNAse as previously
described. The cells were subsequently broken using
a cell
disrupter (Constant Systems Ltd, 1.8 kbar), the cell debris removed
by centrifugation and the cell free extract filtered (0.45 µm). Affinity
chromatography was performed on a NGC Quest 10 system
[
24]
(
Biorad) using XK16/20 columns (GE Healthcare Life Sciences)
be the donor molecule in mixed carbo ligation reactions.
packed with 10 mL Ni-sepharose 6 FF resin (GE Healthcare Life
Sciences). For full details see ESI.
Synthesis of racemic standards. Racemic standards were
Conclusions
[31]
synthesized according to a method previously described.
N-
methylmorpholine (330 µL, 3.0 mmol, 1.0 eq.) was dissolved in
water (40 mL) and the pH was adjusted to 8.0 using 10% HCl.
LiHPA (330 mg, 3.0 mmol, 1.0 eq.) and the corresponding aldehyde
(3.0 mmol, 1.0 eq.) were added and the reaction was stirred
overnight at room temperature. Conversion was monitored by TLC
Creating novel interactions between an active site residue and a
desired substrate should include network of hydrogen
As was shown, this is an effective strategy to
increase the substrate’s affinity towards the active site, although
a polarity based analysis would suggest the opposite. This
alternative approach for the rational mutagenesis of TKs towards
a
[
25-29]
bonds.
(
n-pentane / EtOAc 1:1). Silica powder was added, the water
removed in vacuo and the crude product purified by flash
chromatography (n-pentane / EtOAc 1:1). For full details see ESI.
Dibenzoylation of enantiomers. Dihydroxyketone (1.0 eq.) was
hydrophobic
substrates
was
demonstrated.
While
decarboxylation driven C-C bond formation reactions
2
dissolved in dry dichloromethane (10 mL) under N atmosphere in a
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
flame dried round bottomed flask. Dry triethylamine (10.0 eq.) and
benzoyl chloride (5.0 eq. per hydroxyl) were added and the reaction
mixture was stirred for two hours at room temperature. It was
concentration was followed by its characteristic peaks 4.61 (1H, d,
2
2
J
HH 19.6 Hz), 4.52 (1H, d, JHH 19.6 Hz). L(+) erythrulose was
obtained in the highest quality commercially available (Sigma-
Aldrich) and the calibration curve was corrected mathematically for
a purity of 85%. Enzyme (WT TK, 200 µg, 1.35 nmol) was
quenched by addition of sat. NaHCO
separated and the organic phase was washed (sat. NaHCO
mL, then sat. NH Cl, 1x, 50 mL, then brine, 1x, 30 mL). The organic
phase was dried over Na SO , the solvent was removed in vacuo
3
(30 mL), the phases
3
, 2x, 50
2+
4
incubated with its cofactors (25°C, 20 min, ThDP: 5 mM, Mg : 18
mM, 5 mM sodium phosphate buffer pH = 7.0). LiHPA driven
conversion: glycolaldehyde and LiHPA were added to achieve final
concentrations of 100 mM each and the reaction volume was
adjusted to 500 µL. One-substrate reaction: glycolaldehyde was
added to achieve a final concentration of 200 mM and the reaction
volume was adjusted to 500 µL.
2
4
and the crude product was purified by flash chromatography in the
case of racemic standards (petrolether / EtOAc 10:1). Purification
by flash was omitted in the determination of the enantiomeric
excess. For full details see ESI.
[
15]
Glycolaldehyde activity assay. The volumetric activity of cell free
extracts was determined by incubating 50 µL with the cofactors
Computational docking studies. In silico docking studies were
carried out with YASARA (Version 16.2.18) using the crystal
structures 1TRK (free ThDP cofactor) and 1GPU (containing the
activated ketol) for S. cerevisiae TK and 1QGD for E. coli TK. The
simulation box was defined at 10 Å around the thiamine C2 in 1TRK
and around the ylid anion in 1GPU. The substrates were energy
minimized with ChemBio3D Ultra 12.0 (Cambridgesoft) using MM2
energy minimization. The mutation D477E was introduced into
1GPU and the model was subsequently energy minimized using
YASARA before docking. For full details see ESI.
2+
(
25°C, 800 rpm, 20 min, ThDP: 5 mM, Mg : 18 mM). LiHPA and
glycolaldehyde were added to achieve final concentrations of 50
mM in 300 µL total reaction volume
5 mM sodium phosphate buffer, pH = 7.0). The reaction mixture
was shaken (25°C, 800 rpm, 15 min), quenched by addition of TFA
300 µL, 0.2% v/v), the enzyme precipitated by centrifugation and
(
(
2
analyzed by RP HPLC (R = 0.998) to determine the volumetric
activity. Due to considerably varying volumetric activities of cell free
extracts the enzyme content was normalized to 20 U of activity
based on a glycolaldehyde activity assay previously reported.
Computational docking of glycolaldehyde into
[
15]
the
corresponding mutant active sites with YASARA predicted
comparable binding energies for all mutants. It was thus concluded
that none of the mutations are likely to have introduced a major bias
to an activity based analysis using glycolaldehyde as reference. Full
details see ESI.
Acknowledgements
The authors thank Daan F. J. van Overveld, Rosario Medici
and Albert Godoy Hernandez for help with the enzyme
production and purification. Financial support from STW
Preparative scale bioconversions. Cell free extract (20U based on
the glycolaldehyde activity assay) was incubated with its cofactors
(
grant 11142) to L. G. is gratefully acknowledged.
(
7
20 min, room temperature, 5 mM sodium phosphate buffer, pH =
.0, ThDP: 18 mM and Mg : 5 mM). LiHPA (110 mg, 1.0 mmol, 1.0
2+
eq.) and the corresponding aldehyde (1.0 mmol, 1.0 eq.) were
added and the reaction volume was adjusted to 10 mL. The
reaction was carried out in a sealed flask overnight (25°C, 200
rpm). The product was extracted with MTBE (2x, 40 mL) and the
solvent was removed in vacuo.
Keywords: Transketolase • Thermodynamic control • Kinetic
control • water network • aldehyde
[1]
a) S. Hohmann, P. A. Meacock, Biochim. Biophys. Acta, 1998,1385,
01-219; b) R. Wohlgemuth, J. Mol. Catal. B: Enz., 2009, 61, 23-29; (c)
2
Chiral separation. Enantiomers were derivatized by dibenzoylation
and chiral separation was performed on a Shimadzu LC-20AD
prominence system equipped with a Chiralpak AD-H column
R. Kluger, K. Tittmann, Chem. Rev. 2008, 108, 1797-1833.
M. Pohl, C. Wechsler, M. Müller in Science of Synthesis, Biocatalysis in
Organic Synthesis 2, (Eds.: K. Faber, W.-D. Fessner, N. J. Turner),
Georg Thieme Verlag, Stuttgart, 2014, pp. 93-127.
[2]
(
0.46x25 cm, Daicel) using n-heptane / i-PrOH 97:3 as mobile
phase (35°C, 1 mL/min).
[3]
M. Brovetto, D. Gamenara, P. S. Méndez, G. A. Seoane, Chem.
Rev., 2011, 111, 4346-4403.
[
15]
Analytical quantitation.
Dihydroxyketone product concentrations
[
4]
J. Sukumaran, U. Hanefeld, Chem. Soc. Rev., 2005, 34, 530-542.
A. Ranoux, U. Hanefeld in Stereoselective Synthesis of Drugs and
Natural Products, (Eds.: V. Andrushko, N. Andrushko), John Wiley &
Sons, Inc., 2013, pp. 831-857.
were determined by RP HPLC on a Shimadzu LC-20AD prominence
system equipped with an IC-Sep Coregel 87H3 column (0.4x25 cm,
Transgenomic). The absorbance was followed at 210 nm using
[5]
[
6]
A. Cázares, J. L. Galman, L. G. Crago, M. E. B. Smith, J. Strafford,
L. Ríos-Solís, G. J. Lye, P. A. Dalby, H. C. Hailes, Org. Biomol.
Chem., 2010, 8, 1301-1309.
0
.1% (v/v) aqueous trifluoroacetic acid (TFA) pH = 2.5 as mobile
phase (60°C, 0.8 mL/min).
Determining Michaelis-Menten parameters. Individual reaction times
were initially determined to measure the parameters under credible
initial rate conditions (<20% conversion). The buffered reaction
mixture (300 µL, 5 mM sodium phosphate, pH = 7.0) containing
holotransketolase (50 µg / 337 pmol, 1 mM ThDP, 4 mM Mg ),
LiHPA (100 mM) and the corresponding aldehyde at varied
concentrations (5 – 150 mM) were incubated (25°C, 500 rpm) in
duplicate. The reactions were quenched by 1:1 addition of 0.2%
[7]
[8]
[9]
J. L. Galman, D. Steadman, S. Bacon, P. Morris, M. E. B. Smith, J.
M. Ward, P. A. Dalby, H. C. Hailes, Chem. Commun., 2010, 46,
7608-7610.
F. Subrizi, M. Cárdenas-Fernández, G. J. Lye, J. M. Ward, P. A.
Dalby, T. D. Sheppard, H. C. Hailes, Green Chem., 2016, 18, 3158-
3165.
2+
T. Saravanan, M. Reif, D. Yi, M. Lorillière, F. Charmantray, L.
Hecquet, W.-D. Fessner, Green Chem., 2017, 19, 481-489.
(
v/v) TFA, the enzyme was precipitated by centrifugation and the
[10] M. Lorillière, M. De Sousa, F. Bruna, E. Heuson, T. Gefflaut, V. de
Berardinis, T. Saravanan, D. Yi, W.-D. Fessner, F. Charmantray, L.
supernatant was subjected to RP HPLC analysis. A Michaelis-
Menten type non-linear fit was obtained from the Excel built-in
solver successively minimizing the sum of the squared errors
between measured and fitted data points converging towards
Hecquet, Green Chem., 2017, 19, 425-435.
[11] U. Schörken, G. A. Sprenger, Biochim. Biophys. Acta, 1998, 1385,
229-243.
values for K
Equilibrium analysis by NMR.
from a NMR insert capillary was used as reference and its integral
M
and vmax. For full details see ESI.
[12] Y. Kobori, D. C. Myles, G. M. Whitesides, J. Org. Chem., 1992,
[
21,32]
The benzene signal (s, 7.15 ppm)
5899-5907.
[13] J. Bolte, C. Demuynck, H. Samaki, Tetrahedron Lett., 1987, 28, 45,
1
3
(
including C satellites) was normalised to 1000. The erythrulose
5525-5528.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
[
14] I. A. Sevostyanova, O. N. Solovjeva, G. A. Kochetov, Biochem.
Biophys. Res. Commun., 2004, 313, 771-774.
[
15] A. Ranoux, S. K. Karmee, J. Jin, A. Bhaduri, A. Caiazzo, I. W. C. E.
Arends, U. Hanefeld, ChemBioChem, 2012, 13, 1921-1931.
16] A. Ranoux, U. Hanefeld, Top. Catal., 2013, 56, 750-764.
17] a) M. T. Reetz, M. Bocola, J. D. Carballeira, D. Zha, A. Vogel,
Angew. Chem. Int. Ed., 2005, 44, 4192-4196; b) U. T. Bornscheuer,
G. W. Huisman, R. J. Kazlauskas, S. Lutz, J. C. Moore, K. Robins,
Nature, 2012, 485, 185-194.
[
[
[
18] E. Fiedler, S. Thorell, T. Sandalova, R. Golbik, S. König, G.
Schneider, Proc. Natl. Acad. Sci. USA, 2002, 99, 591-595.
19] E. Krieger, G. Vriend, Bioinformatics, 2014, 30, 2981-2982.
20] D. Yi, T. Saravanan, T. Devamani, F. Charmantray, L. Hecquet, W.-
D. Fessner, Chem. Commun., 2015, 51, 480-483.
[
[
[
[
[
[
[
21] T. Purkarthofer, W. Skranc, H. Weber, H. Griengl, M. Wubbolts, G.
Scholz, P. Pöchlauer, Tetrahedron, 2004, 60, 735-739.
22] J. Kua, M. M. Galloway, K. D. Millage, J. E. Avila, D. O. de Haan, J.
Phys. Chem. A, 2013, 117, 2997-3008.
23] B. Rosche, M. Breuer, B. Hauer, P. L. Rogers, Biotechnol. Bioeng.,
2
004, 86, 788-794.
24] R. Westphal, C. Vogel, C. Schmitz, J. Pleiss, M. Müller, M. Pohl,
Angew. Chem. Int. Ed., 2014, 53, 9376-9379.
25] P. Hendil-Forssell, M. Martinelle, P.-O. Syrén, Chem. Commun.,
2
015, 51, 17221-17224.
[
[
26] C. Kürten, B. Carlberg, P.-O. Syrén, Catalysts, 2016, 6, 90.
27] S. Matsuoka, S. Sugiyama, D. Matsuoka, M. Hirose, S. Lethu, H.
Ano, T. Hara, O. Ichihara, S. Roy Kimura, S. Murakami, H. Ishida, E.
Mizohata, T. Inoue, M. Murata, Angew. Chem. Int. Ed., 2015, 20, 39,
1508-1511.
[
[
28] P.-O. Syrén, S. C. Hammer, B. Claasen, B. Hauer, Angew. Chem.
Int. Ed., 2014, 53, 4845-4849.
29] B. Breiten, M. R. Lockett, W. Sherman, S. Fujita, M. Al-Sayah, H.
Lange, C. M. Bowers, A. Heroux, G. Krilov, G. M. Whitesides, J. Am.
Chem. Soc., 2013, 135, 15579-15584.
[
[
[
30] K. G. Morris, M. E. B. Smith, N. J. Turner, Tetrahedron: Asymmetry,
1
996, 7, 8, 2185-2188.
31] M. E. B. Smith, K. Smithies, T. Senussi, P. A. Dalby, H. C. Hailes,
Eur. J. Org. Chem., 2006, 5, 1121-1123.
32] M. Weber, C. Hellriegel, A. Rueck, J. Wüthrich, P. Jenks, J. Pharm.
Biomed. Anal., 2014, 93, 102-110.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
Scheme 1. Natural transketolase catalyzed reaction.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
Scheme 2. Use of LiHPA as
a ketol donor in TK catalyzed synthetic
applications.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
R
O
N
+
HO
OLi
P
P
S
O
O
O
R O
N
O
OH
S
HO
CO2
O
R
H
O
N
OH
OH
OH
HO
P
S
O
OH
HO
R
N
His481
H
OH
P
P
S
O
O
HO
R
N
O
+
H
S
OH
Scheme 3. Proposed mechanism for the formation of the activated ketol
bearing the carbanion by either decarboxylation (top) or catalytic
deprotonation (bottom).
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
Scheme 4. Decarboxylation driven reaction (left) and one-substrate reaction
(right) for the TK catalyzed synthesis of L-erythrulose.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
Figure 1. Overview of substrates (a), products (b) and derivatized products (c)
required for chiral analysis. Products 1-3 (b) and (c) were obtained in the 3-(S)
configuration with TK. Products 4b/c were not accessible enzymatically.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
Figure 2. in silico docking of butanal into the energy minimized mutant active
site D477E using YASARA.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
1
Figure 3. TK reaction producing L-erythrulose followed by H-NMR. 200 µg
2
+
WT TK, ThDP: 5 mM, Mg : 18 mM, 5 mM sodium phosphate buffer pH =
.0. For the one substrate reaction (red): glycolaldehyde: 200 mM, for the
7
decarboxylation driven reaction (blue): glycolaldehyde: 100 mM and
LiHPA: 100 mM. A) Initial 24 hours showing complete conversion in the
decarboxylation driven reaction; B) Extended time course showing
equilibration of both reactions towards the equilibrium concentration of
29.1±0.6 mM for erythrulose. Inset: addition of LiHPA after 650 h showing
retained enzyme activity (triangles).
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
[
a]
Table 1. Isolated product yields and enantiomeric excess (ee) of the (S) configured enantiomer.
WT
D477E
D477T
R528K
R528Q
R528K/
S527T
R528Q/
S527T
1b
2b
3b
4b
11±8%
34±15%
(94%)
8%
(n.d.)
10±8%
(81%)
8±2%
(77%)
8±3%
(73%)
6±4%
(66%)
[
b]
(84%)
7%
91%)
61±13%
(90%)
12±4%
(84%)
6±4%
(82%)
5±1%
(87%)
6±1%
(68%)
5±1
(82%)
(
[
b]
0%
n.d.)
41±20%
(99%)
n.d.
(n.d.)
3±1%
(n.d.)
0%
(n.d.)
0%
(n.d.)
0%
(n.d.)
[
[
b]
b]
[b]
[b]
[b]
[b]
[b]
[b]
(
[
b]
0%
n.d.)
0%
(n.d.)
n.d.
(n.d.)
0%
(n.d.)
0%
(n.d.)
0%
(n.d.)
0%
(n.d.)
[b]
[b]
[b]
[b]
[b]
(
2+
[
a] 20U of ScTK, 5 mM ThDP, 18 mM Mg ,1 mmol LiHPA, 1 mmol aldehyde, 10 mL final volume in 5 mM sodium phosphate buffer, pH = 7.0, 25˚C, 200 rpm, 18h.
[b] not determined.
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
Table 2. Michaelis-Menten parameters.
[
a]
WT
D477E
D477T
R528K
R528Q
R528K/
S527T
R528Q/
S527T
1b
2b
3b
k
K
k
k
cat
1.2
272
4.2
42
163
260
0.5
48
10
0.8
181
4.4
1.5
239
6.1
1.9
260
7.4
0.8
106
7.5
M
-
1
1
1
cat
K
K
K
M
cat
0.8
327
2.4
9.3
40
233
0.4
43
9.9
0.1
16
6.9
2.1
611
3.5
0.3
67
4.2
0.4
42
8.2
K
k
M
-
M
cat
k
K
k
cat
0.4
150
2.9
0.6
66
8.3
0.3
99
2.5
0.3
86
3.7
[
b]
[b]
M
n.d.
n.d.
n.d.
-
M
cat
-
1
-1
-1 -1
[
a] kcat / s , K
M
/ mM, kcat
K
M
/ M s . For errorbars see ESI fig S10-S12.
2+
Purified ScTK: 50 µg, ThDP: 1 mM, Mg : 4 mM, LiHPA: 100 mM, aldehyde: 5-
50 mM, 5 mM sodium phosphate buffer, pH = 7.0, 25˚C, 500 rpm. [b] not
determined.
1
This article is protected by copyright. All rights reserved.

ChemCatChem
10.1002/cctc.201601649
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
Layout 1:
FULL PAPER
Text for Table of Contents
Author(s), Corresponding Author(s)*
Page No. – Page No.
Title
((Insert TOC Graphic here: max.
width: 5.5 cm; max. height: 5.0 cm))
Layout 2:
FULL PAPER
Stefan R. Marsden, Lorina Gjonaj,
Stephen J. Eustace and Ulf Hanefeld*
Page No. – Page No.
Separating Thermodynamics from
Kinetics – A New Understanding of
the Transketolase Reaction
The initially kinetic control of the Transketoase catalyzed LiHPA reaction was until
now mistakenly described as thermodynamic control. A new understanding of this
effect and of the reasons why hydrophobic substrates are converted by an enzyme
that naturally has a high affinity to hydrophilic substrates sheds new light on
textbook biocatalysis.
This article is protected by copyright. All rights reserved.