Substrate binding to Candida tenuis xylose reductase during catalysis

Article abstract of DOI:10.1039/c3ra41448e

Candida tenuis xylose reductase (CtXR) is studied by in situ NMR, saturation transfer difference (STD) NMR, and molecular docking with respect to its substrate and coenzyme binding in ternary complexes. The natural substrate Xyl as well as Glc and methyl-glucosides preferentially bind as α-anomers of the pyranose forms. These α-anomers are transformed faster, predominately leading to STD effects in the formed products, and can be better docked into the CtXR active site than the β-anomer. The reduction is initiated by α-Xylp ring opening prior to hydride transfer from NADH. Binding and transformation of unnatural 2,4-dichloroacetophenone is not as good, although it is reduced with very high catalytic efficiency. STD NMR indicates a reasonable amount to leave the ternary complex in unreduced form. The molecular docking calculation confirms this result, as only a couple of the investigated ternary complexes allow reduction of the substrates.

Full text of DOI:10.1039/c3ra41448e

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Substrate binding to Candida tenuis xylose reductase
during catalysis
Cite this: RSC Adv., 2013, 3, 25997
*
Michael Vogl and Lothar Brecker
Candida tenuis xylose reductase (CtXR) is studied by in situ NMR, saturation transfer difference (STD) NMR,
and molecular docking with respect to its substrate and coenzyme binding in ternary complexes.
The natural substrate Xyl as well as Glc and methyl-glucosides preferentially bind as a-anomers of the
pyranose forms. These a-anomers are transformed faster, predominately leading to STD effects in the
formed products, and can be better docked into the CtXR active site than the b-anomer. The reduction
is initiated by a-Xylp ring opening prior to hydride transfer from NADH. Binding and transformation of
unnatural 2,4-dichloroacetophenone is not as good, although it is reduced with very high catalytic
efficiency. STD NMR indicates a reasonable amount to leave the ternary complex in unreduced form.
The molecular docking calculation confirms this result, as only a couple of the investigated ternary
complexes allow reduction of the substrates.
Received 25th March 2013
Accepted 4th October 2013
DOI: 10.1039/c3ra41448e
www.rsc.org/advances
corresponding xylitol with concomitant oxidation of NADPH or
Introduction
NADH to NADP+ or NAD+, respectively.19–21 The different anomeric
Enzymes are biological catalysts used in many different
synthetic applications. In particular oxidoreductases [E.C.1.1.1]
are oen applied for this purpose.1–3 Their high transformation
rates as well as the asymmetric induction in formation of highly
enantiomeric enriched secondary alcohols cause a growing
awareness of this enzyme group.3–6 Additionally, several oxido-
reductases do not only transform their natural substrates, but
also bind and process articial compounds with high trans-
formation rates.7,8 Hence, there is a growing interest to inves-
tigate details of the enzymatic mechanism. Especially,
comprehensive knowledge of substrate accommodation in the
binding pocket helps to understand the acceptance of unnat-
ural substrates and guides rational design of enzyme variants
with altered substrate specicities.9,10
forms of xylose in the natural aqueous environment complicated a
detailed binding analysis so far. Additionally, many non-natural
substrates are quite well accepted by CtXR. In particular, mono-
and di-chloro-substituted acetophenones are transformed to the
corresponding phenyl ethanols with very high transformation
rates.22,23 Hence, 2,4-dichloroacetophenone is used as non-natural
model substrate for complementary binding studies. The corre-
sponding results from NMR experiments and docking calcula-
tions provide a better understanding of CtXR substrate specicity
in order to make direct mutations in future.
Results and discussion
Binding of different xylose anomeric forms
Many different methods have been established during the
last decades to gain insight into ligand binding for different
enzyme systems.11 In particular, in situ 1H NMR and Saturation
Transfer Difference (STD) NMR are well established to study
product formation and ligand enzyme binding processes,
respectively.11–17 In these techniques the substrates and prod-
ucts are monitored and provide direct information about their
interaction with the active site and surrounding areas of the
enzyme. Furthermore, data gained by these methods can be
visualized and rened using in silico docking methods.18
In the present study xylose reductase [E.C.1.1.1.21; D-xylose
reductase] from the yeast Candida tenuis (CtXR) is investigated. Its
natural substrate xylose is reduced by this enzyme to the
The natural substrate xylose is reported to be reduced by CtXR
as aldehyde in its open chain form.19,20,24 This result, deter-
mined from kinetic investigations, is in good agreement with
the common mechanism of oxidoreductase catalyzed carbonyl
reductions using NADH. We now study the dynamic procedure
of substrate binding by in situ NMR and monitor the concen-
trations of the two different Xylp anomers during trans-
formation. At 303 K, customary used for previous investigations,
a- and b-anomer amounts decreased in the same ratio using
0.36 mM CtXR.14 However, lowering the temperature to 283 K
and using 7.2 mM CtXR, the a-anomer is consumed considerably
faster (Fig. 1(a)). This change is caused by a distinctly reduced
anomerization rate, while the reaction rate of the CtXR cata-
lyzed reaction is not as intensively inuenced by the 20 K
temperature decrease, in particular due to the larger CtXR
concentration.25,26 The anomerisation of xylose at 283 K is,
¨
University of Vienna, Institute of Organic Chemistry, Wahringer Straße 38, A-1090
Wien, Austria. E-mail: lothar.brecker@univie.ac.at
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hence, not fast enough to completely compensate concentration
changes of a- and b-Xylp caused by a preferred CtXR reduction
of the a-Xylp. This anomer is obviously preferentially bound at
the active site and isomerized to the open chain aldehyde form
during the binding process. Therefore, the xylose a-anomer is
mainly reduced to xylitol. The amount of a-xylose, which is
considerable longer bound to CtXR is considerable low. This
caused by the Xyl : CtXR binding which has a Km of 91 mM and
by the molar ratio of the two compounds, which is 150 : 1.
Having NADH in decient proportion, the reduction stops aer
its complete oxidation to NAD+ and the a- to b-Xylp anomeric
ratio slowly recovers to the dynamic equilibrium (Fig. 1(a)).
The nal a- to b-anomeric ratio of Xyl in presence of CtXR is
slightly smaller than in absence of the enzyme at the start of the
reaction. This difference is likely also caused by preferred
binding of the a-anomer, which is then opened to the aldehyde
form but not reduced due to lack of NADH. Release of the not
transformed aldehyde from the Xyl/CtXR and Xyl/CtXR/NAD+
complexes lead to formation of both a- to b-anomeric forms.
Consequently to an access of the b-form is generated in
comparison to the equilibrium at the starting point.
Performing this in situ NMR experiment at 283 K with the also
accepted aldoses glucose, galactose, and arabinose provides very
similar results. The a-anomers are always preferentially bound,
isomerized, and reduced (Fig. 1(b) and (c)). Best accepted
unnatural substrate, 2,4-dichloroacetophenone is transformed
by CtXR with a catalytic efficiency of 1128 molꢀ1 sꢀ1. Taking into
account that CtXR predominantly binds the a-Xyl for reduction,
the unnatural substrate 2,4-dichloroacetophenone is accepted
better.23,27 However, xylose would have been transformed with
higher catalytic efficiency, if it had only been bound and
accepted in the low aldehyde concentration. Hence, comparison
of these natural and unnatural substrates is now made, with
particular focus on their binding.
STD NMR of ternary ligand/coenzyme/CtXR complexes
The STD NMR technique is a well suited method to investigate
ligand protein interactions. Its principle is to measure the
magnetization which is transferred by dipole–dipole interaction
from the protein to the protons of a non covalently bound
ligand. This method was developed to study binding of single
ligands and mixtures to receptor-proteins.11,12,28,29 However, STD
NMR can also be used to study ligand enzyme interactions.14,15
Apart from binding of substrates, inhibitors, and products, the
binding behavior during the catalytic event can also be studied.
In coenzyme depending transformations, however, ternary
substrate/coenzyme/enzyme complexes are formed during the
reactions and both, substrates and coenzymes are structurally
modied at the same time. The resulting reaction mixtures make
binding investigations rather complex, as apart from the produc-
tive complexes other binary and ternary complexes are formed.
They all likely possess slightly different conformations, which
cause different signal intensities in the STD spectra. However,
some of these versatile complexes can be neglected as the products
and transformed coenzymes are only present in very small
concentrations at the beginning or end of an investigated reaction
Fig. 1 a- and b-anomeric ratio of the substrates xylose (a), glucose (b), and
galactose (c) during CtXR catalyzed reduction at 283 K. Starting concentrations of
carbohydrates, NADH, and CtXR were 1.5 mM, 0.5 mM, and 10.3 mM, respectively.
Anomeric ratios during reactions have been determined by in situ NMR moni-
toring all proton signals which were not overlapping. An average value of all
integrals belonging to one compound has been made. Only the initial ratio of
xylose anomers (indicated by squares) has been determined prior to addition of
the enzyme and from a preliminary measurement after enzyme addition, as quite
fast reaction did not allow the determination of accurate starting point during
transformation. Reduction of the a-anomer is obviously faster in all three cases
resulting in a growing excess of the b-anomer during reaction. Then anomeri-
zation results in the equilibrium. The equilibrium at the end differs from the
starting point, as presence of CtXR causes mainly a-Xyl binding and ring opening.
This leads to an increase in ring opening and release of the a-anomer compared
to the b-anomer. The released aldehyde, however, rearranges to the two
anomeric forms without any influence from the CtXR. Consequently the dynamic
equilibrium shifts to a higher amount of b-anomer compared to the starting
point. Addition of CtXR solutions did not cause changes of any other parameters
like temperature, pD,32 or ion concentration in reasonable amount, as all 1H NMR
shifts remained constant.
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(Fig. 2). Suitable short measurement times therefore allow to
record in situ STD NMR spectra, which provide detailed informa-
tion about binding at different stages of the reaction process.
Furthermore, detailed reference STD spectra can be recorded from
binary and ternary complexes, which do not lead to substrate
transformations. In such experiments binding times can be
roughly estimated in particular from STD signal intensities, which
are diminished by reasonably short or long binding times.12,14,15
STD NMR of ternary Xyl/NADH/CtXR complexes
Based on the in situ NMR experiments and preliminary STD
NMR investigations of CtXR catalyzed transformations, a
detailed binding analysis is performed.14 Monitoring the binary
mixture of the enzyme (0.51 mM) and xylose (6.7 mM) not
leading to transformation due to lack of the coenzyme provides
very small STD signals from both xylose anomers (Fig. 3(a),
Table 1(a)). The average STD signal intensities are in good
agreement with the ca. 1 : 2 ratio of the anomers in aqueous
solution. Both anomers are therefore bound and released in
similar amounts. There is no evidence that Xyl undergoes a
temporary rearrangement to the open chain aldehyde form
during the binding period, when no NADH is present in the Fig. 3 Cutout of STD NMR spectra recorded from (a) Xyl/CtXR, (b) Xyl/NADH/
CtXR, and (c) Xyl/NAD+/CtXR, as well as of (d)1H NMR spectrum of Xyl for
active site and hence no transformation occurs.
Hence, in the next step NADH was added as coenzyme (7.5
mM) to study substrate binding during the xylose reduction.
comparison. Shown are all proton signals of both Xyl anomers, except the H-1
signal of a-Xyl. (a) STD effects of xylose binding to CtXR in a binary complex are
reasonable small and indicate that both Xyl anomers likely bind to the enzyme
with quite short binding times.13,28 (b) In the productive Xyl/NADH/CtXR complex
The gained results are in good agreement with earlier reported
data and support this investigation.14 Here the STD-signals of STD effects of b-Xyl are quite distinctive, while a-Xyl is not released from the CtXR,
but transformed to xylitol. Release of this product causes STD signals between 3.5
the a-anomer protons decrease distinctly and only the signals
from the b-anomer are le as shown in Fig. 3(b). The a-Xyl binds
in the ternary complex, gets magnetized, and is completely
and 3.7 ppm indicated in a circle.15 Further STD signals belong to NADH protons.
(c) In the unproductive Xyl/NAD+/CtXR complex STD NMR signals are distinctly
smaller and indicate both anomers to be bound and released from the enzyme.
reduced to xylitol. Then it leaves the enzyme as magnetized
These data portent a spatially close binding of substrate and coenzyme to CtXR.
product xylitol and causes STD signals of this product. Due to Phase error of H-4 from b-Xyl at ca. 3.45 ppm is likely caused by an effect discussed
earlier and based on interference with D2O irradiated by the lock frequence.14
symmetry of xylitol no detailed assignment to single protons is
possible. The concentration of the magnetized a-anomer in the
solution is low and causes only small STD signals, as only a
small amount is released as unreacted xylose from the complex. However, it cannot be excluded that a small amount of b-Xyl is
On the other hand, the b-anomer obviously binds in the ternary also opened and reduced to xylitol, because the substrate
complex and takes over magnetization from the protein. Then it conformer cannot be identied from the symmetrical product.
is released without ring opening leading to the aldehyde. STD effects of xylose anomers are listed in Table 1(b).
Fig. 2 Schematic progress of in situ STD NMR measurements considering binding of substrates and products. At the start of the reaction bound substrate and first
generated product are released from the enzyme showing STD effects. In the middle of the reaction having a mixture of substrate, product, and both forms of the
cosubstrate also unproductive binding is possible. The different ternary complexes lead to average STD signals of substrates and products. At the end of the reaction
only the product and the transformed form of the cosubstrate are present, leading to STD signals caused by this ternary complex.
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Table 1 STD NMR effects of xylose anomers and of glucopyranosid anomers binding to CtXR in binary as well as in ternary complexes with NADH. All spectra are
referenced to 100% for the largest STD effect in the measurement. Comparison between STD effects of the spectra can be estimated from signal to noise ratios. Long
substrate/product binding times leading also to small STD effects can be excluded13,14
b
Substrate
H-1
H-2
H-3
H-4
H-5a
H-5b
H-6a
H-6b
CH3
(a) S/N ¼ ꢁ10
a-Xylose
b-Xylose
20
50
50
100
30
40
35
30
25
20
25
80
—
—
—
—
—
—
(b) S/N ¼ ꢁ19
a-Xylose/NADHc
b-Xylose/NADH
10
70
10
100
15
45
15
20
20
70
10
45
—
—
—
—
—
—
(c) S/N ¼ ꢁ12
a-Glucopyranosid
b-Glucopyranosid
ndd
30
100
100
90
30
40
60
90
60
—
—
50
30
70
60
60
ꢀ30a
(d) S/N ¼ ꢁ15
a-Glucopyranosid/NADH
b-Glucopyranosid/NADH
ndd
100
100
90
40
40
60
90
60
—
—
50
40
60
40
60
70
ꢀ30a
a
b
Phase error are likely caused by an effect discussed earlier and based on interference with D2O irradiated by the lock frequence.14 Value refers to
c
d
all three proton of the methyl group. Partly overlapped from xylitol signals (see Fig. 3). Not determined due to overlap with impurities.
The higher intensities of STD signals from xylose compared binding partners. The release is, however, still fast enough and
to those from xylitol might be explained by a slightly longer not resulting in a decrease of the STD signal intensity, which
binding time of the unreduced b-Xyl. Such faster release of might be caused by longer binding times. The average KD values
xylitol is explicable by the necessity of the enzyme to be ready for of all investigated carbohydrates are in the mM range being by
the next binding process of xylose. However, STD signal inten- far optimal for the generation of STD signals.11
sity might also be caused by a faster release of more b-Xyl
The corresponding anomerically pure ternary Me-Glcp/
molecules, which is however not in accordance to Xyl : CtXR NADH/CtXR complexes have been studied with further STD
binding having a reasonable low Km of 91 mM. Hence, the NMR measurements. In comparison of the two anomers, the
biocatalyst is quite likely optimized to release the product faster STD signals indicate that the O-methyl-a-glucoside again shows
than the untransformed substrate. The transferred magnetiza- larger STD-effects than the b-anomer. However, the difference is
tion during the STD measurement is therefore larger in case of not as distinct as in the absence of NADH (Table 1(d)).
b-Xyl. However, the binding time of b-Xyl is obviously not long
enough to compensate for this effect.12,28
In comparison to the STD effects of the free xylopyranose,
there are signicant consistencies. The protons of b-Xylp and of
O-methyl-b-Glcp show a very similar binding pattern. Hence, the
binding of the two substrate analogues can be assumed to be
quite similar to those of the xylopyranoses at the beginning in
the productive transformation.
STD NMR of ternary O-methyl-Glcp/NADH/CtXR complexes
In another approach a modication of the substrate is made in
order to mimic productive ternary complexes, however, without
leading to the reaction. For that propose we used O-methyl-
glucosides, an analogue of the substrate Glc.24 These substrate
Docking and simulation of Xyl/NADH/CtXR complexes
analogues do not undergo anomerization and cannot be opened A molecular docking of substrates to the binary complex can be
to the aldehyde form. Therefore, in binding studies the result- made by an in silico technique on basis of the previously pub-
ing ternary complex is similar to those that are present at the lished structure of a co-crystallized CtXR/NAD+ complex.30 It
beginning of the Glc or Xyl reduction processes.
allows an illustration of the ternary complex. For this purpose, a
Analyzing the two binary complexes of anomeric O-methyl simulation of the CtXR/NADH complex is made, which is based
glycosides (6.5 mM) and CtXR (0.51 mM), the O-methyl-a-Glcp on the CtXR/NAD+ crystal structure (PDB-entry 1MI3, http://
protons show on average higher STD intensities compared to www.rcsb.org). Such approximation is possible, as the STD
those of O-methyl-b-Glcp. Only protons H-2 of both anomers effects are very similar for both complexes. Based on this
have the same intensities and the proton H-4 of the b-anomer structure a comparison of molecular modeling and NMR based
possess larger intensities than the corresponding protons from results is made.
O-methyl-a-Glcp (Table 1(c)). Hence, the a-anomer obviously
Molecular docking of b-xylose shows that this anomer is
takes over a larger amount of magnetization than the O-methyl- preferentially bound in two regions next to the active side. One
˚
b-Glc. This greater dipole–dipole interaction is likely caused by of these areas is approximately 4 A away from the transferrable
higher kon and koff rates and a better t of the O-methyl-a-Glcp to hydride of NADH. Additionally, Tyr-51 and His-113, which are
the CtXR binding side, which leads to optimal proximity of the supposed to be involved in stabilizing the open from of the
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aldehyde,31 are placed in a distance of about 2.5 A. Such spatial
location does neither allow a rearrangement, nor a hydride
transfer (Fig. 4(a)). However, it is close enough to the protein to
absorb magnetization via dipole–dipole interaction, causing
STD signals. The measured STD signal intensities are in good
accordance to the special closeness between substrate protons
and protons from the protein. The second area is on the
˚
˚
opposite side of NADH, which is also more than 5.0 A away from
the transferrable hydride. Additionally, the important amino
acids are not in this area (Fig. 4(b)). Hence, b-xylose does not t
well into the active site and is not reduced. The only possibility
to enable the hydride transfer is a non CtXR inuenced
anomerization in this position, resulting in the a-anomer.
The a-xylose, however, ts perfectly into the reactive site
close to NADH. The pro-R hydrogen is in hydride-transfer
˚
distance of ca. 1.82 A away from the carbon of the hemiacetal
function, which forms the aldehyde in the rearrangement.
Furthermore, STD data are also in good agreement with this
ternary complex conformation, especially as the a-Xyl protons
showing large STD effects are spatially close to the CtXR
protons. The distances to the nearest amino acids protons of
˚
˚
Tyr-51 is between 2.0 A and 2.6 A and the one to protons of His-
˚
113 is 3.6 A (Fig. 4(c)).
The results of such simple docking experiments should be
treated carefully in general. The exible loop region of the CtXR
is involved in substrate binding and its movability is most likely
responsible for the large substrate acceptance of the enzyme.31
In the used docking calculation, however, only the ligand
structure is optimized in silico while the enzyme is kept rigid.
However, the present results are in excellent agreement with the
in situ NMR and, in particular, with the STD NMR. It can hence
be concluded that the results from docking visualize a correct
binding and indicate a preferential binding and use of a-xylose
prior to ring opening and reduction.
Mechanism of xylose ring opening to provide reducible
aldehyde form
Ring opening prior to binding can be excluded, as both
anomers would be accepted equally. However, for reduction
the open chain aldehyde form is necessary. The molecular
docking provides a tting of the a-xylose, which is a good
starting point for the ring opening. The proton of hydroxyl
˚
group in Tyr-51 is in about 2.8 A distance to the ring oxygen of
a-xylose. Hence, it can initiate the opening procedure as
shown in Fig. 5(a). The resulting free hydroxylate function of
Tyr-51 can be stabilized by the surrounding amino acids or
might be protonated from the close Asp-50. The xylose ring
opening is then nished by the terminal aldehyde formation
and release of the hydroxy proton of position 1. This can be
accepted by one of the nearby amino acids, especially the Tyr-
51, which might return the proton back to Asp-50. Resulting
Fig. 4 Molecular docking of xylose to a complex of NADH and CtXR.18 The xylose
anomers are shown in green, while the NADH is visualized in pink. A couple of
indicative distances between atoms are shown in red (1H–1H distances, responsible
for STD effects) and green (indicative distances between xylose, NADH, and CtXR).
˚
All are indicated with the length given in A. Docking of b-xylose results in two
different orientations shown in panel (a) and (b). Both conformations show that
this anomer does not fit well into the active side. The spatial distance does hence
not allow a reduction. In panel (c) fitting of a-xylose is shown, which matches well
into the reactive site close to NADH. The spatial closeness between enzyme and
substrate are in good agreement with the STD effects shown in Table 1.
˚
aldehyde function is close (ca. 1.8 A) to the transferrable pro-R
hydrogen of NADH (Fig. 5(b)). It is quite likely directly reduced
by NADH to xylitol. A slightly different orientation of the
aldehyde, however, allows a better tting (Fig. 5(c)) and still
enables the pro-R hydrogen transfer.21,31
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Table 2 STD NMR effects of 2,4-dichloroacetophenone binding to CtXR in
binary and in ternary complex with NADH as well as 1-(20,40-dichlorophenyl)-
ethan-1-ol binding to CtXR in ternary complex with NADH (generated NAD+)
directly released after transformation. The latter two are determined from one
measurement. Signal to noise ratio was about 15 in both spectra
Substrate
H-1b H-2 H-30 H-50 H-60
2,4-Dichloroacetophenone
2,4-Dichloroacetophenone/NADH
1-(20,40-Dichlorophenyl)-ethan-1-ol/
NADH
100
100
20 70 30
—
—
30
40
ꢀ10a 20
30 30
40 60
a
Phase error are likely caused by an effect discussed earlier and based
b
on interference with D2O irradiated by the lock frequence.14 Value
refers to all three proton of the methyl group.
Fig. 5 Mechanism of xylose ring opening and reduction in the CtXR active side
(panel (a)). The distance between the OH-proton of Tyr-51 and the ring oxygen
˚
(ca. 2.8 A) is close enough to initiate the ring opening by protonation indicated in
for the rst time. Here STD effects of protons in positions 2, 50,
and 60 are distinctly higher than those of the three methyl
protons (Table 2). This result shows that 2,4-dichlor-
oacetophenone binding in the binary and ternary complexes
differ from each other and that the mode of binding changes
entirely during the reduction. However, the experiment also
shows that ca. 1/4 of the bound 2,4-dichloroacetophenone is
released in the unreduced form, indicated by the presence of 2,4-
dichloroacetophenone STD signals. The intensity of these signals
can be roughly compared, as the binding time of substrate and
product are approximately in the same range. The reason for the
release is likely a not best possible t of the unnatural substrate
into the active site, which can lead to elevated koff rates.
panel (b). Although the distance between the oxygens of position 1 and Tyr-51 is
˚
reasonable large (4.76 A) the proton released from position 1 probably re-
protonates the Tyr-51 hydroxyl function. The resulting sp2 carbon of the aldehyde
˚
is then placed very close to the transferrable pro-R hydrogen of NADH (ca. 1.8 A).
In panel (c) an optimized fit of the open chain aldehyde prior to hydride transfer is
shown.
STD NMR of the 2,4-dichloroacetophenone/NADH/CtXR
complex
Various structures of unnatural substrates, which are reduced by
the CtXR with excellent catalytic efficiencies, oen differ entirely
from those of aldohexoses. In particular, the carbonyl functions
of these compounds do not form half acetals or half ketals in
aqueous solutions. Their binding in the productive mode is
hence entirely different from those of the monosaccharides.
Here, we use 2,4-dichloroacetophenone as a model compound Docking simulation of 2,4-dichloroacetophenone/NADH/
to study binding of aromatic ketones. It is reduced with a high
catalytic efficiency of 1128 sꢀ1 Mꢀ1, which has been shown to be
caused by an intense polarization of the carbonyl group
(Scheme 1).23 STD NMR recorded from the binary mixture of
model substrate (1.5 mM) and enzyme (0.51 mM) shows that the
three protons of the terminal methyl group provide the most
intense signal. Proton at position 30 has a comparable STD effect.
In contrary the protons H-60 and, in particular, H-50 in the
aromatic moiety have smaller STD effects, as shown in Table 2.
Start of the reaction by addition of the coenzyme lead to forma-
tion of the ternary complex and to entire changes in the STD
effects. Signal intensity of H-30 in the substrate increases by a
factor of 1.33 and signal of H-50 reaches the intensity of H-60.
However, the more interesting signals emerge from the resulting
alcohol (Scheme 1), which is released from the ternary complex
CtXR complex
The in silico molecular docking has been performed for the
unnatural model substrate to compare 2,4-dichloroacetophenone/
CtXR binding with Xyl/CtXR binding. For this purpose the
substrate was changed to 2,4-dichloroacetophenone, while all
other parameter remained unchanged. Results indicate that the
model substrate does not t as good as a-xylose and that it can be
located in the active site in two different orientations (Fig. 6). In
these two cases the hydride would be transferred to the pro-R or
pro-S side of the ketone, respectively, resulting in both enantio-
mers of the produced alcohol. In both orientations the distance
between the transferred hydride and the carbonylic carbon is
˚
determined to be about 2.8 A, which is just close enough to
perform the reduction. However, it is known that only the
S-congured alcohols are formed within CtXR catalyzed reduction
of acetophenones.23,27 Hence, the two different orientations must
cause slight variations in activation energy, which cannot be
determined accurately by the applied in silico molecular docking
method. The orientation leading to the S-alcohol is productive
and causes the hydride transfer, while the other one is not
productive and leads to reorientation or to release of the 2,4-
dichloroacetophenone. This result comes along with the observed
effect of the STD NMR where the unnatural substrate shows
relatively high signals compared with those of by far completely
transformed a-xylose.
Scheme 1 CtXR catalyzed reduction of 2,4-dichloroacetophenone to the cor-
responding (S)-1-(20,40-dichlorophenyl)-ethan-1-ol. Indication of positions is used
in Table 2.
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Furthermore, results can be used as guideline for a rational
enzyme redesign to improve the CtXR-catalyzed reduction of
unnatural substrates in order to allow an optimized applica-
tion in the synthesis of pharmaceuticals.27
Experimental
Chemicals
All chemical used were purchased from Sigma-Aldrich-Fluka
(Tauirchen, Germany) or Carbosynth (Berkshire, UK) with the
highest purity available and used without further purication or
drying.
Fig. 6 Molecular docking of 2,4-dichloroacetophenone to the complex of NADH
and CtXR.18 The substrate and NADH are visualized in green and pink, respectively.
Shown are two different orientations (a and b) possessing very similar distances
between the transferred hydride and the carbon of the carbonyl group. A couple
of indicative distances between atoms are shown in red (1H–1H distances,
responsible for STD effects) and green (indicative distances between xylose,
NMR spectroscopy
˚
NADH, and CtXR). All are indicated with the length given in A. However, only the
Samples were prepared in 0.7 mL D2O and contain ꢁ1.0–20.0 mL
of the CtXR (13 mg mLꢀ1) in 50 mM potassium phosphate
buffer solution at a pD-value of 6.6.32 The respective substrates
concentrations are reported for each experiment. Amounts of
CtXR are indicated for the experiments in the results part. Due
to low solubility, the 2,4-dichloroacetophenone was dissolved in
0.07 mL CD3OD rst and then added to the sample. All 1H and
STD NMR spectra were measured on a Bruker (Rheinstetten,
Germany) DRX-600 AVANCE spectrometer at 600.13 MHz using
triple resonance 5 mm inverse probe. Measurement tempera-
ture was 303 K, or 283 K for some indicated in situ NMR
measurements. Anomeric mixtures of Xyl, Glc, or Gal used for
the in situ NMR measurements were equilibrated for 10 days at
the measurement temperature until no more anomerisation
was detectable by 1H NMR. All chemical shis were referenced
to external acetone at 2.225 ppm.
orientation shown in panel (a) represents the position of the substrate during
hydride transformation, which leads to the solely isolated product (S)-1-(20,40-
dichlorophenyl)-ethan-1-ol by transfer of the pro-R hydrogen from NADH.
The results from the molecular docking method have also to be
handled with care, because the exibility of the loop regions in the
active site was not taken into account. For a more detailed analysis
a molecular dynamic calculation can be helpful. Such investiga-
tion may allow to determine the slight differences in activation
energy between the two orientations of 2,4-dichloroacetophenone
in the active site.
Conclusions
Reduction of natural and unnatural substrates by CtXR has
been studied to certain detail, but was mainly discussed in the
context of substrate structures and carbonyl group polariza-
tion.23 However, substrate binding in the ternary complexes also
has an important inuence on the catalytic efficiency. Here, two
STD NMR
different NMR based methods as well as molecular docking STD NMR spectra were recorded as described earlier.15 The
were used to show that transformations of xylose and similar molar substrate–CtXR and cosubstrate–CtXR ratios were in the
aldoses are initiated by binding of the respective a-anomers. range between 2 900 : 1 and 14 700 : 1. Substrate and cosub-
This selective binding and the concomitant broad acceptance of strate concentrations were between 1.5 mM to 7.5 mM. Selec-
various aldoses for reduction suggests that xylose is not the only tive saturation of the protein was achieved by a series of 40
natural aldose, which is transformed by CtXR. The preferential Gaussian pulses of 50 ms length, and 1 ms delay resulting in a
binding of a-anomers suggests an opening of the pyranose form total irradiation times of 2.04 s. On resonance measurements
to an open chain aldehyde, which is consecutively reduced. were performed at ꢀ2.0 ppm and off resonance at 40.0 ppm.
Binding studies with both anomers of O-methyl-glycosides Subtraction of the spectra was performed during the
indicate the preferred binding of a-pyranose form.
measurement via phase cycling and concomitant change of the
Despite the highly exible substrate binding pocket of irradiation frequency. No water suppression was used to avoid
CtXR, docking of the very well accepted unnatural inuences onto intensities of signals close to HDO signal. To
2,4-dichloroacetophenone into a rigid structure of CtXR/ eliminate protein frequencies a 30 ms spin lock was added aer
NADH illustrates a good tting. This nding contributes to the 90ꢂ pulse.
explain the high catalytic efficiency of the reaction. However,
The number of scans for the experiments varied between 128
the used molecular docking method cannot clarify the enan- and 256. Resulting short measurement times allowed to
tioselectivity of the transformation. Hence, better rened monitor the reaction progress with changing concentration and
molecular dynamic studies are necessary to get a more enabled STD NMR measurements during the transformations.
detailed picture of the ternary complexes, in particular, with Only measurements with the same parameters have been
respect to the exible loop region, which is involved in compared, when different complexes have been studied and
substrate xation. These applications should allow to predict matched. For the interpretation the largest signal in all
catalytic efficiencies based on molecular docking as well as on comparable experiments was set to 100% and the relative
determination of the carbonyl group polarization. intensities were determined in steps of 10%.14,15
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Paper
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Computational methods
For the molecular docking calculations the X-ray structure of
the CtXR [1MI3 from the RCSB PDB] is used and further more
optimized. For that purpose a monomer is cut off the tetrameric
enzyme and the NAD+ is changed to NADH. Additionally a
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gens, which are needed to determine proton–proton distances.
The docking calculations were performed using MOE program
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congurations and retain is set to 100. Docking is performed as
‘free’ docking as well as constraining the docking to the binding
side (NADH, Asp-50, Tyr-51, His-113,.). All these calculations
are made three times with default settings. Also redock calcu-
lations were done with the best tted molecule structures to
provide local minima.
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Acknowledgements
We thank Prof. Dr Peter Wolschann for kind help with docking
simulation and Ing. Susanne Felsinger for measuring NMR
spectra. We are grateful to Prof. Dr. B. Nidetzky and Dr Regina
Kratzer (both TU Graz) for providing the CtXR and for valuable
discussions.
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26004 | RSC Adv., 2013, 3, 25997–26004
This journal is ª The Royal Society of Chemistry 2013