An Unexpected Promiscuous Activity of 4-Oxalocrotonate Tautomerase: The cis-trans Isomerisation of Nitrostyrene

Article abstract of DOI:10.1002/cbic.201200371

Serendipitous switch: While exploring cis-nitrostyrene as a potential electrophile in Michael-type addition reactions catalysed by the enzyme 4-oxalocrotonate tautomerase (4-OT), it was unexpectedly found that 4-OT catalyses the isomerisation of cis-nitrostyrene to trans-nitrostyrene (k_cat/K_m= 1.9×10³M^-1s^-1).

Full text of DOI:10.1002/cbic.201200371

DOI: 10.1002/cbic.201200371
An Unexpected Promiscuous Activity of 4-Oxalocrotonate Tautomerase:
The cis–trans Isomerisation of Nitrostyrene
Ellen Zandvoort, Edzard M. Geertsema, Bert-Jan Baas, Wim J. Quax, and Gerrit J. Poelarends*[a]
The tautomerase superfamily consists of homologous proteins
with a characteristic bab structural fold and a conserved cata-
lytic N-terminal proline.[1] 4-Oxalocrotonate tautomerase (4-OT)
from Pseudomonas putida mt-2 is a member of this superfami-
ly, and catalyses the enol-keto tautomerisation of 2-hydroxy-
hexa-2,4-dienedioate (1) to 2-oxohex-3-enedioate (2;
Scheme 1).[2] In this natural activity of 4-OT, the Pro1 residue
ing the scope of this reaction, we assessed whether cis-nitro-
styrene (6; Scheme 3) can serve as an alternative substrate for
4-OT. Here, we report the unexpected finding that 4-OT can
promiscuously catalyse the cis–trans isomerisation of nitrostyr-
ene. Furthermore, we describe the characterisation of this reac-
Scheme 3. Non-natural cis–trans isomerisation of nitrostyrene catalysed by
4-OT.
tion, and propose two possible mechanisms by which the con-
version of 6 to 4 catalysed by 4-OT might proceed.
Scheme 1. The natural tautomerisation reaction catalysed by 4-OT.
The absorbance spectra of 4 and 6 largely overlap, and both
have an absorbance maximum at around 320 nm, which corre-
sponds to the vinylic C=C double bond (Figure 1A). However,
functions as the catalytic base and shuttles the 2-hydroxyl
proton of 1 to the C5 position to yield 2.[3] Indeed, Pro1 has
the correct protonation state (the pKa of Pro1 in 4-OT is ~6.4)
to be able to act as a catalytic base at cellular pH.[4] Intriguing-
ly, 4-OT can also catalyse several CÀC bond-forming reactions,
including aldol condensation and Michael-type addition reac-
tions.[5–7] In these unnatural activities of 4-OT, Pro1 likely func-
tions as a nucleophile, analogously to the role of the amino
acid l-proline and derivatives thereof in the field of organo-
catalysis.[8]
We have previously shown that 4-OT catalyses the Michael-
type addition of acetaldehyde (3) to trans-nitrostyrene (4) to
yield 4-nitro-3-phenylbutanal (5) (Scheme 2).[7] While investigat-
Scheme 2. Non-natural Michael-type addition catalysed by 4-OT.
[a] E. Zandvoort, Dr. E. M. Geertsema, B.-J. Baas, Prof. Dr. W. J. Quax,
Prof. Dr. G. J. Poelarends
Department of Pharmaceutical Biology
Groningen Research Institute of Pharmacy, University of Groningen
Antonius Deusinglaan 1, 9713 AV Groningen (The Netherlands)
E-mail: g.j.poelarends@rug.nl
Figure 1. A) UV absorbance spectra of cis-nitrostyrene (6, cNS, 2 mm, e6 =
0.31 mmÀ1 mmÀ1) and trans-nitrostyrene (4, tNS, 2 mm, e4 =1.44 mmÀ1 mmÀ1
in 20 mm NaH2PO4 buffer (pH 7.3) containing 10% EtOH; B) UV monitoring
of the incubation of cis-nitrostyrene (6) in the presence of acetaldehyde (3),
with 4-OT, or without 4-OT (blank).
)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200371.
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the shapes of the absorbance spectra differ slightly, and the
e320 values for 4 and 6 differ significantly, thus making it possi-
ble to distinguish between the two compounds when they are
present at the same concentration (e6 =0.31 mmÀ1 mmÀ1, e4 =
1.44 mmÀ1 mmÀ1). To examine whether 4-OT catalyses the Mi-
chael-type addition of 3 to 6, 4-OT (73 mm) was incubated with
3 (50 mm) and 6 (2 mm) in NaH2PO4 buffer (20 mm, pH 7.3).
The reaction was monitored by UV spectroscopy, and a de-
crease in A320 was observed (Figure 1B). However, the starting
absorbance was 2.8, which is about 4 times higher than ex-
pected (0.7) for the concentration (2 mm) of 6 used (Fig-
ure 1A). Furthermore, the value of 2.8 accurately corresponds
to the A320 value expected for 4 (2 mm; Figure 1A). When 6
and 3 were incubated without 4-OT, the A320 value was stable
at 0.7, which is in agreement with the expected A320 value for
6 (2 mm; Figure 1). Taken together, these observations suggest
that 4-OT is able to rapidly catalyse the isomerisation of 6 to 4
(within the time of mixing 4-OT, 3 and 6; Scheme 3).
To further investigate the ability of 4-OT to catalyse the iso-
merisation of 6 to 4, the enzyme (7.3 mm, monomer concentra-
tion) was incubated with 6 (2 mm) in 20 mm NaH2PO4 buffer
(pH 7.3), and the reaction was monitored by UV spectroscopy
(Figure 2B). A fast increase in A320 and a change in the shape
of the absorbance spectrum were observed, thus indicating
formation of 4. To confirm that the conversion of 6 to 4 was
enzyme-catalysed, a control reaction was performed without 4-
OT; this did not result in an increase in A320 (Figure 2A). Al-
though the 4-OT sample used in this assay was of high purity
(>95%, as assessed by SDS-PAGE), the possibility existed that
a contaminating enzyme from the expression host (rather than
4-OT) was catalysing the isomerisation reaction. Therefore, a
4-OT sample free of cellular proteins was prepared by total
chemical synthesis.[9] Incubation of this synthetic 4-OT (7.3 mm,
monomer concentration) with 6 (2 mm) in 20 mm NaH2PO4
buffer (pH 7.3) gave an increase in A320 similar to that observed
for the incubation of 6 with recombinant 4-OT (Figure 2C).
To verify that 4 is formed during incubation of 4-OT with 6,
1
the reaction was analysed by H NMR spectroscopy. 4-OT (4.4ꢁ
10À3 mm, 0.06 mol%) was incubated with 6 (7 mm) in NaD2PO4
buffer (pD 7.6) containing 30% (v/v) deuterated methanol
(Figure 3). At the start of the reaction, characteristic signals
(7.04 and 7.15 ppm) corresponding to the vinyl protons of 6
were detected (Figure 3A). These signals gradually diminished,
and signals assigned to the vinyl protons of 4 (7.87 and
8.11 ppm) appeared. Furthermore, the signals corresponding
to the phenylic protons of 6 (7.40 ppm) were decreasing over
time, while signals for the phenylic protons of 4 (7.52 and
7.66 ppm) emerged (Figure 3B–D). The final spectrum, record-
ed after completion of the enzymatic conversion of 6 (Fig-
ure 3D; t=15 min), is virtually identical to the spectrum of an
authentic standard of 4 (Figure 3E), thus confirming that 4 is
indeed the product of the 4-OT-catalysed isomerisation of 6.
Integration of the signals indicated that the enzymatic conver-
sion of 6 to 4 is relatively fast, with 44 and 70% conversion
after 2 and 5 min, respectively,
and near full conversion after
15 min. A control reaction (with-
out 4-OT, but under otherwise
identical conditions) showed no
conversion of 6, which indicates
that nonenzymatic isomerisation
of 6 into 4 does not occur under
the assay conditions used.
Having established that 4 is
the product of the enzymatic
isomerisation of 6, kinetic pa-
rameters were determined by
using various concentrations of
6 (0.1–6 mm). 4-OT catalyses the
isomerisation of 4 to 6 with
a kcat of 4.8 sÀ1 and a Km of
2.5 mm, thereby yielding a kcat/
K
m of 1.9ꢁ103 mÀ1 sÀ1.
Next, we evaluated the impor-
tance of residue Pro1 for the iso-
merisation activity of 4-OT, by
mutation of the Pro1 residue to
an alanine. The corresponding
mutant, 4-OT P1A (7.3 mm, mo-
nomer concentration), was incu-
bated with 6 (2 mm) in NaH2PO4
buffer (20 mm, pH 7.3) and the
reaction was monitored by UV
spectroscopy (Figure 2D). 4-OT
Figure 2. UV absorbance spectra monitoring the isomerisation of cis-nitrostyrene (6) to trans-nitrostyrene (4).
A) reaction without enzyme, B) reaction catalysed by recombinant 4-OT, C) reaction catalysed by synthetic 4-OT,
D) reaction catalysed by 4-OT P1A.
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Figure 3. Stack plot of partial H NMR spectra monitoring the conversion of 6 to 4 catalysed by 4-OT. A) t=0; B) t=2; C) t=5, and D) t=15 min; E) reference
(4).
P1A showed almost no activity, thus demonstrating that Pro1
is essential for the 4-OT-catalysed isomerisation of 6 to 4.
Arg11 and Arg39 are key catalytic residues in the natural tauto-
merase activity of 4-OT and interact with, respectively, the C6
and C1 carboxylate groups of 1.[10] To test whether either of
these active site arginines is important for binding the nitro
group (or nitronate) of 6, these arginines were mutated to ala-
nine residues, thereby yielding mutants 4-OT R11A and 4-OT
R39A. These mutations did not affect the ability of the enzyme
to catalyse the isomerisation of 6 to 4 (data not shown). This
raised the question as to whether the active site of 4-OT is
needed for this reaction, or whether the amino acid l-proline
(or a peptide with an N-terminal proline) would be sufficient
for catalysis of this isomerisation reaction. Therefore, two con-
trol experiments were performed. Firstly, l-proline (7.3 mm) was
incubated with 6 (2 mm) in NaH2PO4 buffer (20 mm, pH 7.3).
UV spectroscopic analysis of this incubation indicated that 4
was not formed (Figure S1A in the Supporting Information).
Even with a 100 times higher concentration of l-proline
(730 mm) no isomerisation of 6 into 4 was observed. Secondly,
the same incubation experiment was performed, but with a 4-
OT sample that had been boiled for 20 min. (The enzyme dena-
tures upon boiling, which results in an unstructured peptide
chain bearing a proline residue at the N terminus.) The dena-
tured enzyme almost completely lost its activity (Figure S1B).
Both control experiments led to the conclusion that the active
site of 4-OT is responsible for the isomerisation of 6 to 4.[11]
Given the importance of Pro1 for the isomerisation activity
of 4-OT, two possible mechanisms can be proposed. In the
first, 4-OT catalyses the conversion of 6 to 4 through a covalent
enzyme–substrate intermediate (Scheme 4A). Residue Pro1
acts as a nucleophile and attacks the C2 carbon of 6, thereby
leading to the formation of an aci-nitronate intermediate. In
this covalent intermediate (6 and Pro1) the original C=C dou-
ble bond becomes a CÀC single bond. Around this bond, an
internal rotation takes place to give a more favourable confor-
mation with less repulsion between the nitro group and the
phenyl ring. Subsequent collapse of the aci-nitronate inter-
mediate restores the C=C double bond and releases 4 from
Pro1. Such a covalent mechanism is plausible because Pro1
has the correct protonation state to act as a nucleophile at
pH 7.3, and because mechanisms involving nucleophilic attack
on an unsaturated C=C bond have previously been proposed
for natural isomerases, such as maleate isomerase, maleylace-
toacetate isomerase and maleylpyruvate isomerase.[12–14] In
maleate isomerase the nucleophile is a cysteine residue,
whereas in the case of maleylacetoacetate and maleylpyruvate
isomerases, the essential cofactor glutathione functions as the
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tomerase activity, 4-OT catalyses
the hydrolytic dehalogenation of
trans-3-chloroacrylic acid, which
is the natural activity of another
member of the tautomerase su-
perfamily,
trans-3-chloroacrylic
acid dehalogenase (CaaD).[1,16,17]
4-OT also has promiscuous activi-
ties that are not known to occur
naturally within the tautomerase
superfamily, including aldolase,
dehydratase and Michael-type
addition activities.[5–7] Further-
more, when Pro1 in 4-OT was
mutated to an alanine residue
(4-OT P1A), the mutant enzyme
was capable of the decarboxyla-
tion of oxaloacetate.[18] So far,
the isomerisation activity (kcat
4.8 sÀ1, kcat/Km 1.9ꢁ103 mÀ1 sÀ1
)
Scheme 4. A) A possible mechanism for the 4-OT-catalysed isomerisation of 6 to 4. Pro1 acts as a nucleophile to
form a covalent enzyme–substrate intermediate. B) A possible mechanism for the 4-OT-catalysed isomerisation of
6 to 4 in which Pro1 acts as a general base to activate a water molecule.
appears to be the highest pro-
miscuous activity of 4-OT. We
demonstrated that Pro1 is essen-
tial for this isomerisation activity,
nucleophile. To find further support for this proposed cova-
lent-intermediate mechanism, we performed a labelling experi-
ment to determine whether a covalent intermediate between
4-OT and substrate 6 (or product 4) can be formed. An aliquot
of 4-OT (500 mg) was incubated with 6 (10 mm) in NH4HCO2
buffer (1 mm, pH 7.3) containing 30% (v/v) MeOH, and the
reaction was stopped after 5 min by freezing (À208C). ESI-MS
analysis revealed two major peaks, one corresponding to un-
labelled 4-OT (6811 Da, ~80%) and one consistent with the
expected mass for 4-OT labelled with either 6 or 4 (6959 Da,
~20%). Unfortunately, the labelled amino acid residue could
not be identified.[15]
and that the organocatalyst l-proline is not able to catalyse
the cis–trans isomerisation of nitrostyrene. This can be ex-
plained, at least in part, by the higher pKa of the (secondary)
amino group of l-proline compared to the pKa of Pro1 in 4-OT.
Under the assay conditions (pH 7.3), the amino group of l-pro-
line (pKa ~10.6) exists predominantly in the protonated form.
This renders l-proline unable to act as a nucleophile or base,
which might explain its inability to catalyse the isomerisation
reaction of 6 into 4. In 4-OT, however, the pKa of Pro1 (~6.4) is
lower as a consequence of the low dielectric constant of the
surrounding active site environment.[4,19] This enables Pro1 to
function either as a nucleophile or as a general base, according
to one of the proposed mechanisms. Future mechanistic and
structural studies might shed light on the exact mechanism of
this new promiscuous activity of 4-OT.
In the other possible mechanism, Pro1 acts as a general
base that activates a water molecule (Scheme 4B). Upon addi-
tion of the hydroxide ion to the C=C double bond and con-
comitant electron migration, an aci-nitronate intermediate is
formed in which the unsaturated C=C is turned into a saturated
CÀC bond. Now, the molecule can adapt the more favourable
conformation through internal rotation around the C1À
C2 single bond. Collapse of the aci-nitronate intermediate re-
sults in restoration of the C=C double bond, and the release of
product 4. This possible role of Pro1 (as a water-activating resi-
due) is suggested by a mechanism that was proposed for the
promiscuous dehalogenase activity of 4-OT, in which residue
Pro1 can activate a water molecule for attack at the C3 posi-
tion of trans-3-chloroacrylic acid.[16]
Acknowledgements
We thank Dr. Wesley R. Browne for his assistance in the prepara-
tion of cis-nitrostyrene. Furthermore, we thank C. Margot Jeroni-
mus-Stratingh, Annie van Dam, Ing. Marcel P. de Vries, and Dr.
Hjalmar P. Permentier for their expert assistance in acquiring and
interpreting the MS spectra. This research was financially sup-
ported by the Netherlands Organisation for Scientific Research
(VIDI grant 700.56.421 to G.J.P.) and the European Research
Council under the European Community’s Seventh Framework
Programme (FP7/2007-2013)/ERC Grant agreement no. 242293
(to G.J.P.).
In conclusion, we set out to investigate whether 4-OT can
catalyse the addition of 3 to 6, similarly to the previously re-
ported promiscuous Michael-type addition of 3 to 4.[7] Howev-
er, we found that 4-OT does not catalyse the addition of 3 to
6, but instead catalyses the isomerisation of 6 into 4. This adds
yet another chemically distinct reaction to the list of (promis-
cuous) reactions catalysed by 4-OT. Apart from its natural tau-
Keywords: catalytic promiscuity
isomerization · tautomerism
·
enzyme catalysis
·
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Received: June 4, 2012
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COMMUNICATIONS
E. Zandvoort, E. M. Geertsema, B.-J. Baas,
W. J. Quax, G. J. Poelarends*
&& – &&
An Unexpected Promiscuous Activity
of 4-Oxalocrotonate Tautomerase: The
cis–trans Isomerisation of Nitrostyrene
Serendipitous switch: While exploring
cis-nitrostyrene as a potential electro-
phile in Michael-type addition reactions
catalysed by the enzyme 4-oxalocroto-
nate tautomerase (4-OT), it was un-
expectedly found that 4-OT catalyses
the isomerisation of cis-nitrostyrene
to trans-nitrostyrene (kcat/Km =
s
1.9ꢁ103 mÀ1 À1).
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ChemBioChem 0000, 00, 1 – 5
ÝÝ
These are not the final page numbers!