An artificial oxygenase built from scratch: Substrate binding site identified using a docking approach

Article abstract of DOI:10.1002/anie.201209021

The substrate for an artificial iron monooxygenase was selected by using docking calculations. The high catalytic efficiency of the reported enzyme for sulfide oxidation was directly correlated to the predicted substrate binding mode in the protein cavity, thus illustrating the synergetic effect of the substrate binding site, protein scaffold, and catalytic site.

Full text of DOI:10.1002/anie.201209021

Angewandte
Chemie
DOI: 10.1002/anie.201209021
Artificial Metalloenzyme
An Artificial Oxygenase Built from Scratch: Substrate Binding Site
Identified Using a Docking Approach**
Charlꢀne Esmieu, Mickaꢁl V. Cherrier, Patricia Amara, Elodie Girgenti, Caroline Marchi-
Delapierre, Frꢂdꢂric Oddon, Marina Iannello, Adeline Jorge-Robin, Christine Cavazza,* and
Stꢂphane Mꢂnage*
The need for sustainable chemistry urges the scientific
community to design clean chemical processes. In this
context, the concept of green chemistry has emerged,[1] and
involves twelve principles with respect to avoiding both the
use and the generation of hazardous substances. Biocatalysis
is a promising strategy which fulfills most of these principles
(catalysis, reduction of organic solvent, nondangerous syn-
thesis and selective reactions, atom economy, nontoxic metals,
etc.).[2] One approach to creating new biocatalysts for
a defined reaction is the design of artificial metalloenzymes
which are based on the combination of metal-based catalysis
(by an inorganic complex) and protein-driven reaction
selectivity, thus conferring unnatural activities to biomole-
cules.[3] Compared to natural enzymes, the advantage of these
hybrids resides in an additional degree of optimization based
on structural modifications of the inorganic complex embed-
ded within the protein. A great variety of reactions has been
tackled using artificial metalloenzymes.[5] Among them, the
field of oxidation seems to be promising because artificial
hybrids can overcome the drawbacks of di(mono)oxygenases,
that is, the requirement of multiprotein complexes, including
the redox partner (ferredoxins or NADPH reductase for
example).[6]
the fine control at the active site, through constraints imposed
by the protein environment, on the sequence of the chemical
steps and the orientation of the substrates along the catalytic
cycle. Herein we report an original method for the design of
an artificial monooxygenase by taking charge of the three
essential parameters for enzymatic activity, with special
attention to the substrate binding mode. First, we inserted
inorganic catalysts into the periplasmic nickel-binding protein
NikA, through supramolecular interactions, to convert it from
a transport protein into a metalloenzyme.[7] Here, we have
taken advantage of NikAꢀs ability to bind iron complexes with
N2Py2 ligands,[7,8] complexes which are known catalysts for C
À
=
À
H, C C, and C OH oxidations as well as oxygen transfer to
sulfides.[9] Second, molecular docking calculations have been
conducted to screen sulfides for catalytic oxidation by a series
of iron complexꢀNikA hybrids. A set of six potential
substrates having a common motif was synthesized and the
catalytic properties of each hybrid were determined to
validate the docking simulations. Herein, we describe the
proof-of-concept of our method by designing a substrate
family, thus allowing us to define a new kind of artificial
oxygenase for sulfoxidation.
In addition to Fe/EDTAꢀNikA,[7] four different hybrids
were used in this study: Fe/L1ꢀNikA, Fe/L2ꢀNikA, Fe/
L3ꢀNikA, and Fe/L4ꢀNikA (Figure 1). The Fe/L (L =
ligand) complexes,[10] the resulting hybrids, and the crystal
structures of Fe/EDTAꢀNikA, Fe/L3ꢀNikA, and Fe/
L4ꢀNikA were previously described.[7,8]
The catalytic efficiency of an enzyme depends on an
accurate combination of the catalytic site, the substrate-
binding site, and the protein scaffold. This efficiency relies on
[*] C. Esmieu, Dr. E. Girgenti, Dr. C. Marchi-Delapierre, Dr. F. Oddon,
A. Jorge-Robin, Dr. S. Mꢀnage
The crystal structure of Fe/L1ꢀNikA was solved at 1.7 ꢁ
resolution (PDB code ID: 4I9D, see Figure S1 and Table S1 in
the Supporting Information). As previously observed for all
NikA-based hybrids, Fe/L1 binds to NikA through a salt
bridge between Arg137 and the carboxylate moiety of the
ligand. In addition, Trp100 and Trp398 are involved in CH–p
Laboratoire Chimie et Biologie des Mꢀtaux, Universitꢀ Joseph
Fourier-Grenoble 1, CEA, DSV/iRTSV, LCBM, CNRS, UMR5249
38041 Grenoble (France)
E-mail: stephane.menage@cea.fr
Homepage: http://www-dsv.cea.fr/irtsv/lcbm/bioce
Dr. M. V. Cherrier, Dr. P. Amara, M. Iannello, Dr. C. Cavazza
Metalloproteins Unit, Institut de Biologie Structurale Jean-Pierre
Ebel, CEA, CNRS UMR 5075, Universitꢀ Joseph Fourier-Grenoble 1
41 rue Horowitz, 38027 Grenoble Cedex 1 (France)
E-mail: christine.cavazza@ibs.fr
[**] The authors are grateful to the ANR “Programme Labex” (ARCANE
project n8 ANR-11-LABX-003) for funding and for an ANR 08-CP2D-
12 grant. We also thank the CEA, the University of Joseph Fourier,
and the CNRS for institutional support, and Region Rhꢁne-Alpes for
a CIBLE grant. We also thank the staff from the ID23-eh1 beamline
of the European Synchrotron Radiation Facility in Grenoble, France.
We appreciate the help from the staff at the computing facilities of
the CEA (DSV/GIPSI and CCRT).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201209021.
Figure 1. Selected ligands for hybrid synthesis.
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interactions with different parts of the complex, and in p-
stacking interactions with pyridine rings. As expected, two
different molecules (A and B) are present in the asymmetric
unit. In the case of Fe/L3ꢀNikA and Fe/L4ꢀNikA, the iron
complexes are present with cis-b and trans topologies in
molecules A and B, respectively.[8] For Fe/L1ꢀNikA, the
electron density was much more difficult to explain because
we observed a mixture of different topologies in both
molecules of the asymmetric unit. Such an outcome is
probably due to the absence of the cyclohexyl ring in L1,
thus leading to the loss of multiple CH–p interactions with
tryptophan residues, compared those of Fe/L3. This structural
difference also results in a rotation of Fe/L1 of about 808
around the axis formed by the carboxylate moiety and the
iron.
We decided to use molecular docking to find potential
substrates for NikA, substrates which could bind close to the
inserted catalyst (see Figure S2 in the Supporting Informa-
tion). The crystal structure of Fe/L1transꢀNikA was used and
molecules containing a C6H5-S-CH2-X motif were extracted
from the Zinc Database.[11] The main criterion was the
distance between Fe of the complex and S of the molecule,
Figure 3. Selected sulfides for iron/hybrid sulfoxidation. (*) corre-
sponds to the sulfide precursor of the drug.
amide group and Gln385. Conversely, S5 and S6 were chosen
to test the influence of the absence of the R2 ring on the
binding mode. These six molecules were docked in Fe/
L1transꢀNikA: S1–S4 exhibited similar interactions with the
protein. Compared to S0, the interaction with Glu247 was lost
because of the replacement of the NH2 group on the aromatic
ring of R1. However, their binding modes were not affected
and a favorable Fe-S distance was preserved. For S5, the five
best docking modes were found at a site that was different to
that for S1–S4, and S6 was docked at three different regions of
the cavity (see Figure S3 in the Supporting Information). The
influence of both the iron coordination sphere and complex
topology were also tested (see Figures S4 and S5 in the
Supporting Information). Molecular docking performed on
Fe/L4transꢀNikA showed that the presence of the second
carboxylate moiety on L4 hinders the binding of the S1–S6
(Figure S5). Molecular docking was also performed for Fe/
L3transꢀNikA, with the same results as those obtained for Fe/
L1transꢀNikA. When the docking experiments were per-
formed on Fe/L3cis-bꢀNikA, only one molecule, S1, bound in
the same cavity (Figure S4A), and S2–S6 were present at
a different site or with a different orientation (see the
Supporting Information and Figure S4B). This observation
identified S1 a good candidate for a reference substrate in this
study.
À
a distance which was in accord with the suggested Fe O···S
transition state (see the Supporting Information for details).
The results led to a set of 374 molecules, from which a family
with a R1-S-CH2-CONH-R2 motif (S- and N-substituted
thioglycolamide; R1 and R2 are aromatic substituents) was
identified, the simplest member being S0 (R1 = 3-NH2Ph,
R2 = a-naphthyl). Interestingly, the skeleton of the defined
substrate is comparable to the one of omeprazole or
modafinil, which are major drugs from the pharmaceutical
industry. S0 is stabilized in the cavity of Fe/L1transꢀNikA
through a hydrogen bond between the aromatic amide
function and the Gln385 side chain (Figure 2). An additional
interaction was detected with Glu247.
A set of molecules, namely S1–S6, was designed based on
the following criteria: the steric bulk of R1 and R2, their facile
synthesis, and their expected solubility in aqueous medium
(S0 was excluded because of its high insolubility; Figure 3). In
the first series, S1–S4, the amino substituent of R1 was
replaced by various bulky substituents and R2 was an
aromatic ring so as to maintain the interaction between the
The sulfides S1–S6 were synthesized by a nucleophilic
attack of the chosen substituted aromatic sulfide anion on the
2-chloro-N-phenylacetamide. The corresponding products,
that is, sulfoxides and sulfones, were obtained by direct
oxidation using NaIO4 and KHSO5/wet montmorillonite,
respectively.[12] Under these reaction conditions, a mixture
of the sulfone and sulfoxide products was always obtained,
thus leading, after purification, to a (46 Æ 5)% yield of
sulfoxide and (56 Æ 5)% yield of the corresponding sulfone in
the case of S1. Interestingly, all previously reported proce-
dures also reported product mixtures (please follow our
correction).
Initially, Fe/L1ꢀNikA was tested for the catalytic oxida-
tion of S1 by NaOCl (see Figure S6 in the Supporting
Information). We also tried other oxidants such as dioxygen
or H2O2 but no activity could be detected. The experimental
Figure 2. S0 docked in Fe/L1transꢀNikA.
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The three other Fe/LꢀNikA and Fe/EDTAꢀNikA
hybrids were then tested for their ability to selectively oxidize
S1. Only Fe/L3ꢀNikA was able to provide a high yield and
selectivity comparable to those of Fe/L1ꢀNikA (64% versus
69% for the yield and 163 versus 173 for the TON), and Fe/
EDTAꢀNikA, Fe/L2ꢀNikA, and Fe/L4ꢀNikAwere found to
be inactive (see Table S2 in the Supporting Information). On
one hand, the absence of activity for Fe/EDTAꢀNikA attests
to the fact that the metal ion is not sufficient to gain sulfide
oxidation but that the first coordination sphere, that is the
ligand providing a N4O2 coordination to the iron, is essential
for tuning its electronic properties. On the other hand, the
difference in activity between the different Fe/LꢀNikA
complexes correlates to the presence of a labile site in the
iron coordination sphere and was confirmed by structural
analyses (see Figure S1 in the Supporting Information).[8]
Compared to Fe/L2ꢀNikA and Fe/L4ꢀNikA, Fe/L1ꢀNikA
and Fe/L3ꢀNikA, which are missing one carboxylate moiety
of the ligand, contain a water molecule to complete the
coordination sphere, thus leading to an open-shell coordina-
tion. Consequently, the Lewis acidity of the iron should be
increased, thereby reinforcing its ability to activate the
oxidant (NaOCl) by direct binding to the metal center.[13]
Finally, the initial oxidation rates of the two efficient hybrids,
Fe/L1ꢀNikA and Fe/L3ꢀNikA, are distinct and attest to the
fact that the nature of the ligand affects the kinetics of the
sulfoxidation reaction. This data additionally supports the
proposal that the oxygen-transfer reaction is centered on the
iron complex (see Figure S8 in the Supporting Information).
The substrate analogues S2–S6 (Figure 3) were tested with
Fe/L1ꢀNikA. They were all oxidized to produce the corre-
sponding sulfoxides to a certain extent (Table 1). Indeed, the
sulfoxide yields were dependent on the steric hindrance of the
substrate. The most bulky substrates, S3 and S4, having the
dimethyloxyphenyl groups as R1, gave yields of only 40 and
18%, respectively. In contrast, S2 which has a methylphenyl
group as R1, led to an increased yield of 78%. The selectivity
for S1–S3 was complete but in case of S4 it fell to the same
level as that of the uncatalyzed reaction (18%). Interestingly,
sulfoxides were produced when the corresponding substrates
were shown by molecular docking to be oriented in front of
the embedded iron complex. However, the case of S4 is
puzzling because it docks in the required position but displays
low reactivity. We suggest a lower affinity of the hybrid for S4
in solution. The difference in nucleophilicity of the sulfur
atom would be minimal between S3 and S4 and cannot be
responsible for the difference in reactivity. Therefore, the
difference would arise from a subtle combination of steric
effects and hydrophobicity which allow the formation of
supramolecular interactions between the protein and the
molecule. Such interactions could be favorable in the case of
S1–S3 but disadvantageous in the case of S4. For S5 and S6,
the yield and the selectivity were found to be similar to those
observed in the absence of the hybrid. Here, the absence of
catalytic activity is directly related to a nonspecific binding of
the molecules in the S0 cavity, or even outside (see Figure S3
in the Supporting Information). This nonspecific binding is
probably due to the smaller substrate bulk and the loss of
hydrophobic interactions with the protein. In addition, the
Figure 4. Yield of sulfoxide with S1 as a substrate depending on the
catalytic conditions: gray: NaOCl, black: NaOCl + Fe/L1, light gray:
NaOCl + NikA, solid black: NaOCl + Fe/L1ꢀNikA, Catalyst: 5 nmol,
37 mm in 10 mm HEPES, pH 7.0, stirring at room temperature, 4 h.
conditions were optimized to determine the catalytic proper-
ties of the hybrid by varying the catalyst/substrate/oxidant
ratio (Figure 4). The free inorganic Fe/L1 complex was totally
inactive under each of the reaction conditions tested (see the
Supporting Information), whereas either NikA alone or
L1ꢀNikA catalyzed a low production of sulfoxide. In
addition, a minor by-product, identified as the dichlorinated
sulfoxide 4-AcNH-C6H5-SO-C(Cl)2-CO-NH-C6H5 (see Fig-
ure S7 in the Supporting Information) was observed. How-
ever, this product resulted from an uncatalyzed reaction,
because a higher yield (17%) was obtained in the absence of
any component of the hybrid and with a large excess of
NaOCl. In all cases, the overoxidation product (sulfone) was
never detected. Only the hybrid catalyzed the exclusive
formation of the sulfoxide with good yields after four hours
(up to 86%, Figure 4). However, variations of the substrate/
oxidant ratio drastically affected the yield of the reaction. On
one hand, as expected, increasing the oxidant concentration
led to a better yield for a given substrate concentration. For
example, by changing the catalyst/substrate/oxidant ratio
from 1:255:300 to 1:255:600, the reaction yield increased
from (45 Æ 5) to (69 Æ 5)%. On the other hand, if the
substrate concentration increased for a given oxidant con-
centration, the yield of the sulfoxide decreased. For instance
in the case of an amount of 600 equivalents of NaOCl, the
yield decreased from 86 to 68 %, then to 5% for a substrate
amount increasing from 127 to 255 then 500 equivalents,
respectively. The optimal reaction conditions (1:250:600)
provided a high catalytic efficiency. Under these reaction
conditions, (79 Æ 5)% of the total amount of S1 was
consumed in 4 hours and a TON of 173 with a TOF of
43 hÀ1 were measured, with a sulfoxide yield of (69 Æ 5)%. So
far, our enzyme displays one of the higher TONs for sulfide
oxidation catalyzed by an artificial metalloenzyme.[6c–f]
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Table 1: Catalytic properties of Fe/L1ꢀNikA for the oxidation of S1–S6 by
NaOCl.[a]
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Substrate
R1
R2
Ph
Ph
Ph
Ph
H
Hybrid
Yield
[%]
Sel.
[%]
TON
no
yes
0
69
0
À
S1
S2
S3
S4
S5
S6
4-AcNHC6H4
4-CH3C6H4
87[b]
173
no
yes
10
78
100
100
À
199
no
yes
14
40
20[b]
100
À
2,5-(MeO)2C6H3
3,4-(MeO)2C6H3
2,5-(MeO)2C6H3
3,4-(MeO)2C6H3
102
no
yes
15
18
2
À
18[b]
46
no
yes
12
15
12
15
À
À
no
yes
85
62
85
62
À
À
H
[a] Catalytic conditions: 10 mm HEPES buffer, pH 7.0, Fe-L1ꢀNikA
(5 nmol, 37 mm), catalyst/substrate/NaOCl=1:255:600, stirring at room
temperature for 4 h. Errors were estimated to be about 5% from three
different runs. [b] A dichlorinated by-product was observed.
high solubility observed for S5 and S6 compared to that for
S1–S4 supports our interpretation, thus indicating that it
would be directly attacked in solution by the oxidant to
generate the dichlorinated by-product.[14]
Thus, we have designed a reference substrate for our
artificial enzyme, and it contains the Ph-S-CH2-CONH-Ph
motif. We have also demonstrated that the chemoselectivity is
driven by supramolecular interactions between the protein
and the substrate at a position close to the catalytic center,
thus validating our docking approach. However, this outcome
is essential but not sufficient for enantioselectivity as in our
hands only weak, but measurable ee values were detected
(10% for S1, and 5% for S2 and S3). This data underlines the
importance of the protein scaffold on the enantioselective
control of a reaction. Protein and complex modifications
should be undertaken to optimize the enantioselective con-
trol.
Through this work, we have implemented an original
method to design a selective oxygenase for sulfide oxidation
and highlighted the synergetic effect of each partner. The
docking method allowed identification of a substrate binding
site in a non-enzymatic NikA and selection of a proper
substrate. This work is another example of the power of
synthetic biology, wherein a natural system is redirected to
broader applications for sustainable chemistry.
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Received: November 11, 2012
Published online: && &&, &&&&
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Keywords: computational chemistry · iron · metalloenzymes ·
.
[14] M. Deborde, U. von Gunten, Water Res. 2008, 42, 13 – 51.
oxidation · sulfur
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Communications
Artificial Metalloenzyme
C. Esmieu, M. V. Cherrier, P. Amara,
E. Girgenti, C. Marchi-Delapierre,
F. Oddon, M. Iannello, A. Jorge-Robin,
C. Cavazza,* S. Mꢁnage*
&&& — &&&
An Artificial Oxygenase Built from
Scratch: Substrate Binding Site Identified
Using a Docking Approach
The substrate for an artificial iron mono-
noxygenase was selected by using dock-
ing calculations. The high catalytic effi-
ciency of the reported enzyme for sulfide
oxidation was directly correlated to the
predicted substrate binding mode in the
protein cavity, thus illustrating the syner-
getic effect of the substrate binding site,
protein scaffold, and catalytic site.
Angew. Chem. Int. Ed. 2013, 52, 1 – 5
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!