An Artificial Heme Enzyme for Cyclopropanation Reactions

Article abstract of DOI:10.1002/anie.201802946

An artificial heme enzyme was created through self-assembly from hemin and the lactococcal multidrug resistance regulator (LmrR). The crystal structure shows the heme bound inside the hydrophobic pore of the protein, where it appears inaccessible for substrates. However, good catalytic activity and moderate enantioselectivity was observed in an abiological cyclopropanation reaction. We propose that the dynamic nature of the structure of the LmrR protein is key to the observed activity. This was supported by molecular dynamics simulations, which showed transient formation of opened conformations that allow the binding of substrates and the formation of pre-catalytic structures.

Full text of DOI:10.1002/anie.201802946

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Accepted Article
Title: An Artificial Heme Enzyme for Cyclopropanation Reactions
Authors: Lara Villarino, Kathryn Splan, Eswar Reddem, Cora Gutiérrez
de Souza, Lur Alonso-Cotchico, Agustí Lledós, Jean-Didier
Maréchal, Andy-Mark Thunnissen, and Gerard Roelfes
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201802946
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COMMUNICATION
An Artificial Heme Enzyme for Cyclopropanation Reactions
[
a]
[b]
[a]
[c]
Lara Villarino, Kathryn E. Splan, Eswar Reddem, Lur Alonso-Cotchico, Cora Gutiérrez de
[
a]
[c]
[c]
[d]
Souza, Agustí Lledós, Jean-Didier Maréchal, Andy-Mark W. H. Thunnissen and Gerard
Roelfes*[a]
Abstract: An artificial heme enzyme was created by self-assembly
Regulator (LmrR), capable of catalyzing abiological
enantioselective cyclopropanation reactions. Moreover, we
propose that the structural dynamics of the artificial heme enzyme
are key to its catalytic activity.
from hemin and the Lactococcal multidrug resistance Regulator
(
LmrR). The crystal structure shows the heme bound inside the
hydrophobic pore of the protein, where it appears inaccessible for
substrates. Yet, good catalytic activity and moderate
LmrR is a homodimeric protein with a unique and highly
dynamic structure that is key to its biological function.[28, 29] It
presents an unusually large hydrophobic and promiscuous
binding pocket at the dimer interface, which has proven to be very
suitable for the creation of a novel active site by anchoring of a
catalytically active Cu(II) complex inside. The resulting artificial
enantioselectivity was observed in the abiological cyclopropanation
reaction. We proposed that the dynamic nature of the structure of the
LmrR protein is key to the observed activity. This was supported by
molecular dynamics simulations, which showed transient formation of
opened conformations allowing for binding of substrates and
formation of pre-catalytic structures.
metalloenzymes
enantioselective Lewis acid catalysis.
supramolecular assembly of the artificial metalloenzyme by
combination with Cu(II)-phenanthroline complex proved
have
been
applied
successfully
In particular the
in
[
30-33]
Engineered heme proteins such as Cytochrome P450,
Myoglobin and Cyctochrome C have emerged as excellent
catalysts for new-to-nature reactions,[1, 2] including carbene
a
powerful, giving rise to excellent enantioselectivities in the
[
3-7]
olefinations,[8, 9]
[34]
transfer reactions such as cyclopropanations,
N-H,
Friedel-Crafts alkylation reaction.
The diversity of reactions
[
10, 11]
Si-H, and B-H insertion reactions.[13] These enzymes
[12]
catalysed by LmrR based artificial enzymes led us to believe that
the LmrR scaffold could be extended to other catalyst types,
including heme-based catalysts.
have proven amenable to optimization by both genetic methods
as well as co-factor replacement.[
(
14-20]
A common feature of these
designed) heme enzymes is that they contain a large
hydrophobic substrate binding pocket orthogonal to the plane of
the heme moiety. Alternatively, significant effort has been devoted
to the de novo design of heme proteins, particularly based on 4-
helix bundles,[21-25] antibodies or other proteins,[26, 27] yet none of
these has found application in catalysis of new-to-nature
reactions. A key difference is that these artificial heme enzymes
generally do not present a defined binding site suitable for binding
the often hydrophobic substrates. Here, we report a novel artificial
heme enzyme based on the Lactococcal multidrug resistance
[a]
Dr. L. Villarino, Dr. E. Reddem, C. Gutiérrez de Souza MSc, Prof. G.
Roelfes
Stratingh Institute for Chemistry, University of Groningen
Nijenborgh 4, 9747 AG Groningen, The Netherlands
E-mail: j.g.roelfes@rug.nl
[b]
[c]
[d]
Prof. K.E. Splan
Department of Chemistry, Macalester College
Scheme 1. Schematic representation of the assembly of the LmrR-based
artificial heme enzyme and the catalyzed enantioselective cyclopropanation
reaction.
1600 Grand Avenue, Saint Paul, Minnesota 55105, United States
L. Alonso-Cotchico MSc, Prof. A. Lledós, Prof. J.D. Maréchal
Departament de Química, Universitat Autònoma de Barcelona
Edifici C.n., 08193, Cerdanyola del Vallés, Barcelona, Spain.
Dr. A. M. W. H. Thunnissen
Groningen Biomolecular Sciences and Biotechnology Institute
University of Groningen
The design was based on an LmrR variant that includes a C-
terminal Strep tag and has two mutations in the DNA binding
domain, i.e., K55D and K59Q, which facilitates expression and
purification.[ A second mutant, LmrR_W96A, was prepared to
assess the effect of the tryptophan residues in the hydrophobic
Nijenborgh 4, 9747 AG Groningen, The Netherlands
31]
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
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COMMUNICATION
pore (W96 and W96´, with the prime denoting the dimer related
subunit), which are known to play a role in the binding of guest
molecules.[28]
an LmrR that had the K55 and K59 residues reintroduced. The
overall conformation of LmrR is similar to that observed in
previously published drug-bound complexes.[28] Electron density
in between the indole rings of the central W96/W96’ pair is
attributed to the presence of the bound heme. Clear density is
observed only for the porphyrin ring, owing in part to a
crystallographic 2-fold axis, but also to a lack of specific stabilizing
interactions with the polar peripheral heme substituents. The
heme, lacking the axial chloride ligand, was modelled in the LmrR
crystal structure with four alternate binding modes, differing by
rotations around a central axis perpendicular to the plane of the
heme, and by a flip of the heme due the crystallographic 2-fold
symmetry (Figure S6). In all binding modes, the heme-iron is
shielded at either side of the heme by the indole ring of W96 and
W96’ and lies at an average distance of ~4 Å from the carbon
atoms, suggesting cation-π interactions. Further stabilization is
provided by van der Waals contacts with hydrophobic residues in
the vicinity of the central tryptophan pair, i.e., M8, A11 and V15
The artificial heme enzymes were created through self-
assembly, by addition of iron (III) chloroprotoporphyrin IX (Hemin)
to the corresponding protein LmrR variant in a buffered solution
(
4
50 mM KHPO , 150 mM NaCl, pH 7) (scheme 1). The interaction
of hemin with LmrR was examined by electronic absorption
spectroscopy. The visible spectrum of hemin at pH 7.0 (Figure 1a)
exhibits a broad Soret band and a weak peak c.a. 610 nm in the
Q band region, which is characteristic of a - porphyrin dimer
previously shown to predominate in aqueous hemin solutions at
neutral pH.[35, 36] Addition of LmrR results in a substantial
sharpening in the Soret and Q-band regions, resulting in a
spectrum that is similar to that of hemin in organic solvents.[36]
Clearly, upon addition of the protein, the dimeric porphyrin
structure is disrupted and the hemin is in a monomeric form.
Monitoring spectral changes as a function of added protein
indicates that hemin binds to LmrR with a ratio of one hemin per
dimeric protein. This stoichiometry was further confirmed via
monitoring of tryptophan fluorescence quenching, which was
complete upon the addition of 1 equivalent of hemin per LmrR
dimer (Figure 1b). Fitting of fluorescence titration data yields a
(and their equivalents from the dimer mate).This structure is
unlikely to allow for catalysis, as the catalytic iron is fully shielded
and cannot be accessed by substrates. However, the crystal
structure already suggests considerable dynamics in the binding
of the heme, which may include catalytically viable conformations.
This was supported by MD simulations (vide infra).
dissociation constant (K ) of 38  27 nM, indicating that the affinity
D
of the protein for hemin is quite high, and is in fact comparable to
or stronger than previously measured for other flat, planar
molecules.[28]
Figure 2. Crystal structure of LmrR⊂heme (PDB 6FUU). The protein
crystallized in a tetragonal crystal form with one polypeptide chain occupying
the asymmetric unit; the functional dimer (here shown in cartoon representation)
being formed by a crystallographic dyad. In the electron density maps, the
polypeptide chain is well defined, except for the tip region of the β-wing
(residues 70–73) and the N- and C-termini (residues 1–4 and 109–131,
including the C-terminal strep-tag). These regions show a high degree of
disorder and were excluded from the final model. The heme is stacked in
between the side chains of W96/W96’ (for clarity only one of the alternate heme
binding orientations is shown).
Figure 1. (a) Electronic absorption spectra upon the addition of LmrR to 5 µM
hemin. Inset: Changes in absorption values as
a function of protein
concentration. Buffer: 50 mM phosphate buffer/150 mM NaCl, pH 7.0. (b)
Fluorescence spectra upon the addition of hemin to 1 µM LmrR dimer. Inset:
Fraction of LmrR bound to hemin as a function of added hemin concentration.
Buffer: 50 mM phosphate buffer/150 mM NaCl, pH 7.0.
In contrast, UV/vis titration of hemin with LmrR_W96A results
in a loss of spectral intensity, but no substantial change in spectral
line shape, likely owing to non-specific binding on the protein
surface (Figure S4). These results clearly show that W96/W96’
play a key role in the interaction of hemin with LmrR that leads to
a monomeric hemin structure, and indicate that hemin lies either
partly or entirely within the hydrophobic pocket.
The potential of the artificial heme enzymes in catalysis was
examined in the cyclopropanation of o-methoxystyrene (1a) with
ethyldiazoacetate (EDA, 2) under anaerobic conditions as the
benchmark reaction (Tables 1, S4). In the absence of LmrR, the
reaction was sluggish and diethyl fumarate (4), which results from
the dimerization of EDA, was found as the major product (entry
1). The artificial heme enzyme (LmrR⊂heme) was assembled in
situ by addition of 1 mol% of hemin to a slight excess (1.1 equiv)
of LmrR in previously deoxygenated 50 mM KPi buffer pH 8.0.The
LmrR⊂heme catalyzed reaction requires addition of sodium
Hemin:LmrR stoichiometry and the importance of W96/W96’
in complex formation are further supported by the crystal structure
of LmrR co-crystallized with hemin (Figure 2, S6). Crystals
diffracting to sufficiently high resolution could be obtained only for
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COMMUNICATION
dithionate as reductant to generate the Fe(II)-heme, which is the
active complex required for carbene generation. The reaction was
started by addition of 30 mM of 1a and 10 mM of 2. After 18 h at
significant increase in activity was observed in case of the
mutants D100A and M8A, with the latter also giving rise to the
highest enantioselectivity, that is, 44% ee (entry 7).
4
ºC, the trans isomer of the cyclopropanation product 3a
Further optimization of the reactions conditions was
performed with this mutant (Tables S5-6). It was found that a
threefold excess of styrene over EDA and pH 7 were the optimal
conditions in terms of activity and selectivity (entry 9)
(trans/cis=92:8) was obtained as the major product in 25% yield,
which corresponds to a total turnover number (TTN) of 247, and
with an ee of 17%. (entry 2). The only detectable side product was
diethyl fumate (4), with 28 turnovers. These results clearly show
the acceleration and chemoselectivity due to the LmrR scaffold
compared to the reactions catalyzed by hemin alone, under the
same conditions.
The activity of LmrR_M8A⊂heme was compared for
substrates 1a-c. (entries 8-13). In all cases, the artificial heme
enzyme significantly outperformed the free hemin cofactor. The
enantioselectivity observed ranged from 25% ee in the case of 3c
(entry 13) to 51% ee in the case of 3a (entry 9).
Table 1. Results of cyclopropanation reaction of styrene derivatives 1a-c with
EDA (2) catalyzed by LmrR⊂heme.[a]
The observed catalytic activity and enantioselectivity are
difficult to rationalize based on the crystal structure, which shows
the catalytic iron center sandwiched between the two tryptophan
residues where it is inaccessible for the substrates. To gain more
insight into the origin of the catalytic activity, computational
studies were performed.
entry
LmrR
-
1[c]
1a
1a
1a
1a
3
Yield %
5 ± 2
TTN
51
3/4
0.8
11
9
ee %[b]
-
1
2
3
4
3a
3a
3a
3a
LmrR
25 ± 11
23 ± 2
247
232
17 ± 5
11 ± 1
LmrR _F93A
LmrR
Calculations focused on determining the conformational
rearrangement required for LmrR⊂heme to allow substrate
binding and the reaction to take place. Local and global
rearrangements of the protein were assessed for the binding of
the heme as well as in presence of the substrates by combining
protein-Ligand Docking, Quantum Mechanics calculations and
large scale Molecular Dynamics simulations.
38 ± 8
375
276
15
20
8
24 ± 5
< 5
_D100A
5
6
7
LmrR
W96A
1a
1a
3a
3a
28 ± 13
_
1.5
.5
±
LmrR _V15A
3
17 ± 1
0
LmrR _M8A
1a
1a
1a
1b
1b
1c
1c
3a
3a
3a
3b
3b
3c
3c
36 ± 13
5 ± 2
359
51
15
0.8
6
44 ± 12
Protein-ligand dockings of both the heme (5) and its adduct
with the carbene intermediate (6) (Figure S6) were carried out on
LmrR, starting from the LmrR⊂heme crystal structure. Local
rearrangements were assessed by introducing rotameric flexibility
for all the amino acids at the dimer interface. For 5 and 6, four
binding poses with good predicted affinity (ChemScore values
higher than 50 units) were found for both cases (Table S3). They
correspond to different orientations of the heme group as a result
of rotations around the axis perpendicular to the average plane of
the heme passing through the metal. The best docking solutions
of LmrR⊂5 show the heme sandwiched between W96/W96’,
similar to the crystal structure. However, in the case of the
carbene complex (LmrR⊂6), some low energy solutions present
W96’ rotated towards the solvent, thus providing space to
accommodate the co-substrate (Figure S1). To assess the
structural rearrangement of LmrR upon heme binding, the four
predicted structures of LmrR⊂5 and LmrR⊂6 were submitted to
8[c]
-
-
9[c]
LmrR _M8A
45 ± 9
6 ± 0
449
59
51 ± 14
1
1
1
1
0[c]
1[c]
2[c]
3[c]
-
n.d
n.d
0.2
3
-
LmrR _M8A
-
39 ± 13
1 ± 0
391
12
38 ± 5{d}
-
LmrR _M8A
35 ± 13
351
25 ± 5
[
a] Conditions: 1 (30 mM), 2 (10 mM), hemin (1 mol%; 10 μM), LmrR_X (1.1
mol%; 11 μM) in 50 mM phosphate buffer (pH 8.0), under Ar, at 4°C for 18h;
Results are the average of at least two independent experiments, both carried
out in duplicate. [b] of the trans product; trans:cis > 85:15. [c] pH 7.0. [d] of the
[
37]
1
R, 2R enantiomer.
The role of the protein scaffold was explored further by
100ns MD simulations.
mutagenesis of residues in and around the hydrophobic pore. In
addition to W96 (vide supra), residues M8, V15, F93, and D100
were mutated to alanine (see ESI). With the exception of V15A,
all mutants showed significant protein enhanced catalytic activity
that was comparable or greater than LmrR itself (entry 3-7).
Surprisingly, this was also the case for LmrR_W96A, albeit with a
complete loss of enantioselectivity. We attribute this to hemin-
LmrR_W96A association away from the hydrophobic pore, which
may result in assemblies that are catalytically active but lack the
Cluster analysis shows a high stability of the LmrR-heme
complex (Figure S8), with W96 and W96’ generating
a
hydrophobic patch that sandwiches the heme. Some of the most
populated clusters of the LmrR⊂5 system (clusters 1 and 3-6)
show high similarity with the crystal structure, with the iron atom
inaccessible to solvent (Figure 3a). On the other hand, clusters 0
and 2 (see SI for details), show changes in the orientation of the
α helix containing W96’ (α4) and a flip of the W96’ indole towards
the outside of the pore (Figures 3b, S10). These predicted
defined chiral interactions to induce enantioselectivity.
A
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COMMUNICATION
conformational changes are in agreement with previous reports
that flexibility in the orientation of α4 is a major contributor to the
ability of LmrR to structurally adapt to different drug molecules.[29]
These changes in the protein structure are accompanied by a
significant displacement of the heme towards the solvent. The
result is an opened conformation that has significant free space
on the axial face of the heme group. This appears of key
importance to catalysis, since the iron site becomes accessible to
bind the carbene, which allows the reaction occur.
Since the M8A mutant showed the best results in catalysis,
the dynamic behaviour of this artificial metalloenzyme was also
studied. For consistency, all the simulations were carried out in
the same conditions as before. The system resulting from the
docking of the heme linked to the TS structure for the 1R,2R
enantiomeric product into the LmrR protein was selected as
starting point for a 100ns MD simulation, which suggests that the
effect of this mutation is mainly steric: the aromatic moiety of the
styrene now occupies the free space resulting from the change of
the large methionine into the much smaller alanine. Also, W96’ is
flipped towards the solvent and thus contributes to binding of the
TS structure into the dimer interface (Figure 3f).
MD simulations of the LmrR-heme-carbene complex
(LmrR⊂6) show that this opened conformation is indeed capable
of accommodating the carbene moiety. The simulations further
underline the importance of W96’ in controlling the accessibility of
the heme-carbene complex. With W96’ pointing towards the
hydrophobic core, the heme-carbene is directed towards the
solvent, where it will be accessible for the styrene co-substrate
The combined results demonstrate unequivocally that the
cyclopropanation reaction occurs in the hydrophobic pocket of
LmrR. Initially, this is counterintuitive: heme enzymes such as
P450 usually present a large hydrophobic cavity for binding
substrates whereas, in contrast, upon binding of hemin, the
pocket of LmrR is fully occupied. Yet, the artificial enzyme exhibits
good activity and shows enantioselectivity in catalysis. This is
attributed to the dynamic nature of the LmrR based artificial heme
enzyme involving substantial geometric rearrangement of the
heme environment to allow for binding of the substrates and their
interaction with the heme. Indeed, the MD simulations show that
the LmrR-heme complex undergoes significant conformational
changes, giving rise to transient open conformations that make it
possible to reach a pre-catalytic state, allowing the reaction to
proceed.
(Figure 3c). Rotation of W96’ to the outside of the pore causes the
heme-carbene (6) to remain at the dimer interface (Figure 3d, S8).
Finally, the effect of the second substrate (styrene) was
studied. First, the transition state geometry that leads to the
generation of the 1R,2R cyclopropane was obtained by QM
calculations (Figure S7) and then subjected to the same analysis
as before (Table S3). The majority of the structures (clusters 0, 2
and 3) correspond LmrR with a broader dimer interface where the
two helices containing the W96/W96’ residues appear further
separated and are thus capable of accommodating the catalytic
complex (Figure 3e). W96’ appears to be involved in stabilizing
the TS structure through π-stacking with the phenyl group of the
styrene.
In conclusion, we have created an artificial heme enzyme
based on the protein LmrR, which shows good activity and
moderate enantioselectivity in an abiological reaction, that is, the
catalytic cyclopropanation of styrenes. This represents the first
example of organometallic catalysis with an LmrR based artificial
metalloenzyme and thus illustrates the versatility of LmrR as a
scaffold for artificial metalloenzymes design. A key finding is that
enzyme is active despite the fact that the crystal structure shows
a tightly bound heme that appears inaccessible to substrates. It is
proposed that the artificial enzyme can open up to allow formation
of precatalytic strutures, as a result of the dynamics of the protein-
heme assembly. Molecular dynamics studies add support to this
hypothesis. This work suggests that dynamics has to be taken into
account in the design of artificial enzymes and may be key to
achieving catalytic activity.
Experimental Section
Representative procedure for the catalytic cyclopropanation: The
following final concentrations were typically used in catalysis: 30 mM
styrene, 10 mM EDA, 10 mM Na
a final volume of 720 L. 50 mM potassium phosphate buffer and 80 L of
a 100 mM Na solution were combined in a sealed vial equipped with
a stir bar and degassed by bubbling argon through the mixture for 10 min.
Next, a buffered LmrR solution was added carefully. Degassing was
continued by flushing Ar without bubbling. In a separate vial, 80 L of a
solution of hemin (100 M, pre-dissolved in buffer/DMSO, final fraction of
2 2 4
S O , 10 M hemin and 11 M LmrR in
Figure 3. Representative structures resulting from MD simulations of: the LmrR-
heme system ((a) cluster 3, RMSD with crystal 1.634 Å and (b) cluster 2 RMSD
with crystal 2.471 Å, 400 ns MD simulation), the LmrR-heme-carbene system
2 2 4
S O
(
(c) cluster 0, RMSD with crystal of 0.548 Å and (d) cluster 2, RMSD with crystal
of 1.000 Å, 400ns MD simulation) and the cyclopropanation transition state ((e)
into LmrR, (f) into LmrR_M8A, 100ns MD simulation).
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COMMUNICATION
DMSO 5% v/v) was deoxygenated by bubbling argon through the solution
for at least 10 min, and added into the reaction via cannula. After
incubation for 30 min, fresh stock solutions of 1a (20 μL, 1.2 M in DMSO)
and 2 (20 μL, 0.4 M in DMSO) were added and the reaction was stirred at
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4
°C for 18h under a positive argon pressure. The reactions were analyzed
by adding 100 μL of internal standard (2-methylanisole, 1 mM in EtOAc) to
the reaction mixture, followed by extraction with ethyl acetate (800 L) after
vortex for 1 min. The organic layers were dried over Na SO while
2 4
vortexing for 1 min. Yields, enantio- and stereoselectivity were determined
by HPLC or GC-FID.
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