Supramolecular Assembly of Artificial Metalloenzymes Based on the Dimeric Protein LmrR as Promiscuous Scaffold

Article abstract of DOI:10.1021/jacs.5b05790

Supramolecular anchoring of transition metal complexes to a protein scaffold is an attractive approach to the construction of artificial metalloenzymes since this is conveniently achieved by self-assembly. Here, we report a novel design for supramolecular artificial metalloenzymes that exploits the promiscuity of the central hydrophobic cavity of the transcription factor Lactococcal multidrug resistance Regulator (LmrR) as a generic binding site for planar coordination complexes that do not provide specific protein binding interactions. The success of this approach is manifested in the excellent enantioselectivities that are achieved in the Cu(II) catalyzed enantioselective Friedel-Crafts alkylation of indoles.

Full text of DOI:10.1021/jacs.5b05790

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Supramolecular Assembly of Artificial Metalloenzymes based
on the Dimeric Protein LmrR as Promiscuous Scaffold
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Jeffrey Bos†, Wesley R. Browne†, Arnold J. M. Driessen‡, and Gerard Roelfes†*
†Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands
‡Groningen Biomolecular Sciences and Biotechnology Institute, and the Zernike Institute for Advanced Materials
University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands
KEYWORDS artificial metalloenzyme, enzyme design, supramolecular assembly, asymmetric catalysis, Friedel-Crafts
reaction
ABSTRACT: Supramolecular anchoring of transition metal complexes to a protein scaffold is an attractive approach to the
construction of artificial metalloenzymes, since this is conveniently achieved by self-assembly. Here, we report a novel
design for supramolecular artificial metalloenzymes that exploits the promiscuity of the central hydrophobic cavity of the
transcription factor Lactococcal multidrug resistance Regulator (LmrR) as a generic binding site for planar coordination
complexes that do not provide specific protein binding interactions. The success of this approach is manifested in the
excellent enantioselectivities that are achieved in the Cu(II) catalyzed enantioselective Friedel-Crafts alkylation of indoles.
Artificial metalloenzymes open the way to engage the
molecular recognition properties of biomolecules, such as
proteins or DNA, in achieving enzyme-like activities and
selectivities in transition metal catalysis.1 While tremen-
dous advances have been made already, the rational de-
sign of an artificial metalloenzyme for a given reaction
requires knowledge of the relation between structure and
activity that is still beyond the state of the art.2 Indeed,
the design of artificial metalloenzymes at present is fo-
cused first and foremost on the (re-)design of the metal
binding site.3 A popular approach involves the judicious
positioning of a catalytically active transition metal com-
plex within an existing protein scaffold.1 This is often
realized through supramolecular anchoring of the catalyst
at a specific site in the protein, using metal complexes
tethered via their ligand to high affinity binding motifs.
This approach has been applied successfully in the design
of a variety of artificial metalloenzymes, most notably the
LmrR is a transcriptional repressor that is involved in
the antibiotic resistance response of Lactococcus lactis.6 It
is a homodimeric protein with a size of 13.5 kDa per mon-
omer and contains an unusual large hydrophobic pore at
the dimer interface. Here, flat aromatic organic molecules
can bind as is shown in X-ray and NMR structures of
LmrR with various planar drugs bound.7 Two tryptophan
residues, one from each subunit, i.e. W96 and W96’, play
a key role in binding by sandwiching the organic mole-
cules using π-stacking interactions (Figure 1). The LmrR
structure has been reported to exhibit a broad and shal-
low conformation energy surface, in which the confor-
mation can shift readily to accommodate structurally
unrelated compounds without prerequisite specific bind-
ing interactions.7b
Recently, we have reported two new classes of artificial
metalloenzymes containing a covalently linked Cu(II)
complex at the dimer interface of LmrR.8 These artificial
metalloenzymes were shown to catalyze Diels-Alder,
hydration and Friedel-Crafts alkylation reactions, with
excellent ee’s in the first and moderate ee’s in the latter
two reactions. These results demonstrated that the hy-
drophobic cavity of LmrR can accommodate many diverse
substrates and reaction types. Encouraged by these re-
sults, it was decided to explore the supramolecular as-
sembly of an LmrR-based artificial metalloenzyme, taking
advantage of the promiscuity of the hydrophobic pocket
at the dimer interface. We envisioned that Cu(II) com-
plexes of planar aromatic ligands would be capable of
binding partly or completely within the LmrR pocket,
with the result that the catalyzed reaction takes place
inside the chiral microenvironment provided by the LmrR
hydrophobic pocket.
,
streptavidin/biotin system.4 5 Yet, the nature of the tether
and its position on the ligand place restrictions as to the
structural flexibility and dynamics of the metal complex.
As a consequence, the conformational space and second
coordination sphere interactions that can be accessed
during catalysis are also restricted.
Here, we report a novel design for supramolecular arti-
ficial metalloenzymes that exploits the extensive promis-
cuity of the central hydrophobic cavity of the protein
Lactococcal multidrug resistance Regulator (LmrR) as a
generic, moderate affinity binding site for planar coordi-
nation complexes that do not provide specific protein
binding interactions. The success of this approach is man-
ifested in the excellent enantioselectivities that are
achieved in the Cu(II) catalyzed enantioselective Friedel-
Crafts alkylation of indoles.
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the Kd was determined to be 1 order of magnitude lower,
i.e. 45 µM. Analytical size exclusion chromatography of
the LmrR_LM Cu(II)-L1 and LmrR_LM_W96A
Cu(II)-L1 hybrids was performed. Both eluted as a single
band with an apparent molecular weight of 29 kDa, show-
ing that the addition of Cu(II)-L1 does not disrupt the
homodimeric structure (Figure S7).8
1
2
3
4
5
6
⊂
⊂
7
8
9
The binding of Cu(II)-L1 to LmrR_LM was studied fur-
ther by fluorescence decay lifetime experiments. The
tryptophan residues can be divided into two groups: those
within the hydrophobic pocket (W96/W96’) and those
distant from the hydrophobic pocket (W67/W67’and
W119/W119’). The observed fluorescence lifetime is a
weighted sum of the individual lifetimes of both groups
(note that these are too similar to be resolved reliably by a
bi- or multi-exponential decay fit, given the much greater
emission intensity of the tryptophans within the pocket,
see SOI for further details), and was 4.4 ns in case of
LmrR_LM (Figure 2, Figure S8). Upon saturation of
LmrR_LM with Cu(II)-L1, i.e. with the latter at 44 µM, a
decrease beyond experimental uncertainty in the fluores-
cence lifetime to 3.5 ns was observed. The final lifetime is
close to the lifetime observed for LmrR_LM_W96A, i.e.
the lifetimes for W67/W119, which is 3.7 ns (Figure 2).
Combined, these data indicate that Cu(II)-L1 binds pre-
dominantly in proximity to W96/W96’, which strongly
suggest it is bound at or near to the hydrophobic pore of
LmrR (for a detailed discussion, see SOI)
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Figure 1. a) Schematic representation of the design of the
supramolecular artificial metalloenzyme in which a Cu(II)
complex is bound at the dimer interface of LmrR, followed by
application in a catalyzed Friedel-Crafts alkylation. b) Space
filled representation of dimeric LmrR (pdb 3F8B) and ribbon
representation of the ligand binding pocket (inset). Trypto-
phan residues W96 and W96’ are shown in gray. Pictures
were generated with Pymol.
The LmrR variant used in this study, referred to as
LmrR_LM, contains a C-terminal Strep tag to facilitate
purification and two mutations in the DNA binding do-
main, i.e. K55D and K59Q, which facilitated expression
b
and purification.8 A second mutant was prepared in
which the tryptophan residues at the dimer interface were
replaced by alanine, i.e. LmrR_LM_W96A. Both mutants
were expressed in E. coli BL21(DE3)C43. Typical yields of
purified protein were 20-30 mg L-1 culture.
The artificial metalloenzymes were prepared through
self-assembly, by combining LmrR_LM with a Cu(II)
complex of a planar aromatic ligand L1-7 (Figure S1) in a
buffered solution (20 mM MOPS, 150 mM NaCl, pH 7.0).
The binding affinity of the Cu(II) complex to LmrR_LM
was determined by titration through monitoring of the
quenching of tryptophan fluorescence, taking into ac-
count the quenching of fluorescence of the tryptophan
residues at the exterior of the protein, i.e. W67 and W119,
(Figure S2), by using LmrR_LM_W96A as control (Figure
S6). A dissociation constant (Kd) of 2.6±2 µM was deter-
mined for binding of Cu(II)-L1 to LmrR_LM, which is
significantly higher than the Kd reported for the tight-
binding drugs H33342 and daunomycin (21±8 nM and
236±53 nM, respectively).6 The binding of Cu(II)-L1 to
LmrR_LM_W96A mutant which does not contain trypto-
phan in the hydrophobic pocket, was significantly weaker:
Protein
[Cu(II)-L1) (µM)
Lifetime (ns)
LmrR_LM
0
4.4
3.5
3.7
3.7
LmrR_LM
44
0
LmrR_LM_W96A
LmrR_LM_W96A 44
Figure 2. Representative fluorescence decays (in black),
IRF corrected exponential fits (in red) and residuals for
LmrR_LM and LmrR_LM
⊂
Cu(II)-L1. In both cases the chi2
values for the fits are ca. 1.01. Further addition of Cu(II) re-
duces emission intensity but has no additional effect on
lifetime as expected for static quenching. The instrumental
response function (IRF) was recorded using BaSO4 (in gray).
The binding of a series of copper complexes of nitrogen
based bidentate ligands (Cu(II)-(L2-L7)) (Table S1) to
LmrR was studied also. The Kd for binding of these Cu(II)
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responding products 3e and 3f, respectively (Table 1, entry
complexes to LmrR_LM was found to be within the same
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order of magnitude as for Cu(II)-L1, suggesting that in-
deed, the hydrophobic pore of LmrR acts as a generic
binding pocket and that significant specific interactions
to the metal complex are not required,
7-9). The double substituted indole 2g gave results com-
parable to 2a, albeit that additional unidentified products
were detected (Table 1, entry 10, Figure S4). The ee’s
achieved are significantly higher than those obtained with
our previous artificial metalloenzyme design containing a
covalently linked bipyridine ligand.8a
The potential of these new artificial metalloenzymes in
catalysis was evaluated in the enantioselective vinylogous
Friedel-crafts (FC) alkylation of 5-methoxy-1H-indole (2a)
with 1-(1-methyl-1H-imidazol-2-yl)but-2-en-1-one (1) to
generate 3a.9 This is a reaction that we previously showed
to be compatible with dynamic catalytic assemblies.9a
Artificial metalloenzymes were prepared in situ by self-
assembly from 9 mol% of Cu(II)-L1 (90 µM) with a slight
Table 1. Results of Friedel-Crafts alkylation reactions
catalyzed by LmrR-based artificial metalloenzymes
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excess
(1.3
equiv.)
of
LmrR_LM
in
3-(N-
morpholine)propanesulfonic acid buffer (MOPS) (20 mM,
pH 7.0), containing 150 mM NaCl, Based on the Kd more
than 90% of Cu(II)-L1 is bound to LmrR_LM under these
conditions. The catalytic reaction was started by addition
of 1 mM 1 and 5 mM indole 2a. After 16 hours at 4 °C, a
conversion of 57 % was observed and 3a was obtained
with an ee of 61 % in the (+) enantiomer (Table 1, entry 1).
Interestingly, a strong dependence of the results of cataly-
sis on the indole concentration was observed. When the
concentration of 2a was decreased 5 fold, i.e. to 1 mM, the
conversion and ee increased to 84% and 75%, respectively
(Table 1, entry 2). Further decreasing the concentration of
indole to 0.5 mM, i.e. using a ratio of 1:2a of 1:0.5, resulted
in an ee of 84% (Table 1, entry 3) This suggests that the
indole competes with Cu(II)-L1 for binding to LmrR_LM,10
i.e., at higher indole concentrations Cu(II)-L1 is displaced
from the hydrophobic pore of LmrR_LM. This results in
an increased fraction of unbound Cu(II)-L1 that catalyzes
the reaction in a racemic fashion, causing a decrease in ee
of 3a.
eq.
indole
conversion
(%)b
entry indole
LmrR_LM
product
ee (%)c
⊂
Cu(II)-L1:
1
2a
5
3a
3a
3a
3b
3c
3d
3e
3f
57±7
84±5
90±2d
94±4
19±8
79±2
full
61±8(+)
75±13(+)
84±3(+)
82±1
2
3
2a
1
2a
2b
2c
2d
2e
2f
0.5
4
5
6
7
8
9
10
1
1
64±7(+)
18±2(-)-R
94±0(+)-R
93±1
1
1
1
full
2f
0.5
1
3f
fulld
56±25e
93±1
2g
3g
81±1
Artificial metalloenzymes based on Cu(II)-(L2-L7) pro-
vided similar enantioselectivies in the catalyzed reaction.
The presence of a ligand is required, since ee was not
obtained when using LmrR_LM + Cu(NO3)2 (Table S2).
LmrR_LM_W96A
11 2a
⊂
1
Cu(II)-L1:
3a
53±28
<5
a Typical conditions: 75% Cu(II)-L1 (9 mol%; 90 µM) load-
ing with respect to LmrR_LM or LmrR_LM_W96A, 1 mM 1,
0.5-5 mM 2, in 20 mM MOPS buffer (pH 7.0), 150 mM NaCl,
at 4°C for 16 h (2a) or 64 h (other indoles). Conversions and
ee values are an average of at least two independent experi-
Using LmrR_LM_W96A
⊂
Cu(II)-L1 as catalyst in the
reaction of 1 and 2a resulted in moderate conversion and
<5% ee of 3a. (entry 11). This is in agreement with the
weaker binding affinity of Cu(II)-L1 in the absence of
W96/W96’, i.e. under the conditions of catalysis only a
small fraction of the Cu(II) complex is actually bound to
the LmrR_LM_W96A.
b
ments, both carried out in duplicate. Conversions are de-
c
termined by HPLC and are based on substrate 1. Sign of
rotation and absolute configuration based on elution order in
chiral HPLC.9 d Conversion is based on substrate 2.11 e Addi-
tional unidentified products were detected (Figure S4).
The substrate scope of LmrR_LM
⊂
Cu(II)-L1 was eval-
uated by variation of the 2-acyl imidazole and of the in-
dole. The artificial metalloenzyme did not tolerate larger
subsitutents on the β position of the enone (Table S3). In
contrast, a broad scope of indoles 2 were accepted. Re-
placing the methoxy group at the 5 position of the indole
with an N-morpholine moiety (2b) resulted in a similar
ee, whereas a chloride at this position (2c) resulted in a
lower ee (Table 1, entries 4-5). With N-methyl indole (2d)
the ee decreased to 18% (table 1, entry 6). The best results
were obtained with nonsubstituted indole (2e) and 2-
methyl indole (2f), that is, full conversion of 1 and an
excellent ee of 94% (R-enantiomer) and 93% for the cor-
The combined data of the binding studies, fluorescence
lifetime experiments and catalysis demonstrate that the
catalysis takes place in the hydrophobic pore of LmrR-LM
and that tryptophan residues W96 and W96’ in the newly
created active site are of key importance to the catalysis.
Possibly, they are involved in intercalation-like binding of
the Cu(II) complex.12 Alternatively, instead of binding the
metal complex, it is also possible that either the enone or
the indole substrates are bound between the tryptophan
residues via π stacking, analogous to the observed binding
mode of H33342 and daunomycin6, and are thus posi-
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tioned for the reaction, resulting in enantioselectivity.
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This model is in agreement with the observed preference
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and unsubstituted indole 2e. The observed competition
between 2a and Cu(II)-L1 in binding LmrR implies that
both substrates and Cu(II) complex prefer to bind, at least
partly, in the same location, which suggests significant
dynamics during catalysis.
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(10) The binding constant of indole 2a to LmrR_LM could not
be established due to spectral overlap.
In conclusion, we have introduced a novel design of an
artificial metalloenzyme, created by supramolecular bind-
ing of a catalytically active Cu(II) complex in the hydro-
phobic cavity the dimer interface of LmrR. A key element
of the present design is the promiscuity of the hydropho-
bic cavity of LmrR that can accept many compounds,
including substrates and catalytically active transition
metal complexes, without requiring specific ligand - pro-
tein interactions, as is the case in most supramolecular
artificial metalloenzyme designs. The success of this ap-
proach was manifested in the catalyzed asymmetric vi-
nylogous Friedel-Crafts alkylation of indoles in water,
giving rise to excellent ee’s. The results of this study sug-
gest that precise design of second coordination sphere
interactions may not always be the most appropriate
approach a priori to achieving high selectivity in artificial
metalloenzyme catalysis, provided that substrates and
metal complexes have enough freedom to find themselves
the optimum orientation and interactions in a promiscu-
ous chiral space. The present design is highly flexible and
is envisioned to be adapted readily for binding other met-
al complexes and catalysis of other reactions. It is notable
also that LmrR is the first scaffold that has been used
successfully in multiple anchoring strategies, thus under-
lining the versatility of LmrR as a scaffold for artificial
metalloenzymes.
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ASSOCIATED CONTENT
Supporting Information. Detailed experimental proce-
dures, characterization data and additional results of cataly-
sis experiments. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*j.g.roelfes@rug.nl.
ACKNOWLEDGMENT
We wish to thank P. Dijkstra, I. Drienovská and L. Villarino
Palmaz for assistance with protein expression and the fluo-
rescence decay lifetime experiments. Support from the
NRSC-Catalysis, the European Research Council (starting
grant no. 280010) and the Ministry of Education, Culture and
Science (Gravitation program no. 024.001.035) is gratefully
acknowledged. AJMD acknowledges the research program on
biobased ecologically balanced sustainable industrial chemis-
try (BE-BASIC) for support.
(11) Due to the strong dependence on the indole (2) concen-
tration, relatively large errors were sometimes observed in the
conversions.
(12) Draksharapu, A.; Boersma, A. J.; Leising, M.; Meetsma, A.;
Browne, W.R.; Roelfes G. Dalton Trans. 2015, 44, 3647.
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59
60
85x29mm (300 x 300 DPI)
ACS Paragon Plus Environment