Enantioselective artificial metalloenzymes by creation of a novel active site at the protein dimer interface

Article abstract of DOI:10.1002/anie.201202070

A game of two halves: Artificial metalloenzymes are generated by forming a novel active site on the dimer interface of the transcription factor LmrR. Two copper centers are incorporated by binding to ligands in each half of the dimer. With this system up to 97% ee was obtained in the benchmark Cu^II catalyzed Diels-Alder reaction (see scheme). Copyright

Full text of DOI:10.1002/anie.201202070

Angewandte
Chemie
DOI: 10.1002/anie.201202070
Artificial Metalloenzymes
Enantioselective Artificial Metalloenzymes by Creation of a Novel
Active Site at the Protein Dimer Interface**
Jeffrey Bos, Fabrizia Fusetti, Arnold J. M. Driessen, and Gerard Roelfes*
Natural metalloenzymes are a continuing source of inspira-
tion for the design of bio-inspired catalyst. Key to their high
catalytic efficiencies and excellent (enantio)selectivities are
the second coordination sphere interactions provided by the
protein scaffold. The emerging concept of hybrid catalysis is
an effort to impart enzyme-like characteristics to homoge-
neous transition-metal catalysts by embedding catalytically
active transition-metal complexes in a biomolecular scaffold,
resulting in an artificial metalloenzyme.[1–4] Several elegant
examples of artificial metalloenzymes, some of which are
capable of performing highly enantioselective reactions, have
been reported. However, the majority of these examples rely
on a limited number of protein scaffolds that have a binding
pocket that is large enough to bind the catalyst and still leave
space for the substrates. Examples include scaffolds, such as
avidin, streptavidin, bovine serum albumin (BSA), and apo-
myoglobin.[5] An alternative approach to the design of
artificial metalloenzymes involves creation of a new active
site at an appropriate position in a protein scaffold, which is
not necessarily an existing active site or binding pocket.[6,7]
Herein we present a novel concept for the creation of artificial
metalloenzymes, which involves the creation of an active site
on the dimer interface of the transcription factor LmrR. With
this artificial metalloenzyme up to 97% ee was achieved in
the benchmark copper(II)-catalyzed Diels–Alder reaction
(Figure 1a).
Dimerization of proteins is an important phenomenon in
nature and is used as a convenient way to increase the
complexity of the protein structure and to control activity.[8]
Indeed, quite regularly, active sites of dimeric proteins are
Figure 1. a) Schematic representation of the proposed artificial metalloenzyme in
which a CuII complex is grafted on the dimer interface of a protein scaffold.
b) Ligands used for grafting on the dimer interface. c) Pymol representations of
dimeric LmrR in a ribbon and a space-filling model (protein data bank (pdb)
code: 3F8B). Either position 89 (red) or 19 (yellow) were used for the covalent
attachment of ligand 1 or 2.
[*] J. Bos, Dr. G. Roelfes
Stratingh Institute for Chemistry, University of Groningen
Nijenborgh 4, 9747 AG Groningen (The Netherlands)
E-mail: j.g.roelfes@rug.nl
composed of residues from both monomeric polypeptides.
Protein dimerization is the result of a combination of
intermolecular interactions, with a key contribution made
by hydrophobic interactions. This situation makes the protein
dimer interface an attractive microenvironment for the
creation of a new active site: the chirality of the second
coordination sphere, which is composed of the dimer inter-
face residues, combined with the hydrophobic nature of the
dimer interface, which will facilitate the binding of organic
substrates, should allow high catalytic activity and enantiose-
lectivity in the catalyzed reaction. In a first design we have
attempted the creation of an artificial metalloenzyme using
a dimeric hormone peptide as the scaffold.[9] Despite achiev-
ing good enantioselectivities using this metallopeptide as
catalyst, the introduction of the transition-metal catalyst into
Homepage: http://roelfes.fmns.rug.nl
Dr. F. Fusetti
Department of Biochemistry, Groningen Biomolecular Sciences and
Biotechnology Institute, and Netherlands Proteomics Centre (NPC),
University of Groningen
Nijenborgh 4, 9747 AG Groningen (The Netherlands)
Prof. Dr. A. J. M. Driessen
Department of Molecular Microbiology, Groningen Biomolecular
Sciences and Biotechnology Institute, and the Zernike Institute for
Advanced Materials, University of Groningen
Nijenborgh 7, 9747 AG Groningen (The Netherlands)
[**] We wish to thank R. Megens for the synthesis of substrate 3a and
3b. Financial support from the NRSC-Catalysis is gratefully
acknowledged.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201202070.
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the scaffold caused a significant disruption of the structure
and loss of the dimerization affinity, that is, the catalysts
predominantly existed in the monomeric form.
nitrogen at 48C. After the reaction, excess of ligand was
removed by preparative size exclusion chromatography. The
coupling efficiencies were determined by the Ellmanꢀs test.[13]
Typical coupling efficiencies were in the range of 80–90%.
The conjugation of compound 1 or 2 to LmrR M89C/N19C
was further supported by electrospray ionization (ESI) mass
spectrometry (Figure S4). In addition, the selective conjuga-
tion of ligand 1 to LmrR M89C was confirmed by a trypsin
digest experiment, which shows that only the Cys89 residue
was functionalized with ligand 1 (Figure S5).
For the present study, the Lactococcal multidrug resist-
ance Regulator (LmrR), a transcriptional repressor that
regulates the production of a major multidrug ABC trans-
porter in Lactococcus lactis, was selected as the protein
scaffold.[10,11] LmrR is a dimeric protein, with a size of
13.5 kDa per subunit, and contains a typical b-winged helix-
turn-helix domain with an additional C-terminal helix
involved in dimerization. A particularly interesting aspect of
LmrR is that at the dimer center a large flat hydrophobic pore
is present (Figure 1c). It was envisioned that a new active site
could be created in this hydrophobic pore, which is large
enough to incorporate a metal complex while still leaving
enough space for the substrates to bind.
For the construction of the artificial metalloenzyme
a covalent anchoring strategy utilizing cysteine conjugation
was chosen.[12] Since the protein is a homodimer, covalent
attachment will lead to two metal complexes being incorpo-
rated in the protein: one on each monomer. Based on the
crystal structure of LmrR, positions 19 and 89 were selected
for the covalent attachment of a CuII complex because these
positions are located at the far ends of the hydrophobic pore,
thus minimizing the chance that the metal complexes will
interfere with each other (Figure 1c). In computer modeling,
manual docking of the Diels–Alder product 4a (see
Scheme 1) bound to the CuII phenanthroline into LmrR
suggested that the pore is indeed large enough for the
reaction to occur inside (Supporting Information, Figure S6).
The LmrR N19C and M89C mutants were prepared by
standard site-directed mutagenesis techniques (Quick-
Change). These cysteine residues are unique because no
naturally occurring cysteine residues are present in LmrR. In
addition, a sequence encoding a Strep-tag was added to the
LmrR gene for purification purposes. The resulting constructs
were expressed in E. coli BL21(DE3)C43 and subsequently
purified by affinity chromatography (Strep-Tactin Sepharose
column). Because LmrR is a transcription factor, an addi-
tional ion-exchange chromatography step using a Heparin
column was necessary to remove residual bound DNA. The
purity of the protein samples was determined by analytical
SDS-PAGE (Figure S2). Typical yields of purified protein
were 5–10 mgLÀ1 culture.
The effect of the conjugation of ligands 1 or 2 to the LmrR
scaffold on the quaternary structure was evaluated by
analytical size exclusion chromatography. The LmrR ligand
conjugates (LmrR_X) eluted as single band from a Superdex-
75 10/300 GL size exclusion column with an apparent size of
29 kDa comparable to wild-type LmrR containing a Strep-
tag. Also LmrR M89C_1 containing CuII (LmrR
M89C_1_CuII), in 3-(N-morpholine)propanesulfonic acid
buffer (MOPS) containing 150 mm NaCl and in the presence
of substrate 3a (catalytic conditions) eluted with the same
apparent size (Figure S3). These data show that the quater-
nary structure does not change significantly upon functional-
ization and thus LmrR_X is still dimeric.
The catalytic potential of LmrR_X_CuII was evaluated in
the Diels–Alder reaction of azachalcone (3a) with cyclo-
pentadiene as a benchmark reaction (Scheme 1).[14] The
reactions were performed with 3 mol% of Cu(H2O)6(NO3)2
Scheme 1. Asymmetric Diels–Alder reaction catalyzed by LmrR_X/
Cu(NO3)2 complexes in a MOPS buffered solution.
(30 mm) and with a slight excess (1.1 equiv) of the LmrR_X
conjugate (taking into account the coupling efficiency) in
MOPS buffer solution (20 mm, pH 7.0) containing 150 mm
NaCl. These catalyst concentrations are significantly lower
than what was used before in our DNA-[15] and peptide-based
catalytic asymmetric Diels–Alder reactions.[9] The reaction
was carried out for 3 days at 48C, and the Diels–Alder
product 4a was obtained as a mixture of endo and exo isomers
as the only detectable products.
Phenanthroline- and bipyridine-based ligands (1 and 2,
Figure 1b), containing a bromo acetamide group for selective
conjugation to cysteine, were synthesized. Ligand 1 was
prepared by reaction of 1,10-phenanthroline-5-amine with
bromoacetyl bromide (Scheme S1). For the synthesis of
ligand 2 a classical Krçhnke synthesis was employed to
make 5-methyl-2,2’-bipyridine, which was converted into 5-
(aminomethyl)-2,2’-bipyridine by bromination, reaction with
hexamethylenetetramine, and hydrolysis. Finally, reaction
with bromoacetyl bromide resulted in ligand 2 (Scheme S1).
The conjugation of the phenanthroline- or bipyridine-
based ligands to the LmrR mutants was achieved by treating
LmrR with an eight-fold excess of ligand 1 or 2. The reaction
was carried out in the dark in degassed potassium phosphate
buffer (50 mm, pH 7.75) containing 150 mm NaCl under
The conversion of 3a using a CuII phenanthroline complex
without the presence of LmrR was 20%. When the reaction
was carried out with LmrR_N19C_1 an ee value of 50% was
observed of the endo product (endo/exo 92:8), albeit also with
a low conversion. Anchoring of the phenanthroline ligand to
the 89 position, that is, using LmrR_M89C_1, resulted in
a conversion of 93% and an excellent ee of 97% for the
(+) enantiomer of the endo product (endo/exo 95:5) (entry 2,
2
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Table 1: Results of Diels–Alder reactions catalyzed by copper LmrR_X complexes
Two residues in the pocket of LmrR_M89C_1,
(Scheme 1).[a]
that is, Val15 and Asp100, were selected for a limited
mutagenesis study because these residues are most
likely situated in close proximity to the CuII com-
plex. This selection was based on an X-ray structure
of LmrR with a manually docked phenanthroline
Entry Catalyst
Product Conversion endo:exo ee (endo)
[%]
[%]
1
2
LmrR_N19C_1_CuII
4a
4a
4a
4b
4c
4a
4a
24Æ3
93Æ4
98Æ1
56Æ9
97Æ1
55Æ8
89Æ4
38Æ4
14Æ2
30Æ5
23Æ1
29Æ8
20Æ5
full
92:8
95:5
53Æ5 (+)
97Æ1 (+)
95Æ1 (+)
93Æ1 (+)
<5
66Æ2 (À)
97Æ1 (+)
40Æ2 (+)
21Æ3 (+)
<5
LmrR_M89C_1_CuII
LmrR_M89C_1_CuII
LmrR_M89C_1_CuII
LmrR_M89C_1_CuII
LmrR_M89C_2_CuII
LmrR_M89C_V15A_1_CuII
3[b]
4
90:10
96:4
(Figure S6).
The
LmrR_M89C_V15A
and
LmrR_M89C_D100E mutants were prepared using
standard QuickChange mutagenesis methods and
the corresponding conjugates with ligand 1 were
prepared and characterized as described above. The
V15A mutation did not have an influence on the
enantioselectivity of the catalyzed Diels–Alder
reaction (entry 7, Table 1). In contrast, with the
structurally conservative D100E mutation a signifi-
cantly decreased conversion and ee value was
obtained (entry 8 and 9, Table 1) suggesting that
the catalyzed reactions are sensitive to the structure
of the microenvironment of the pore of the LmrR
scaffold.
5
6
7
8
n.a.[c]
63:37[d]
96:4
LmrR_M89C_D100E_1_CuII 4a
LmrR_M89C_D100E_1_CuII 4b
88:12
84:14
90:10
88:12
90:10
93:7
9
10
11[e]
12[f]
13
14
LmrR_M89C_CuII
LmrR_9_CuII
4a
4a
13Æ4 (+)
28Æ7 (+)
0
LmrR_Phenanthroline_CuII 4a
Phenanthroline_CuII
Phenanthroline_CuII
4a
4c
95:5
0
[a] Typical conditions: 90% Cu(H2O)6(NO3)2 (3 mol%; 30 mm) loading with respect
to LmrR_X in 20 mm MOPS buffer (pH 7.0), 150 mm NaCl, for 3 days at 48C.
Conversions and ee values are an average of two independent experiments, both
carried out in duplicate. [b] Reaction carried out at room temperature. [c] exo peak
could not be observed. [d] ee exo 90Æ2%. [e] Compound 9 was added 2:1 with
respect to LmrR (wild-type). [f] Copper phenanthroline was added 2:1 with respect
to LmrR (wild-type).
The tolerance towards variation in the structure
of the substrate was investigated using the a,b-
unsaturated 2-acyl pyridine substrates 3b and 3c.
When R = m-methoxyphenyl (3b) the same trend
was observed as with azachalcone (3a): excellent
Table 1). Performing the reaction at room temperature only
caused a small decrease in ee value (entry 3, Table 1).
The higher conversions obtained with the artificial
metalloenzyme compared to CuII-phenanthroline alone indi-
cate that the reaction is accelerated by the protein scaffold.
Indeed, following the consumption of 3a by UV/Vis spec-
troscopy showed that the reaction catalyzed by the artificial
metalloenzyme is significantly faster than the reaction
catalyzed by CuII phenanthroline alone (Figure S7). Addition
of a fresh aliquot of reagents after the reaction was complete
showed that LmrR_M89C_2 was still active, albeit slightly less
than before, indicating a small degree of inactivation over
time (Figure S7b).
Interestingly, LmrR_M89C_2, which contains a conju-
gated 2,2’-bipyridine instead of a phenanthroline ligand, gave
rise to 66% ee of the opposite, that is, the (À) enantiomer of
the endo isomer of the Diels–Alder product 4a (entry 6,
Table 1). Hence, by judicious choice of the CuII binding
ligand, both enantiomers of the Diels–Alder product can be
accessed. Furthermore, with this artificial metalloenzyme
significant amounts of the exo product were obtained also
(endo/exo 63:37), with the exo product having 90% ee. No
significant enantioselectivity was found in the control reac-
tion performed with LmrR M89C having a free cysteine
(without ligand 1 or 2, entry 10, Table 1). Additionally, low
enantioselectivities and conversions were found with wild-
type LmrR supplemented with an analogue of 1, that is, the
propionamide derivative of 1,10-phenantroline-5-amine (9),
and CuII phenanthroline (entry 12 and 13, Table 1). This result
demonstrates that the covalent linkage of the ligand to the
protein scaffold is required for selective catalysis. Using an
excess of Cu(H2O)6(NO3)2 with respect to LmrR_M89C_1
(ratio 2:1 CuII/monomer LmrR_M89C_1) resulted in precip-
itation of the protein.
ee values with LmrR_M89C_1 and a significantly lower
ee values with the D100E mutant (entries 4 and 9, Table 1).
Using 3c, which contains a methyl group at the b-position, full
conversion was achieved but the Diels–Alder product was
obtained without any significant ee value. The data obtained
with this limited set of substrates suggests some substrate
selectivity of artificial metalloenzyme, but more research is
required to understand its structural origin.
The results show that the protein scaffold is not only
a source of chirality for the reaction, but that it also causes an
acceleration of the reaction rate, that is, the reaction is protein
accelerated. Moreover, there appears to be a correlation
between the rate and the enantioselectivity of the reaction,
that is, the most enantioselective enzymes also gave rise to the
highest conversions. Furthermore, both the conversion and
ee values depend on the nature of the ligand, the position at
which the ligand is anchored to the protein scaffold, and is
also sensitive to mutation in the hydrophobic pocket. This
demonstrates that the catalysis is dependent on the structure
of the microenvironment around the catalytic CuII ion and
these combined data strongly suggest that the reaction indeed
takes place in the newly created active site in the hydrophobic
pocket at the LmrR dimer interface. At present, the effect of
the individual changes on the catalysis is difficult to ration-
alize, since LmrR shows some plasticity.[10] For this reason we
are currently performing a detailed structural study of the
LmrR-based artificial metalloenzyme.
In conclusion, we have presented a novel strategy towards
the creation of an artificial metalloenzyme, which involves
grafting a new active site onto the dimer interface of the
protein LmrR by conjugation of a bidentate ligand capable of
binding CuII ions. The dimer interface provides the chiral
microenvironment for the catalyzed reaction. The artificial
metalloenzyme is capable of catalyzing Diels–Alder reaction
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was shown that the hydrophobic pocket of LmrR indeed
functions as the active site where the reaction takes place.
These results represent the first example of an artificial
metalloenzyme in which the metal complex is linked cova-
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Experimental Section
Representative procedure for LmrR_X_CuII catalyzed reactions. To
a solution of Cu(H2O)6(NO3)2 (30 mm) in 20 mm MOPS buffer
(pH 7.0) at 08C was added LmrR_X (1.1 equiv based on Cu(H2O)6-
(NO3)2, variable concentrations depending on the coupling efficiency)
to a final volume of 300 mL. A fresh stock solution (10 mL) of
substrate in MOPS/CH3CN was added (1 mm). After addition of
freshly distilled cyclopentadiene (0.8 mL, 33 mm) at 08C, the reaction
was mixed for 3 days by continuous inversion at 48C. The product was
isolated by extraction with Et2O (3 ꢁ 0.5 mL). The organic phases
were dried (Na2SO4) and evaporated under reduced pressure to give
the product. The conversion and ee value were determined by HPLC.
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Published online: && &&, &&&&
Keywords: artificial metalloenzyme · asymmetric catalysis ·
.
copper · Diels–Alder reactions · dimeric protein
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4
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Communications
Artificial Metalloenzymes
J. Bos, F. Fusetti, A. J. M. Driessen,
G. Roelfes*
&&&& — &&&&
Enantioselective Artificial
Metalloenzymes by Creation of a Novel
Active Site at the Protein Dimer Interface
A game of two halves: Artificial metal-
loenzymes are generated by forming
a novel active site on the dimer interface
of the transcription factor LmrR. Two
copper centers are incorporated by bind-
ing to ligands in each half of the dimer.
With this system up to 97% ee was
obtained in the benchmark CuII catalyzed
Diels–Alder reaction (see scheme).
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