Robust and Versatile Host Protein for the Design and Evaluation of Artificial Metal Centers

Article abstract of DOI:10.1021/acscatal.9b02896

Artificial metalloenzymes (ArMs) have high potential in biotechnological applications as they combine the versatility of transition-metal catalysis with the substrate selectivity of enzymes. An ideal host protein should allow high-yield recombinant expres

Full text of DOI:10.1021/acscatal.9b02896

Research Article
pubs.acs.org/acscatalysis
Cite This: ACS Catal. 2019, 9, 11371−11380
Robust and Versatile Host Protein for the Design and Evaluation of
Artificial Metal Centers
Johannes Fischer,†,‡,⊥,∇ Dominik Renn,†,‡,∇ Felix Quitterer,‡ Anand Radhakrishnan,§,# Meina Liu,†
Arwa Makki,†,¶ Seema Ghorpade,† Magnus Rueping,† Stefan T. Arold,*,§,∥ Michael Groll,*,‡
,†
̈
and Jorg Eppinger*
†KAUST Catalysis Center (KCC), Division of Physical Sciences & Engineering and Computational Bioscience Research Center
(CBRC), Division of Biological Sciences & Engineering, King Abdullah University of Science and Technology (KAUST), 23955
Thuwal, MK, Saudi Arabia
§
‡Center for Integrated Protein Science, Department Chemie, Lehrstuhl fu
D-85747 Garching, Germany
̈
̈
̈
r Biochemie, Technische Universitat Munchen (TUM),
∥
́
Centre de Biochimie Structurale, CNRS, INSERM, Universite de Montpellier, 34090 Montpellier, France
S
* Supporting Information
ABSTRACT: Artificial metalloenzymes (ArMs) have high
potential in biotechnological applications as they combine the
versatility of transition-metal catalysis with the substrate
selectivity of enzymes. An ideal host protein should allow
high-yield recombinant expression, display thermal and
solvent stability to withstand harsh reaction conditions, lack
nonspecific metal-binding residues, and contain a suitable
cavity to accommodate the artificial metal site. Moreover, to
allow its rational functionalization, the host should provide an
intrinsic reporter for metal binding and structural changes,
which should be readily amendable to high-resolution
structural characterization. Herein, we present the design,
characterization, and de novo functionalization of a fluorescent ArM scaffold, named mTFP*, that achieves these characteristics.
Fluorescence measurements allowed direct assessment of the scaffold’s structural integrity. Protein X-ray structures and
̈
transition metal Forster resonance energy transfer (tmFRET) studies validated the engineered metal coordination sites and
provided insights into metal binding dynamics at the atomic level. The implemented active metal centers resulted in ArMs with
efficient Diels−Alderase and Friedel−Crafts alkylase activities.
KEYWORDS: artificial metalloenzyme, protein engineering, protein design, biocatalysis, metalloproteins, metalloenzyme,
biohybrid catalysis
enantioselectivities53−55 or benefit from coactivation through
1. INTRODUCTION
the second coordination sphere within a protein specificity
According to a recent estimate, more than half of all proteins
pocket.56,57 However, when compared to molecular catalysts,
are associated with metal ions in their active form.1 It is,
ArMs suffer from low stability toward elevated temperatures,
pH changes, and/or solubility in organic solvents. Additionally,
therefore, important to understand the factors that govern
metal binding and reactivity in biological systems. Expanding
their synthesis and catalytic activity typically require specific
the scope of nature’s toolset through introduction of artificial
chemical modifications or ligands. These limitations preclude
metal centers and coordination geometries holds the promise
to provide new techniques for chemical synthesis,2−8
simple recombinant production and complicate in vivo
biocatalysis,9−11 and biological research.6,12−19 Various meth-
applications. To attenuate these limitations, several groups
developed various techniques58−60 to enhance the efficacy and
ods to generate artificial metalloenzymes (ArMs) were
applicability of ArMs, in particular based on computational
reported. These methods include domain-based directed
methods61−65 and large-scale (high-throughput) screen-
ing.53,66−70 For instance, an artificial copper binding site was
incorporated inside the central cavity of the thermophilic TIM-
evolution strategies12,13,20−24 and strategies for engineering
transition-metal binding sites by implementing (metal-
chelating) unnatural amino acids25−28 or by replacing the
active site metal ions in naturally occurring metalloen-
zymes.28−30 Other approaches use covalent31−47 or supra-
molecular insertion of metal-containing artificial cofac-
tors.48−53 Some systems achieve impressive efficiencies and
Received: July 9, 2019
Revised: October 16, 2019
© XXXX American Chemical Society
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Figure 1. (a) Strategy used: starting from mTFP1, in silico designed point mutations were ranked according to their calculated stability (1), then
assessed in silico for thermal stability (2), the resulting constructs were experimentally tested (3), yielding the stable and inert mTFP*, and scaffold
functionalization was used to derive metal binding ArMs (4). (b) Mutations associated with the different design steps: residues in red have been
removed, bold residues indicate mutation sites, where green indicates positions to achieve the inert host, and blue achieves the rationally designed
ArMs.
barrel glycerol phosphate synthase Thermotoga maritima,
tHisF, through mutational analysis.71 This strategy of
engineering metal binding sites from canonical amino acids
is highly appealing as the generation of a suitable metal
coordinating geometry only requires conventional site-specific
mutagenesis, eliminating complicated synthesis, bioconjuga-
tion, and purification steps to add ligands and cofactors. In
parallel, other groups advanced the individual steps required to
produce a catalytically active ArM based on canonical amino
acids, namely, (i) engineering the affinity and selectivity of
metal binding toward peptides72−76 and proteins,18,58,77,78 (ii)
elucidating the three-dimensional architectures of metal
coordination by proteins,7,79−82 and (iii) altering the catalytic
activity of the existing metalloproteins.18,83,84 Nevertheless, it
remains challenging to introduce de novo metal binding sites,
based on canonical amino acids, to allow functionalization of
an inert scaffold for catalytical chemistry applications.67,71,85−88
In particular, one of the remaining difficulties in the design
of such an ArM is to predict its affinity and selectivity for
different metals.56,67 Hence, it is uncertain under which
conditions a potential (natural or engineered) metal binding
site coordinates with metal ions and to what extent this site is
selective. A host protein should therefore have a reporter
mechanism, which allows direct observation of metal binding
and changes in protein structure. Also, to facilitate
quantification of metal coordination, the host should display
a least amount of (nonspecific) metal binding residues on its
surface and comprise a cavity to accommodate the artificial
metal site. Additionally, the ArM protein needs to express well,
be purified easily, and show a high stability toward temper-
ature, pH, and organic solvents.
Intrinsic protein fluorescence can be used to determine the
metal binding properties if the introduced metal association
modifies the protein’s fluorescence features. This principle was
̈
adopted to introduce transition metal ion Forster resonance
energy transfer (tmFRET).89,90 Given a sufficient spectral
overlap, the light emission of the chromophore of fluorescent
proteins is quenched when a transition metal is coordinated at
a distance less than the Forster radius.91 Thus, the fluorescence
̈
alterations predominantly result from resonant energy transfer
between the metal ion and the chromophore, whereas static
quenching, electron transfer, or perturbation of the protein’s
structure typically results in only minor changes.8,85,86
Moreover, dynamic collision quenching (described by a
Stern−Volmer constant KSV) becomes important at high
metal concentrations. Because the tmFRET intensity depends
on the occupation of the binding site, the metal−chromophore
distance, and the spectral overlap of fluorophore emission and
metal absorption bands, tmFRET data can quantify protein−
metal affinities and provide insights into the nature of the
binding event.
We demonstrate the design, characterization, and applica-
tion of a novel fluorescent ArM scaffold, named mTFP*. The
intrinsic fluorescence of this exceptionally stable host allowed
fast quantification of metal binding thermodynamics and
revealed subtle changes in coordination geometries for
different mutants. Additionally, mTFP* crystallizes easily and
therefore facilitates rational design of metal binding pockets
based on high-resolution X-ray structures. Because mTFP* was
engineered to minimize background metal ion binding, it
presents an attractive system to introduce novel metal binding
sites. We further prove the suitability of the mTFP* scaffold as
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a versatile template for rationally designed biocatalysts through
producing the mTFP*-derived artificial copper enzymes
mTFPCHH and mTFPEHH, which have Friedel−Crafts
alkylase92−95 and Diels−Alderase activity.96−100
2. ENGINEERING THE HOST PROTEIN mTFP*
To identify an ArM host protein that allows quantification of
metal binding through the tmFRET concept,89,90 we
investigated highly stable fluorescent proteins. Based on the
available literature data, structural information and homology
modeling,101,102 we chose mTFP1103 as a parent scaffold.
mTFP1 is a monomeric, bright, and photostable version of the
Clavularia cyan fluorescent protein that combines temperature
and pH stability with a bright fluorescence (ε = 64 000 M−1
cm−1, Φ = 64) and spectral characteristics applicable for
tmFRET (absorbance: 462 nm, emission: 492 nm).103
Moreover, mTFP1 can be produced recombinantly in large
amounts, which exhibits a cavity with dimensions similar to
catalytic centers of natural metalloenzymes.104 Hence, the
pocket’s second coordination sphere can interact with a
substrate bound to the metal center and thus might control
substrate orientation and coactivation.
Incorporating catalytically active late transition metals,
which are not available to nature, is a particular motivation
in ArM design.8 However, such metals preferentially
coordinate with intermediate or soft Lewis bases. These
nonspecific interactions compete with engineered binding sites
and therefore complicate ArM development. Thus, hosts for de
novo design should be depleted of surface-exposed His, Met,
and Cys. Accordingly, we replaced in silico six surface-exposed
amino acid side chains (H30Y, M118L, H128Y, H177Y,
H178Y, and H209Y) within mTFP1 (PDB: 2HQK).103
Analysis of the hydrogen-bonding networks and evolutionary
conservation indicated structural tolerance of these mutations
(see the Supporting Note and Table S1). In order to select
substitutions that do not reduce the stability of mTFP1, we
performed an in silico design strategy (Figure 1a).
Figure 2. (a) Comparison of B-factors obtained after 50 ns of MD
simulation for mTFP1 (based on the PDB 2HKQ103 as a starting
model) and for mutant1 (named mTFP* hereafter). (b) Crystal
structure of mTFP* at 1.00 Å resolution. The antiparallel sheet is
represented in green, connecting loops in blue, and the α-helix in
orange. The chromophore is shown as balls and sticks (purple).
Modified residues are highlighted in red. (c) Chromophore of mTFP*
with coordinating residues T58, W89, R91, S142, and H159. The
2Fo−Fe electron density map of the chromophore is shown in blue
and contoured at 1σ. (d) Structural superposition of mTFP1 (blue)
and mTFP* (green) match onto each other with a root-mean-square
deviation (rmsd) of Cα atoms <0.2 Å. (e) B-Factor comparison of
mutants representing the overall low thermal mobility and structural
integrity, as indicated by dashed lines. (f) Surface representation of
the catalytic cavity in mTFP* (PDB: 4Q9W). Surface colors indicate
positive and negative potentials contoured from 20 kT/e (intense
blue) and −20 kT/e (intense red). Residues Y200 and Y204, used to
build the coordination site, are shown.
We used FoldX105,106 to computationally evaluate the
stability of 30 mTFP1 mutants (Table S2) and submitted
the four best scorings to molecular dynamics (MD)
simulations (Table S3 and Supporting Note). Although all of
these mutants did not display increased thermal mobility
(Figures 2a, S1), we noted that the N- and C-terminal
sequences (MVSKEETTM and GMDELYK, respectively)
(Table S4) of mTFP1 showed high flexibility in simulations
in agreement that they are not resolved in the X-ray structure
(PDB: 2HQK).103 Thus, both short sequences were removed
to avoid unwanted interference or potential metal coordina-
tion. After the successful completion of molecular cloning and
protein expression of these candidates, mutant-1 revealed the
highest purification yields [500 mg of protein per liter culture
of BL21 (DE3) gold cells] and was accordingly selected as our
host protein scaffold, which we named mTFP* (Figure S2).
artificial (metallo)proteins can easily be determined using
standardized fluorescence and absorbance measurements.
Notably, mTFP* displayed a marked decrease in circular
dichroism ellipticity at 205 nm between 87 and 90 °C (Figure
S3b,c). At 80 °C, the addition of chaotropic reagents such as
urea (10 M) did not decrease the fluorescence signal over a
time period of 30 min (Figure S4a,b). At 25 °C, circular
dichroism and fluorescence data indicated that the structural
integrity of mTFP* is preserved within a pH range from 2.3 to
12.6 (Figure S4c). Moreover, the protein is soluble up to
concentrations of 2 mM in buffer and can withstand high
concentrations of organic solvents (Figure S4d,e).
Next, we determined the X-ray structure of mTFP* to a
resolution of 1.0 Å (RFree = 15.0%; PDB: 4Q9W). The
coordinates of mTFP1 (PDB: 2HQK)103 acted as a template
to perform Patterson search calculations using the software
PHASER.108 The well-defined electron density map depicted
the entire mTFP* protein (Table S5). The monomer adopts a
β-barrel fold composed of 11 antiparallel β-sheets around the
3. mTFP* SCAFFOLD IS HIGHLY STABLE AND CAN
ACT AS A FLUORESCENT REPORTER
mTFP* had an absorbance maximum at 468 nm and a
fluorescent emission peak at 495 nm (Figure S3a). In
comparison to mTFP1, these values are red-shifted by about
3 nm. Because the emission signal of the chromophore serves
as an intrinsic probe to estimate the amount of correctly folded
mTFP* protein in solution,107 the stability of mTFP*-derived
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central A62−Y63−G64 chromophore (Figure 2b), which is
stabilized by the H-bonding network including residues T58,
W89, R91, S142, and H159 (Figure 2c). None of the six
mutations we introduced in mTFP* caused significant
structural perturbations relative to mTFP1 (Figure 2d).
However, as predicted by the MD simulations, B-factor
comparison revealed that the neighboring loops D150−G151
and K180−Y188 in mTFP* are conformationally constrained,
while the mobility of the loops surrounding the cleft between
sheets 7, 8, and 10 (L163−G167 and Y-200−Y204) is
markedly increased (Figure 2e). This higher flexibility is
favorable for ArMs, as we will show below, by allowing the
mutated residues within the cleft to rearrange during metal
binding.
fully restored after dialysis, demonstrating that metal
coordination was weak and reversible (Figure 3a,b). Soaking
experiments carried out for 10 min further confirmed these
observations (Figure S5). Conversely, the tmFRET quenching
effect was markedly enhanced for mTFP1, which persisted
after dialysis (Figure 3).
To determine the binding affinity of mTFP* to Cu2+, we
performed tmFRET titration experiments (Figure 4a,b and
4. mTFP* DISPLAYS A MARGINAL BACKGROUND
AFFINITY TO TRANSITION-METAL IONS
To assess unspecific metal binding affinity of mTFP*, we
incubated 0.2 mM mTFP* with Cu2+, Ni2+, Rh3+, and Pd2+
ions (sulfates or nitrates) for up to 24 h (Figure S5a,b) and
attempted to revert quenching through dialysis (Figure 3).
Figure 4. (a) Metal coordination at pH 6.0 indicates copper and zinc
binding for mTFPCHH and mTFPEHH along with the ancestor mTFP1.
No metal binding occurred in the mTFP* scaffold. (b) Metal
coordination at pH 7.5 reveals copper and zinc binding for mTFPCHH
and mTFPEHH, whereas no metal binding was measured for the
mTFP* scaffold.
Supporting NotetmFRET). At the high metal concen-
trations used in these experiments (up to 0.1 mM), dynamic
collision quenching becomes important, which can be
described by a Stern−Volmer constant KSV. Curve fitting to
a single-site binding model (Supporting Note) revealed
dynamic quenching at high Cu2+ concentrations with a KSV
of 0.09 0.01 mM−1 at pH = 6 and 1.12 0.14 mM−1 at pH
= 7.5 (Table S7). Conversely, tmFRET data of mTFP1
required at least two specific binding constants Ksdite of 0.02
0.01 μM and Kbdack 330 151 μM (pH = 6) or 2.14 0.29 and
268 125 μM (pH = 7.5) to achieve a satisfying curve fit (R >
0.95) (Table S7), revealing additional nonspecific binding
events for mTFP1, but not for mTFP*.
Figure 3. (a,b) Fluorescence measurement of mTFP1 (a) and
mTFP* (b). The samples were soaked in 1, 5, 10, and 25 equiv of
Cu2+, Pd2+, Rh2+, and Ni2+, respectively, for 24 h. Binding
reversibilities were confirmed throughout signal recovery after dialysis.
Note that the quenching efficiency depends on the specific spectral
overlap between metal and protein.
5. INTRODUCING SPECIFIC TRANSITION-METAL
BINDING SITES INTO mTFP*
Our X-ray structures revealed that residues from β-sheets 7, 8,
10, 11, and the central helix constitute a cavity that is about 1.1
nm wide, 1.3 nm deep, and 1.7 nm long. These dimensions are
typical for catalytic centers of metal enzymes.104 Moreover, the
distance between the chromophore and the cleft (1.05−2.30
nm) is within the appropriate range for tmFRET studies.109
Therefore, we chose this cavity for our engineering approach.
Residue Y200 sits in the center of this cavity (Figure 2f).
Metal binding below 25 equiv of Cu2+, Rh3+, or Pd2+ ions was
neither detected by electrospray ionization mass spectrometry
(ESI-MS) nor by matrix-assisted laser desorption/ionization-
time-of-flight MS (MALDI-TOF MS). At high equivalents
(1:5 and above), a correlated decrease of mTFP* fluorescence
indicated the emergence of a tmFRET signal (Figures 3a,b, S5,
and Table S6). Ultimately, the fluorescence of mTFP* was
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Because this position corresponds to H209 in mTFP1 (Figure
1b, Table S4), we chose mutation Y200H as our starting point
to engineer an artificial metal binding site. By inspecting the
homology model of this mutant, we identified positions D54,
Y55, I197, and Y204 as potential sites for the introduction of
additional metal binding His, Glu, or Cys residues. An in silico
point mutation analysis by FoldX predicted the mutants
I197C−Y200H−Y204H (mTFPCHH) and I197E−Y200H−
Y204H (mTFPEHH) as the most stable variants. These changes
were introduced using site-directed mutagenesis (Figure 1b),
and the heterologous protein expression yielded >200 mg/L of
highly pure protein (Figure S2) with spectroscopic character-
istics and heat/pH stabilities similar to those of mTFP*
(Figure S6).
19.6%; PDB: 6QSM), in which residue H200 was turned
outward to reduce the repulsive columbic interaction of the
two positively charged side chains H200 and H204 (Figure
5c). In the following, we refer to the pH 6.0 conformation as
“open” and pH 7.5 structures as “closed”. This “open”
conformation underlined the flexibility of residues 197−206
of β-strand 10 (Figure 5e,f) which was stabilized by a hydrogen
bond between the protonated H200 and T208. This structural
rearrangement reduced the distance between C197 and H204
from 5.7 to 5.2 Å. Next, we determined the crystal structure of
mTFPCHH with a 1:1.1 excess of CuSO4 at pH = 7.5 (Table S5,
RFree = 19.4%; PDB: 4R6D). This structure closely resembled
mTFP* with a root-mean-square deviation (RMSD) of 1.1 Å
and one Cu2+ ion in the center of the protein cleft, 1.5 nm
away from the AYG62−64 chromophore (Figure S7a). For both
H200 and H204, the short N−Cu2+ distances of 2.1 Å
indicated a dative bonding interaction between the metal atom
center and the two histidine side chains. The Cu2+
coordination, which is completed by two water molecules,
reduced the thermal flexibility of the residues 195−210
(Figures 5e and S6b) and caused a minor rearrangement of
the flexible part of strand 10, enhancing the Cu−S distance to
5.7 Å. Therefore, the Cu2+ coordination only involved H200
and H204 (Figure 5d), contrary to our force field-based
calculations which suggested an implication of C197.
mTFPCHH and mTFPEHH crystalized under conditions
similar to those established for mTFP* at pH = 7.5 (Table
S5, mTFPCHH: RFree = 18.4%; PDB: 6QSL; mTFPEHH: RFree
=
19.8; PDB: 6QSO). In both X-ray structures, the side chains of
the introduced metal chelating amino acids point toward the
inside of the cavity (Figure 5a,b).
Conversely, mTFPCHH crystals grown from acidic conditions
(pH = 6.0) revealed a second conformation (Table S5, RFree
=
To verify the metal coordination of those crystal forms for
which we did not obtain copper complex structures (open
mTFPCHH and closed mTFPEHH), we used their respective apo
structures to establish the models of these complexes (Figure
S7b,c). In these crystal structures, C197 and H204
(mTFPCHH/open), as well as E197, H200, and H204
(mTFPEHH), revealed a promising preorientation for metal
binding [d(SC197−NH204) = 5.2 Å; d(OE197−NH200) = 4.6 Å,
d(OC197−NH204) = 3.0 Å, and d(NH200−NH204) = 3.7Å] (Figure
5a,b). In silico introduction of a copper center induced only
minor rearrangements of these residues during energy
minimization. Thus, our X-ray data and in silico analysis
confirmed that Cu2+ coordination is possible for all forms,
whereby mTFPCHH involves either residues H200 and H204
(closed form) or C197 and H204 (open form). Conversely,
the copper binding motif of the mutant mTFPEHH involves all
three residues E197, H200, and H204 (Figure 5a−d).
6. tmFRET ASSAYS REVEALED METAL
COORDINATION AND pH-DEPENDENT
REARRANGEMENTS
In agreement with our structural data, fluorescence measure-
ments via tmFRET revealed a stable metal coordination in
mTFPCHH and mTFPEHH at both pH 6.0 and 7.5. Curve fitting
of tmFRET experiments (Figure 4) allowed us to determine
the apparent Kdsite values of Cu2+ for mTFPCHH to be 0.56
0.06 μM at pH 6.0 and 0.05 0.01 μM at pH 7.5, whereas for
mTFPEHH, they were 8.2 1.5 μM at pH 6.0 and 4.5 2.7
μM at pH 7.5 (Table S7).
Figure 5. (a−d) Crystal structures of the catalytic clefts of mTFPCHH
(closed configuration) at pH 7.5 (a), mTFPEHH (b), mTFPCHH (open
configuration) at pH 6.0 (c) with open configuration undergoing
structural rearrangement in the course of pH change (a,c), and
cocrystallized structure of mTFPCHH coordinating a Cu2+ ion (d).
Surface colors indicate positive and negative electrostatic potentials
contoured from 20 kT/e (blue) and −20 kT/e (red). The
coordination residues are represented in sticks (a−d). (e) B-factor
comparison of mTFP*, mTFPCHH, and mTFPCHH/Cu2+ indicating a
stabilized coordination throughout the copper coordination by His−
His-motive. (f) Superposition mTFP*(green), mTFPCHH (orange),
and Cu2+−mTFPCHH, visualizing the structural rearrangements of the
cleft flanking loop, especially upon Cu2+ ion coordination (* indicates
the shift of residue Y/H200).
The signals indicated a 1:1 complex of the mTFPCHH or
mTFPEHH mutant protein and Cu2+, which was verified by
liquid chromatography (LC)−ESI−MS and inductively
coupled plasma (ICP)-MS data (Figure S8, Tables S6 and
S8). Conversely, these methods did not show Cu2+
coordination by mTFP* (Tables S6 and S8). The metal
binding to both artificial sites proved to be highly stable,
preserving a metal atom occupancy above 50% following 24 h
of dialysis in buffer without metal ions (Figure S5c). In
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addition, fluorescence quenching was observed after 24 h of
dialysis for other transition-metal ions, which increased in the
order Ni2+ < Rh3+ < Pd2+ < Cu2+ for mTFPCHH (with a
fluorescence recovery of 99, 81, 85, and 67%, respectively),
indicating that mTFPCHH can also coordinate with other
transition metals than copper. As expected, the fluorescent
recovery was close to 100% for mTFP* for all these metals
(Figure S5, Supporting NotetmFRET).
Titrations of copper-loaded mTFPCHH or mTFPEHH in the
presence of Zn2+ fully restored the fluorescence of the apo
protein, with affinities of 128 32 μM at pH 6.0 and 4.01
0.14 μM at pH 7.5, while mTFPEHH titrations resulted in 288
118 μM at pH 7.5 (Figure 4, Table S7). Thus, through
competition experiments with copper-loaded mTFP* variants,
tmFRET can be used to study the binding process of transition
metals, for which the spectral overlap integral is negligible.
7. ARTIFICIAL CU−PROTEINS ARE CATALYTICALLY
ACTIVE AND ACHIEVE HIGH STEREOINDUCTION
To evaluate the potential catalysis of our artificial Cu−proteins,
we conducted [4 + 2] cycloadditions, commonly known as
Diels−Alder reactions (Figure 6a, Table S9). The activity
assays displayed a distinct pH profile for the copper-
coordinated cysteine mutant Cu2+−mTFPCHH (Figure 6b,
Table S9), while the glutamate variant mTFPEHH delivered
quantitative yields over a pH range from 3.5 to 7.5 with low
enantioselectivities (Table S9). In the next series of experi-
ments, we analyzed the Friedel−Crafts alkylation with our
engineered mTFPCHH and mTFPEHH copper proteins. We
measured excellent enantioselectivities in the reaction of α,β-
unsaturated 2-acylimidazole with indole (Figure 6c, Table S9).
Notably, mTFPCHH produced stereoselectivities of up to
92% ee (Figure 6d, Table S9), while mTFPEHH achieved an ee
between 40 and 93%. Background reactions without added
Cu(NO3)2 showed no considerable conversions. In compar-
ison, nonchelated Cu2+ ions achieved over 90% conversion,
however, without enantio- or stereocontrol (Table S9,
Supporting Note). The lower rate of the artificial enzyme
may be due to (passive) steric restrictions.110,111 For the
Friedel−Crafts alkylation, the pH variation from 5.0 to 7.5 had
only a minor impact on both the catalytic activity and the
stereoinduction of mTFPCHH or mTFPEHH, suggesting that the
Cu2+ ion is firmly bound and shielded in the protein pocket.
Following this demonstration of basic catalytic functionality,
we tested whether changes within the second coordination
sphere would further enhance the Diels−Alder activity of the
mutant, mTFPCHH. From our X-ray analysis and additional
MD simulations, we concluded that a smaller, more hydro-
phobic, and sterically restricted binding site would improve the
substrate coordination. Moreover, the selectivity might
increase and presumably cause a stronger coordination of the
Diels−Alder substrate (Figure S9a). Consequently, we
replaced residues at the edge of the cleft containing the
metal binding motif with more polar and hydrophobic amino
acids (K135A, T137S, E164G, G165K, I197Y, N199Y, D201K,
and K202Y) (Figure S9a). The corresponding optimized
mTFPCHH mutant, mTFPCHH opti, showed catalytically en-
hanced stereoselectivities and accelerated reaction rates, for
example, doubled for pH 6.0 (Figure S9). The most obvious
increase was at pH 6.0, where the introduced mutations
consistently accounted for a boost from 2 to 26% enantiomeric
excess. Thus, catalysis of our ArM can be further enhanced
through modifications of its second coordination sphere.
Figure 6. (a) Scheme of catalyzed Diels−Alder reaction with the
following conditions: [azachalcone] = 1.0 mM; [cyclopentadiene] =
90 mM; [Cu(NO3)2] = 0.1 mM; [protein] = 0.12 mM; T = 4 °C; t =
20 h; and reaction volume = 4 mL. Reactions were run at the given
pH in 50 mM MES buffer containing 50 mM NaCl. Conversions and
ee values were determined by HPLC. (b) Diels−Alderase activity of
mTFPCHH and mTFPEHH, through various pHs, which revealed
appropriate catalytic activity, and up to 17% ee (Table S9). (c)
Reaction scheme of catalyzed Friedel−Crafts alkylation with the
following conditions: [imidazole] = 1.0 mM; [indole] = 5.0 mM;
[Cu(NO3)2] = 0.1 mM; [protein] = 0.12 mM; T = 4 °C; t = 48 h;
and reaction volume = 4 mL. Experiments were run at the given pH in
50 mM MES buffer containing 50 mM NaCl. Conversions and ee
values were determined by HPLC. (d) Friedel−Crafts alkylase activity
of mTFPCHH and mTFPEHH, through various pHs, which resulted in
catalytic activity, up to 26% conversion, and up to 92% ee (R
enantiomer) (Table S9).
8. DISCUSSION
Since the initial attempt in 1978,48 numerous strategies were
used to produce ArMs. However, only few ArMs were based
on the natural amino acid anchoring strategy. In addition,
although metal binding motifs have previously been identified
within the existing protein scaffolds,69 the integration of such
binding sites within a non-native scaffold, using only natural
amino acids, remained unsuccessful. Herein, we present the
design and structural and functional characterization of such a
novel ArM. Starting from a highly stable fluorescent protein,
we engineered a low metal affinity variant, mTFP*, based on
selection of surface-exposed metal binding residues, by using
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ACS Catal. 2019, 9, 11371−11380

ACS Catalysis
Research Article
FoldX prediction of changes in folding energy for mutants and
stability evaluation of highest scoring mutants with MD
simulations.
tmFRET titrations revealed that mTFP* possessed only a
low, unspecific metal affinity (Kd > 1 mM), whereas the
original mTFP1 scaffold had at least two metal binding sites
AUTHOR INFORMATION
Corresponding Authors
*E-mail: stefan.arold@kaust.edu.sa (S.T.A.).
*E-mail: michael.groll@tum.de (M.G.).
*E-mail: jorg.eppinger@kaust.edu.sa (J.E.).
■
ORCID
with Kds of 2.14
0.29 and 268
125 μM. Furthermore,
Seema Ghorpade: 0000-0001-5576-1390
Stefan T. Arold: 0000-0001-5278-0668
Michael Groll: 0000-0002-1660-340X
mTFP* tolerates temperatures up to 89 °C, pH values between
2.3 and 12.6, is stable in 10 mM urea at 80 °C, and withstands
high organic solvent concentrations. In combination with the
crystal structure of mTPF*, we combined computational and
experimental approaches to rationally introduce two different
metal binding sites into a selected surface pocket (mTFPCHH
and mTFPEHH). Both mTFPCHH and mTFPEHH revealed high
affinities for several metal atoms, with single site Ksdite for Cu2+
and Zn2+ in the μM range. The high enantioselectivities
achieved for the Friedel−Crafts alkylation of indole demon-
strated that the pocket shape and size of mTFPCHH and
mTFPEHH are appropriate to induce sufficient energy differ-
ences of the diastereotopic transition states. The initially low
stereoselectivity and reaction rates for the Diels−Alder
reaction were improved via the modification of the second
coordination sphere. Thus, we achieved a catalytic function
from a noncatalytic scaffold by rationally introducing amino
acid motifs capable of hosting a catalytically active metal atom
in a well-defined geometry.
Collectively, we could elucidate that low expression yields,
weak protein stability, low recyclability, unspecific cofactor/
metal atom ligation, and limited bioavailability can be
overcome by developing novel mutants of mTFP*, a derivate
from a well-characterized fluorescent protein. We provide a
promising fundament on which ArMs can now be advanced
based on the natural amino acid anchoring strategy. The
absence of artificial cofactors combined with the intrinsic
fluorescence of our scaffold markedly facilitates improving or
amending implemented catalytic activities (e.g., through
directed evolution) and therefore expands the repertoire of
ArM templates. Hence, our approach may provide a roadmap
for engineering other catalytically active ArMs.
̈
Jorg Eppinger: 0000-0001-7886-7059
Present Addresses
⊥L.E.K. Consulting, Brienner Str. 14, 80333 Munich, Germany.
#Division of Digital Science, Thermo Fisher Scientific, Im
Steingrund 4, 63303 Dreieich, Germany.
¶Department of Biochemistry, Faculty of Science, Center for
Science and Medical Research, University of Jeddah, 80203
Jeddah, Saudi Arabia.
Author Contributions
∇J.F. and D.R. contributed equally to this work.
Funding
The research reported in this publication was supported by
King Abdullah University of Science and Technology
(KAUST), through the baseline funds and the office of
sponsored research under award no. 1974-01, and the TUM/
KAUST Joint Doctoral Degree Program.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The staff of the Beamline X06SA at the Paul Scherrer Institute,
SLS, Villigen, Switzerland, is acknowledged for assistance
during data collection. The authors thank S. Abdul Rajjaka for
help with the bioinformatic setup.
ABBREVIATIONS
■
ArMs, artificial metalloenzymes
ArM, artificial metalloenzyme
tmFRET, transition metal FRET
MD, molecular dynamics
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acscatal.9b02896.
■
S
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