Semi-synthesis of an artificial scandium(iii) enzyme with a β-helical bio-nanotube

Article abstract of DOI:10.1039/c2dt31030a

We have succeeded in preparing semi-synthesized proteins bound to Sc ³⁺ ion which can promote an epoxide ring-opening reaction. The Sc³⁺ binding site was created on the surface of [(gp5βf) ₃]₂ (N. Yokoi et al.,

Full text of DOI:10.1039/c2dt31030a

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PAPER
Semi-synthesis of an artificial scandium(III) enzyme with a β-helical
bio-nanotube†
Hiroshi Inaba,a Shuji Kanamaru,b Fumio Arisaka,b Susumu Kitagawaa,c and Takafumi Ueno*b,c
Received 11th May 2012, Accepted 26th July 2012
DOI: 10.1039/c2dt31030a
We have succeeded in preparing semi-synthesized proteins bound to Sc3+ ion which can promote
an epoxide ring-opening reaction. The Sc3+ binding site was created on the surface of [(gp5βf)3]2
(N. Yokoi et al., Small, 2010, 6, 1873) by introducing a cysteine residue for conjugation of a bpy moiety
using a thiol–maleimide coupling reaction. Three cysteine mutants [(gp5βf_X)3]2 (X = G18C, L47C,
N51C) were prepared to introduce a bpy in different positions because it had been reported that Sc3+ ion
can serve as a Lewis-acid catalyst for an epoxide ring-opening reaction upon binding of epoxide to bpy
and two –ROH groups. G18C_bpy with Sc3+ can accelerate the rate of catalysis of the epoxide ring-
opening reaction and has the highest rate of conversion among the three mutants. The value is more than
20 times higher than that of the mixtures of [(gp5βf)3]2/2,2′-bipyridine and L-threonine/2,2′-bipyridine.
The elevated activity was obtained by the cooperative effect of stabilizing the Sc3+ coordination and
accumulation of substrates on the protein surface. Thus, we expect that the semi-synthetic approach can
provide insights into new rational design of artificial metalloenzymes.
originally existed in the proteins, the possibilities of artificial
Introduction
metalloenzymes will be expanded.
Scandium ion (Sc3+) is a potent candidate for anchoring to a
In nature, metalloenzymes promote enzymatic reactions by using
various metal ions such as Fe, Mn, Cu, Co, Ni, Zn, among
others.1 Metal ion coordination complexes constructed within
the unique molecular environments provided by protein scaffolds
help them to overcome their relative instability in aqueous
media. The coordination structures are stabilized by ligation of
amino acid residues such as His, Cys, Asp and/or natural cofac-
tors such as porphyrin and chlorophyll.2 In preparation of biomi-
metic systems, various synthetic metal complexes have been
fixed within protein scaffolds in order to construct artificial
metalloenzymes that can achieve various catalytic reactions such
as hydrogenation, sulfoxidation, and the Diels–Alder reaction.3–8
Anchoring of the synthetic metal complexes has been achieved
by using natural host–guest interactions of heme/apo-myoglobin
and biotin/(strept)avidin9–12 or selective conjugation reactions of
cysteinyl thiol/maleimide and lysinyl amine/succinimidyl
ester.13–16 Furthermore, if synthetic metal complexes conjugated
to proteins are activated by coordination of amino acid residues
protein because of its unique catalytic activity. It can function as
a Lewis-acid in catalysis of various reactions such as formation
of C–C and C–A bonds (where A = N, O, S, P, and Si).17 Sc3+
retains activity in water, in contrast to other Lewis acids such as
AlCl3, BF3, TiCl4 and SnCl4, which are unstable in water. This
suggests that anchoring of Sc3+ to a protein would provide a
promising strategy for construction of a new class of Lewis-acid
metalloenzymes. However, an enzyme containing Sc3+ has not
yet been reported because it has proven difficult to stabilize the
Sc3+ coordination structure within a protein.
Herein, we report the construction of an artificial Sc3+ enzyme
by combining a conjugated synthetic ligand and natural co-
ordinating amino acids on surface of a protein as shown in
Fig. 1a. The synthetic ligand is covalently anchored to a position
adjacent to the coordinating amino acids by chemical modifi-
cation of a cysteinyl thiol which is introduced by site-directed
mutagenesis. Natural coordinating amino acids on the host
protein are used as supporting ligands. It was expected that the
coordination structure of Sc3+ would be stabilized by the coop-
erative coordination of the synthetic ligand and the amino acid
ligands. The host protein must meet certain criteria as follows;
(1) it must have chemical and thermal stability under chemical
modification and catalytic reaction conditions, and (2) coordinat-
ing amino acids and the synthetic ligand must be located at an
appropriate position to promote metal binding. Thus, we employ
our previously reported tubular protein [(gp5βf)3]2 which
satisfies these requirements. It is a chimeric protein assembly
aGraduate School of Engineering, Kyoto Univ., Katsura, Kyoto 615-
8510, Japan
bGraduate School of Bioscience and Biotechnology, Tokyo Institute of
Technology, Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
E-mail: tueno@bio.titech.ac.jp; Fax: +81 45 924 5806;
Tel: +81 45 924 5806
cInstitute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto
Univ., Yoshida, Kyoto 606-8501, Japan
†Electronic supplementary information (ESI) available: Additional
figures and experimental methods. See DOI: 10.1039/c2dt31030a
11424 | Dalton Trans., 2012, 41, 11424–11427
This journal is © The Royal Society of Chemistry 2012

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Fig. 1 (a) Scheme of semi-synthesis of a metal-binding site on a
protein surface. (b) A schematic drawing indicating conjugation of 2,2′-
bipyridine (bpy) to [(gp5βf)3]2. Cys is introduced to a position near Thr
and Ser by site-directed mutagenesis (construction of [(gp5βf_X)3]2,
X = G18C, L47C, N51C). Bpy was coupled to the Cys residues by a
selective reaction between cysteinyl thiol and a bipyridine-maleimide
derivative (bpy-MI) (construction of X_bpy).
Fig. 2 Side view of the crystal structure of [(gp5βf)3]2.21 One of the
six sites is extended. Green sticks represent G18, L47, and N51. Yellow
and orange sticks represent Thr and Ser, respectively. S61 has two
alternative conformations.
formed by genetic fusion of the β-helical motif of gene product
5 (gp5β)18–20 and the foldon domain (f) isolated from bacterio-
phage T4.21 [(gp5βf)3]2 adopts
a trimer–dimer structure
T53/S61 of [(gp5βf)3]2 which have O–O distances of 4.2, 5.0,
and 4.9 Å, respectively (Fig. 2). We introduced cysteine residues
by site-directed mutagenesis at G18, L47 and N51. These sites
are geometrically adjacent to the alcohol pairs. The introduced
cysteine residues were selectively conjugated with bpy because
[(gp5βf)3]2 itself has no cysteine residues. Three cysteine
mutants [(gp5βf_X)3]2 (X = G18C, L47C, N51C) were prepared
for conjugation of the 2,2′-biprydine-maleimide derivative
(bpy-MI) (Fig. 1b).
Modification of [(gp5βf_X)3]2 (X = G18C, L47C, N51C)
with bpy-MI was carried out using our previously reported
method.23 A dimethylsulfoxide solution of bpy-MI (6 mM,
2.2 mL) was added to [(gp5βf_G18C)3]2 (5.3 μM, 41.4 mL) in
20 mM potassium phosphate buffer pH 7.0. After stirring for
15 h at 25 °C, the mixture was purified with Sephadex G-25
(10 mM MOPS buffer pH 7.5). G18C_bpy was obtained with
an isolated yield of ∼100%. The MALDI-TOF mass spectrum of
G18C_bpy shows a mass increment of 279 Da, which is
assigned to bpy-MI (calcd 279 Da), with complete loss of the
peak of the original gp5βf_G18C monomer (14 842 Da)
(Fig. S1†). The result indicates that all of the Cys residues of
G18C_bpy were conjugated with bpy-MI molecules. The
tubular structure of G18C_bpy was confirmed by gel permeation
chromatography (GPC). The elution profile of G18C_bpy had a
peak at 13.5 min. This peak is the same as that of
[(gp5βf_G18C)3]2 (Fig. S2†). This indicates that the tubular
assembly structure was maintained even after conjugation of
bpy-MI. These results show that six bpy-MI units were comple-
tely conjugated to six cysteine residues of [(gp5βf_G18C)3]2
which retains its original tubular folding. L47C_bpy and
N51C_bpy were also prepared with a ∼100% modification yield
(89 kDa) which is composed of six gp5βf monomers (14.8 kDa).
Compared to other host tubular proteins such as tobacco
mosaic virus (TMV),15 M13 bacteriophage,16 and potato virus
X (PVX),22 [(gp5βf)3]2 has a tubular structure with excellent
stability. The crystal structure of [(gp5βf)3]2 indicates regular
alignment of amino acids on the surface of the β-helix.21 We
anchored catalytic metal complexes on the surface of [(gp5βf)3]2
by chemical modification of Lys and Cys residues.21,23 This indi-
cates that [(gp5βf)3]2 can serve as an ideal host protein for cata-
lytic reactions with Sc3+ ion.
Results and discussion
The Sc3+ binding site was created on the surface of [(gp5βf)3]2
by the introduction of a cysteine residue to conjugate a 2,2′-
bipyridine (bpy) moiety using the thiol–maleimide coupling
reaction (Fig. 1b). A previously reported Sc3+ ligand containing
bpy and two –ROH groups, [bpy–(ROH)2], was employed as a
model to coordinate the Sc3+ ion in a cooperative manner.24–27
Tetradentate coordination of the bpy and two –ROH groups to
the Sc3+ ion is essential to produce the complex, Sc[bpy–(ROH)2],
which is capable of catalyzing the epoxide ring-opening reac-
tion.24,27 Hydroxy protons of the –ROH moieties serve an essen-
tial role for the high catalytic activity.27 The two –ROH groups
can be altered by alcohol groups of Thr or Ser residues of
[(gp5βf)3]2 because the alcohols (pKa ∼13) retain –ROH form
under the catalytic conditions. The crystal structure of Sc[bpy–
(ROH)2] shows that the O–O distance between the two –ROH
groups is 4.2 Å.25 We expect that a similar coordination structure
can be reconstructed with pairs of T21/T29, T45/T53, and
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and retaining the tubular folding under the same conditions
(Fig. S1†). The MALDI-TOF mass spectrum of G18C_bpy with
Sc(OTf)3 shows new peaks assigned to adducts of a single Sc3+
ion to the G18C_bpy monomer, although no such new peaks
were observed when Sc(OTf)3 was added to unmodified
[(gp5βf)3]2 under same conditions (Fig. S3†). These results indi-
cate that a single Sc3+ ion can coordinate to the bpy moiety on
G18C_bpy without any random binding of Sc3+ ions on the
surface of G18C_bpy.
between bpy and the residues to act the ion as a Lewis acid to
activate the epoxide for nucleophilic attack. With respect to the
catalytic reactions in aqueous solution, the conversion by
G18C_bpy (42%) is higher than that by a synthetic ligand
reported previously (15%),26 although the higher conversion of
the synthetic ligand was achieved in CH2Cl2 (95%).24 The re-
action catalyzed by the previously reported synthetic Sc3+
complex was accelerated in water by utilization of Sc(DS)3
(DS = dodecyl sulfate) instead of Sc(OTf)3 due to the formation
of a colloidal dispersion of DS that accumulates catalysts and
confines substrates to the inside.26 In the case of G18C_bpy,
factors responsible for the higher conversion are thought to be
the increase in both coordination stability of the Sc3+ center and
easy accessibility of substrates at the exposed surface of
G18C_bpy as indicated in our previous report which describes
accumulation of substrates at the catalytic center exposed on the
surface of [(gp5βf)3]2.21
The differences among the catalytic activities of X_bpy
(X = G18C, L47C, N51C) (entries 1–3, Table 1) are explained
by the crystal structure of [(gp5βf)3]2. The high b-factor of Cα
of G18 (55 Å2) relative to the b-factors of Cα of L47 (23 Å2)
and N51 (22 Å2) (Fig. S4†) suggests that bpy of G18C_bpy has
a high level of flexibility. Flexible amino acid residues are
known to accommodate demanding conformations that would
favor the coordination structure more than rigid residues.28 It is
expected that a bpy conjugated to a cysteine residue at a position
18 can form more suitable Sc3+ coordination structure for the
catalytic reaction than that at the other positions although all the
cysteine residues of the mutants are located at the positions
adjacent to two threonine or serine residues which are enable to
bind to a Sc3+ ion with a bpy ligand as that of a synthetic
Sc[bpy-(ROH)2] (Fig. 2).25 The combination of appropriate
location of Thr or Ser residues and a bpy moiety anchored to a
flexible cysteine residue is essential for the creation of the semi-
synthesized Sc3+ center on G18C_bpy.
To evaluate the catalytic activity of X_bpy (X = G18C, L47C,
N51C) with Sc3+, ring-opening reactions of cis-stilbene oxide
(1) with aniline derivatives (2a, 2b) were examined in 10 mM
MOPS buffer pH 7.5, 10% acetonitrile for 48 h at 40 °C in the
dark and under an Ar atmosphere. Conversion yields were deter-
1
mined by H-NMR. G18C_bpy shows the highest rate of con-
version of aniline (2a) among the reactions listed in Table 1
(42%, entry 1). This value is more than 20 times higher than
that of the mixtures of [(gp5βf)3]2/2,2′-bipyridine (entry 5) and
L-threonine/2,2′-bipyridine (entry 6). The rates of conversions by
L47C_bpy (entry 2) and N51C_bpy (entry 3) were found to be
3 and 5 times lower, respectively, than that of G18C_bpy
(entry 1). G18C_bpy also catalyzed the reaction with the aniline
derivative p-toluidine (2b) with a conversion rate comparable to
that of 2a (entries 1 and 4). Enantioselectivities were determined
by HPLC using a Chiralcel AD column. G18C_bpy was found
to have higher ee selectively ((1R, 2R), 17%, entry 1), than the
ee selectivity values of L47C_bpy ((1S, 2S), 9%, entry 2) and
N51C_bpy ((1S, 2S), 1%, entry 3).
G18C_bpy has higher catalytic activity than that of a mixture
of each of the ligands (entry 1 vs. entries 5 and 6, Table 1). This
demonstrates the advantage of the approach. It is estimated that
Sc(bpy)(Thr)2 complex is not stably formed by simple mixing of
bpy and L-threonine in water (entry 6). This observation is con-
sistent with our previous report that a flavin ligand anchored on
[(gp5βf)3]2 stabilizes the Cu+-flavin structure.21 These results
clearly indicate that spatially adjacent bpy and two Thr residues
on G18C_bpy coordinate the Sc3+ ion in a cooperative manner
Conclusions
A Sc3+ binding site was semi-synthesized by chemical modifi-
cation to place 2,2′-bipyridine in a position adjacent to two
alcohol groups of Thr aligned on surface of the β-helical tubular
protein. The Sc3+ binding site can accelerate the rate of catalysis
of the epoxide ring-opening reaction. The high activity was
accomplished by taking advantage of the cooperative effect of
stabilizing the Sc3+ coordination and accumulation of substrates
on the protein surface. This is the first example of construction
of an artificial Sc3+ enzyme by cooperative coordination of bpy
and threonine residues. Although there is still scope for improve-
ment of catalytic activity and selectivity, this work indicates that
the semi-synthetic approach is one of the useful strategies for
construction of new artificial metalloenzymes. We are under-
going further optimization of the artificial enzyme.
Table 1 Results of Sc3+ catalytic ring-opening reaction
Entry Ligands
Substrates Conversion/% ee/%
1
2
3
4
5
G18C_bpy
L47C_bpy
N51C_bpy
G18C_bpy
[(gp5βf)3]2/2,2′-
bipyridine
2a
2a
2a
2b
2a
42
13
8
36
17(1R, 2R)
9 (1S, 2S)
1 (1S, 2S)
0.5 (1R, 2R)
—
Trace
6
L-Threonine/2,2′-
bipyridine
2a
2
0.4 (1R, 2R)
Experimental
Reaction conditions: [2,2′-bipyridine] = 0.20 mM, [[(gp5βf)3]2] =
33 μM, [Sc(OTf)3] = 0.20 mM, [L-threonine] = 1.0 mM, [1] = 4.0 mM,
[2a, 2b] = 4.0 mM in 10 mM MOPS buffer pH 7.5, 10% acetonitrile,
under dark and Ar, 40 °C, 48 h. Each entry was examined three times.
Physical measurement
UV-vis spectra were recorded on a SHIMADZU UV-2400PC
UV-vis spectrometer. 1H-NMR spectra were recorded on a
11426 | Dalton Trans., 2012, 41, 11424–11427
This journal is © The Royal Society of Chemistry 2012

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Bruker UltraShieldTM 500 Plus spectrometer. MALDI-TOF mass
spectra were recorded on an Autoflex III (Bruker Daltonics).
Acknowledgements
This work was supported by a Research Fellowship for Young
Scientists of JSPS for H.I. (No. 4240) and Grant-in-Aids for
Scientific Research (No. 20350063 for T.U.) from MEXT and
TOKUYAMA SCIENCE FOUNDATION, Japan.
Materials
Unless otherwise stated, all chemicals were purchased from
commercial suppliers and used without further purification.
4-ethylmaleimide-2,2′-bipyridine (bpy-MI) were prepared
according to the literature methods.23 Cys mutants of [(gp5βf)3]2
were prepared by using a Quick Change site-directed mutagen-
esis kit (Qiagen). DNA sequences of the mutants were deter-
mined by an ABI3100 (Applied Biosystems). Expression and
purification of the mutants were performed as described pre-
viously.21,23 In the measurement of MALDI-TOF, samples were
dialyzed against 5 mM ammonium acetate aqueous solution and
then mixed with an equal volume of 70% v/v acetonitrile–water
solution containing 0.03% w/v sinapic acid and 0.1% v/v tri-
fluoroacetic acid. GPC was carried out with a HPLC system and
columns (Asahipack GF-510HQ, Shodex, Tokyo, Japan), at
room temperature with 20 mM Tris buffer pH 8.0 as the eluent.
HPLC analyses to determine enantioselectivities were performed
with a chiral stationary phase column (Chiralcel AD purchased
from Dicel Co., Ltd).
Notes and references
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Modification of cysteines with bpy-MI
G18C_bpy; A DMSO solution of bpy-MI (6 mM, 2.2 mL) was
slowly added to an aqueous solution of [(gp5βf_G18C)3]2
(5.3 μM, 41.4 mL in 20 mM potassium phosphate buffer
pH 7.0) and the mixture was gently stirred at 25 °C for 15 h.
G18C_bpy was purified by Sephadex G-25 equilibrated with
10 mM MOPS buffer pH 7.5. L47C_bpy and N51C_bpy were
prepared by the same procedure. The protein recovery after the
modification was ∼100%. UV/Vis: λmax 283 nm (ε·M−1 cm−1
255 000). MS (MALDI-TOF) (Fig. S1†): G18C_bpy mono-
mer [gp5βf_G18C + bpy-MI]+, calcd,15 118; found, 15121,
L47C_bpy monomer [gp5βf_L47C + bpy-MI]+, calcd,15 062;
found, 15076, N51C_bpy monomer [gp5βf_N51C + bpy-MI]+,
calcd,15 061; found,15 065.
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Ring-opening reaction of meso-epoxide
Under Ar atmosphere, to a solution of Sc(OTf)3 (90 μL, 1.2 ×
10−7 mol) X_bpy (450 μL, 1.2 × 10−7 mol of 2,2′-bipyridine) or
L-threonine (450 μL, 6.0 × 10−7 mol) in 10 mM MOPS
buffer pH 7.5 were added. Subsequently acetonitrile (30 μL) or
2,2′-bipyridine (30 μL, 1.2 × 10−7 mol) in acetonitrile were
added. After addition of cis-stilbene oxide (1) (15 μL, 2.4 ×
10−6 mol) and aniline derivative (2a, 2b) (15 μL, 2.4 × 10−6 mol)
in acetonitrile, the mixture was incubated for 48 h at 40 °C with
stirring under dark and Ar atmosphere. The substrates and pro-
ducts were extracted with ethyl acetate (1 mL) three times after
addition of saturated NaHCO3 aqueous solution (1 mL). Conver-
sions of 2a and 2b to 3a and 3b, respectively, and enantio-
1
selectivities of 3a, 3b were determined by H-NMR and HPLC
analysis, respectively, as reported previously.24,26,29
This journal is © The Royal Society of Chemistry 2012
Dalton Trans., 2012, 41, 11424–11427 | 11427