A Well-Defined Osmium-Cupin Complex: Hyperstable Artificial Osmium Peroxygenase

Article abstract of DOI:10.1021/jacs.7b00675

Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os) peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (T_m ≈ 120 °C). The spectroscopic analysis and atomic resolution X-ray crystal structures of Os-bound TM1459 revealed two types of donor sets to Os center with octahedral coordination geometry. One includes trans-dioxide, OH, and mer-three histidine imidazoles (O₃N₃ donor set), whereas another one has four histidine imidazoles plus OH and water molecule in a cis position (O₂N₄ donor set). The Os-bound TM1459 having the latter donor set (O₂N₄ donor set) was evaluated as a peroxygenase, which was able to catalyze cis-dihydroxylation of several alkenes efficiently. With the low catalyst loading (0.01% mol), up to 9100 turnover number was achieved for the dihydroxylation of 2-methoxy-6-vinyl-naphthalene (50 mM) using an equivalent of H₂O₂ as oxidant at 70 °C for 12 h. When octene isomers were dihydroxylated in a preparative scale for 5 h (2% mol cat.), the terminal alkene octene isomers was converted to the corresponding diols in a higher yield as compared with the internal alkenes. The result indicates that the protein scaffold can control the regioselectivity by the steric hindrance. This protein scaffold enhances the efficiency of the reaction by suppressing disproportionation of H₂O₂ on Os reaction center. Moreover, upon a simple site-directed mutagenesis, the catalytic activity was enhanced by about 3-fold, indicating that Os-TM1459 is evolvable nascent osmium peroxygenase.

Full text of DOI:10.1021/jacs.7b00675

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Article
A Well-Defined Osmium-Cupin Complex:
Hyperstable Artificial Osmium Peroxygenase
Nobutaka Fujieda, Takumi Nakano, Yuki Taniguchi, Haruna Ichihashi, Hideki
Sugimoto, Yuma Morimoto, Yosuke Nishikawa, Genji Kurisu, and Shinobu Itoh
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b00675 • Publication Date (Web): 24 Mar 2017
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A Well-Defined Osmium-Cupin Complex: Hyperstable Artificial
Osmium Peroxygenase
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Nobutaka Fujieda,a,* Takumi Nakano,a Yuki Taniguchi,a Haruna Ichihashi,a Hideki Sugimoto,a Yuma
Morimoto,a Yosuke Nishikawa,b Genji Kurisu,b and Shinobu Itoha,*
aDepartment of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka
565-0871, Japan
bInstitute for Protein Research, Osaka University, 3-2 Yamada-oka, Suita, Osaka 565-0871, Japan
ABSTRACT: Thermally stable TM1459 cupin superfamily protein from Thermotoga maritima was repurposed as an osmium (Os)
peroxygenase by metal-substitution strategy employing the metal-binding promiscuity. This novel artificial metalloenzyme bears a
datively bound Os ion supported by the 4-histidine motif. The well-defined Os center is responsible for not only the catalytic activ-
ity but also the thermodynamic stability of the protein folding, leading to the robust biocatalyst (Tm ≈ 120 ºC). The spectroscopic
analysis and atomic resolution X-ray crystal structures of Os-bound TM1459 revealed two types of donor sets to Os center with
octahedral coordination geometry. One includes trans-dioxide, OH, and mer-three histidine imidazoles (O3N3 donor set), whereas
another one has four histidine imidazoles plus OH and water molecule in a cis position (O2N4 donor set). The Os-bound TM1459
having the latter donor set (O2N4 donor set) was evaluated as a peroxygenase, which was able to catalyze cis-dihydroxylation of
several alkenes efficiently. With the low catalyst loading (0.01% mol), up to 9100 turnover number was achieved for the dihydrox-
ylation of 2-methoxy-6-vinyl-naphthalene (50 mM) using an equivalent of H2O2 as oxidant at 70 C for 12 h. When octene isomers
were dihydroxylated in a preparative scale for 5 h (2 % mol cat.), the terminal alkene octene isomers was converted to the corre-
sponding diols in a higher yield as compared with the internal alkenes. The result indicates that the protein scaffold can control
the regio-selectivity by the steric hindrance. This protein scaffold enhances the efficiency of the reaction by suppressing dispro-
portionation of H2O2 on Os reaction center. Moreover, upon a simple site-directed mutagenesis, the catalytic activity was en-
hanced by about three-fold, indicating that Os-TM1459 is evolvable nascent osmium peroxygenase.
is the metal-substitution of natural metalloenzymes based on
INTRODUCTION
To meet industrial demands,1 tremendous efforts have
their metal binding promiscuity to generate novel activity.19,20
There are several lines of evidence that the replacement of
been recently made to develop highly regio- and/or ste-
copper center of blue copper protein by HgII and CdII can lead
reo(enantio)-selective artificial metalloenzymes by combining
to the large thermal stabilization of protein folding because of
a
biomolecule (protein/DNA) with a synthetic metal
stronger donor-acceptor combination (soft donor (Cys and
Met) and soft metal ion) and their strong coordination bond
of heavy metal ion.21 Thus, the replacement of native metal
ion to preferable different metal ion will be one of powerful
methods to endow the protein scaffold with robustness as
well as novel catalytic capability. In this context, platinum-
group elements (Ru, Os, Rh, Ir, Pd, and Pt) might be suitable
for this purpose, although these elements are hardly em-
ployed in natural enzymes due to their scarcity in usual envi-
ronments.
Independently, Kazlauskas group22 and Soumillion group23
have accomplished the metal replacement of 3-His metal
binding site of carbonic anhydrase, that is a Zn-containing
hydrolase, by Mn to create a stereo-selective alkene epoxi-
dase. Kazlauskas group further developed this strategy by
employing Rh ion to create a stereo-selective alkene hydro-
genase24 and a regio-selective hydroformylase.25 Hartwig
complex.2–4 For the purpose of linking of a protein matrix and
a metal complex, various strategies have been pursued in-
cluding covalent attachment, supramolecular interaction, and
dual linking methods of them with dative anchoring by the
amino acid residues to the metal center of complex.5–11 Alt-
hough the rates of the reactions catalyzed by these systems
have been much slower than those of natural enzymes, Ward
group and Hartwig group demonstrated that artificial metal-
loenzymes could be controlled by genetic manipulation of the
protein scaffold12 and consequently possess the fundamental
characteristics of natural enzymes.13,14 On the other hand, the
another approach has been employed based on the direct
complexation of bare metal ions with either natural-15 or non-
natural-16 amino acids as the ligands. More recently, the en-
zyme repurposing’s have been revealed to be promising, via
self-assembly of monomeric protein17 and by exploring the
latent metal binding site.18 Far more well-organized strategy
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group report the efficient synthesis of oraganometallic com- catalytic activity of Os-bound TM1459 can be evolved by the
plexes with this hydrolase.26 These results demonstrate that
such methodology is promising for construction of biocata-
lysts. However, the elimination of non-specific binding of Rh
ion to other His residues seems to be fairly difficult.24,25 By
and large, the precise interaction and location of metal bind-
ing is rarely established.
amino acid alteration.
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RESULTS AND DISCUSSION
Incorporation of Osmium Ion into Well-defined Metal Bind-
ing Site of Apo-TM1459. TM1459 was over-expressed in E. coli
BL21(DE3) with the expression plasmid containing the rare
codon-modified TM1459 gene (Table S1-S2 and Figure S1-S3)
and isolated as homogenous state on SDS-PAGE (Figure S4).
Its metal content was determined to be less than 0.02 metal
ions (Mn, Fe, Co, Ni, Cu, Zn each) per protein subunits by the
Inductively Coupled Plasma-Atomic Emission Spectrometry
(ICP-AES) analysis, indicating that EDTA efficiently eliminated
the contamination of metal ions in the wash step of affinity
chromatography (see SI Experimental Section). The crystal
structure of as-isolated TM1459 (apo-TM1459) was deter-
mined at 1.20 Å resolution by the molecular replacement
method using the reported coordinate of Mn-TM1459 (Figure
1AB, PDB code: 1VJ2) as a search model (Table S3 and Figure
S5).27 The overall structure is nearly identical to the reported
structure (chain A of the Mn-bound form vs chain A of apo-
form, RMSD = 0.414 (98 C atoms)).27 No electron density
corresponding to metal ions was observed in consistent with
the results of ICP-AES analysis (Figure 2A).
In this study, we have succeeded to develop a nascent
mononuclear osmium enzyme with a cupin-type fold, where
well-defined amino acid residues for Os-coordination are dis-
posed in the small cone-shaped cleft of protein matrix. For
this purpose, we employed the TM1459 gene from Thermo-
toga maritima encoding a homodimeric Mn-binding protein
with a molecular weight of 12,977 Da (residues 1–114, Figure
1A).27 Currently, the biological function of Mn-bound TM1459
(Mn-TM1459) has yet to be identified, although it was found
to show the C=C bond cleavage activity in the presence of
alkylperoxide as oxidant in vitro.28 The metal binding site con-
sists of 4-histidine residues in a similar geometry to that of
the tris(2-pyridylmethyl)amine (TPA) ligand system (Figure
1BC), the Fe-complexes of which have been extensively stud-
ied as the model complexes of the active sites of nonheme
metalloenzymes.29 We have already achieved the efficient cis-
1,2-dihydroxylation reactions with alkene derivatives by using
Os-TPA complexes.30 The several alkene dihydroxylation sys-
tems of Os ion with the protein molecule as additives have
been reported.31–33 In all these cases, the turnover number
(TON) and the regio-selectivity for the substrate are still lim-
ited. Moreover, straightforward redesign of the well-ordered
catalytic- and substrate-binding site around metal center re-
mains unachieved, because of non-specific binding of Os ions
to protein surface as well.
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Figure 1. (A) The overall structure and (B) Mn center of TM1459
protein (PDB code: 1VJ2). (C) The schematic representation of
TPA-metal complex
Herein, in a spirit of “chemomimetic biocatalysis”,34,35 we
have developed an atom economic and selective cis-1,2-
dihydroxylation reaction system by using thermostable pro-
tein as the ligand instead of TPA. Os-cupin (TM1459) com-
plexes have characterized by X-ray crystallographic analysis at
atomic resolution and spectroscopic methods, and their sta-
bilities were confirmed by differential scanning calorimetry
(DSC) and circular dichroism (CD) spectroscopy. In order to
evaluate the chemical function of the Os-bound TM1459, the
alkene dihydroxylation reaction with H2O2 has been examined
in detail. Furthermore, the dihydroxylation reactivity of sev-
eral mutants has been investigated to clarify whether the
Figure 2. Closed view of the metal binding 4-histidine motif of
atomic resolution crystal structures of TM1459s. (A) Apo-form,
PDB code: 5WSD; (B) Form I, PDB code: 5WSE; (C) Form II, PDB
code: 5WSF; (D) Superimposed structure of 4-histidine motif of
three form (Apo vs Form I vs Form II). The protein main chain is
displayed as ribbon and key amino acid residues are highlighted
as sticks. 2Fo-Fc map, omit map (Fo-Fc), anomalous map con-
toured at 1.5, 4.0, and 8.0  are shown in gray, green, and ma-
genta mesh, respectively. Omit map was calculated around Os
and O center. Selected bonds, angles, and their estimated errors
(Table S3–S6): Form I (Panel B, chain A), Os–N (His52),
2.11(0.02) Å; Os–N (His58), 2.09(0.02) Å; Os–N (His92),
2.10(0.02) Å; Os–O1, 1.76(0.02) Å; Os–O2, 1.88(0.02) Å; Os–O3,
1.77(0.02) Å; O1–Os–O3 168.2(0.8); O2–Os–N (His92),
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and the imidazole of His54 rotated further away due to the
178.9(0.8), Form II (Panel C, chain C), Os–N (His52), 2.14(0.02)
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3
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Å; Os–N (His54), 2.11(0.04) Å; Os–N (His58), 2.10(0.02) Å; Os–
N (His92), 2.15(0.03) Å; Os–O1, 1.81(0.02) Å; Os–O2, 2.05(0.02)
Å; O1–Os–O2 89.4(1.1); O2–Os–N (His92), 175.9(1.2); O1–Os–
N(His54), 175.2(0.9).
steric repulsion with the Os-coordinating O3 (Figure 2D). The
coordinating three histidines have the metal-to-N distances of
2.11(0.02), 2.09(0.02), and 2.10(0.02) Å, respectively. Besides
these N donors (one imidazole of His54 is uncoordinated), the
Os center is ligated by three oxygen atoms taking an octahe-
dral geometry. Two oxygen atoms (O1 and O3) with shorter
Os–O distances (1.76(0.02) and 1.77(0.02) Å) and another
oxygen atom (O2) with longer Os–O distance (1.88(0.02) Å)
could be assigned to oxide and hydroxide ligands, respective-
ly. From structural analogy to the reported trans-dioxide-OsVI-
tetrapyrazole complex,36 the Os center in form I is likely to be
a trans-dioxide-OsVI(OH) species retaining its high-valency
after complexation with three imidazole. This is consistent
with the previous observation that the trans-dioxide-OsVI is
5
The apo-TM1459 was incubated with 1.2 equivalents of
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K2[OsVI(O)2(OH)4] for
4
days in 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) buffer at pH 7.0 and 70
C under N2. The color of the solution (purple) has changed to
yellow within 16 h and then gradually turned into reddish
brown, suggesting presence of an intermediate (Figure S6A).
In order to scrutinize this transformation, the yellow species
(form I) and the reddish brown species (form II) were collect-
ed after 16 h and 96 h incubation, respectively, and were
subjected to size-exchange chromatography to remove the
excess amount of Os ions. The metal content of each species
was determined to be 1.0  0.1 Os ions per protein subunits
by the ICP-AES analysis. The results suggested that both forms
I and II accommodate an Os ion in each subunit. Then, the
valence state and the coordination structures were examined
as follows. Form I exhibited an absorption spectrum with
poorly resolved shoulder peaks between 300 and 450 nm,
whereas form II showed an absorption band at 373 nm (or-
ange and red spectra, respectively, in Figure 3A). Further-
more, it’s noteworthy to note that both form I and form II
exhibited the broad ESR signal centered at g = 2.1 (Figure
S6B). Whereas form I exhibited several additional minor sig-
nals was observed, there was no such signal in the case of
form II (red spectrum in Figure S6B).
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much more stable as compared to the cis-dioxide-OsVI 37
.
In stark contrast, in form II crystal, the Os center is support-
ed by four histidine residues (His52, 54, 58, and 92) with met-
al-to-N distances of 2.14(0.02), 2.11(0.03), 2.10(0.02), and
2.15(0.03) Å, respectively (Figure 2C). Thus, the imidazole ring
of His 54 flipped toward the Os center (O3 position in the
form I crystal) and rotated away from the mouth of cavity in
the crystals of form I and apo-form (Figure 2D). Further struc-
tural inspection lead to identification of the two labile sites in
a cis-position occupied by two oxygen atoms, taking octahe-
dral geometry. According to the bond length (2.05(0.02) and
1.81(0.02) Å), one oxygen atom (O2) could be water molecule
and the other one (O1) could be hydroxide ion. This coordina-
tion geometry is reminiscent of the structure of
[OsIII(H2O)(OH)(TPA)]2+ complex, UV-vis spectrum of which
shows the peak at 350 nm, that is similar to that of form II
(373 nm, Figure 3A).30 In the reported Mn-bound structure,
the distances of water molecules to Mn ion are 2.24 (Mn–O1)
and 2.06 (Mn–O2) Å, which are significantly longer than that
of Os–O1 in form II (Figure S7E), supporting the assignment of
O1 as an hydroxide oxygen.27 Taken together, Os ion initially
attaches to the three His imidazoles (His52, 58, and 92) with
keeping the OsVI oxidation state and the trans-dioxide geome-
try (form I, Scheme 1B). Then both oxide oxygens as well as
OsVI are reduced during the incubation at 70 C for 4 days to
OsIII, hydroxide, and water molecule, respectively, and ligand
replacement occurs concomitantly (form II, Scheme 1C). To
avoid confusion of these two forms (form I and II), hereafter,
form II (reddish brown species) is called as Os-TM1459.
Figure 3. (A) UV-vis spectra of 0.1 mM apo-form (black), form I
(orange), and form II (red) in 10 mM potassium phosphate buffer
at pH 7.0 and room temperature. Inset: Extended UV-vis spectra
of apo-form (black), form I (orange), and form II (red). (B) Calori-
metric scans of apo-form (black), Mn-bound form (green), and
Os-bound form (red) in 50 mM potassium phosphate buffer at pH
7.0.
Scheme 1. Schematic Representation of [OsVI(O)2(OH)4]2+
Incorporation in Apo-form of TM1459 Protein
Crystal Structures of Osmium-incorporated TM1459. For fur-
ther information about form I and form II, the crystal struc-
tures of both Os-bound forms were determined at atomic
resolution of 1.12 and 1.11 Å, respectively (Figure 2, Figure S7
and Table S3–S6). The overall structures of form I and form II
are almost identical to that of the apo-form (Figure S7AB). In
each chain of both form I and form II, one intense anomalous
peak due to Os ion was found around the N atoms of His52,
58, and 92 (Figure 2BC, magenta mesh). In the form I crystal,
the positions of imidazoles of His58 and His92 deviated from
those of the apo-form because of the coordination to Os ion,
Stability of Os-TM1459. The stability of apo-, Mn- and Os-
TM1459 is examined by DSC analysis (Figure 3B). Apo-, Mn-,
and Os-TM1459 exhibited the several overlapping transitions.
Since all of these forms didn’t show any reversible profiles
due to the corresponding refolding transitions in the reverse
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scan, it was difficult to determine detailed thermodynamic
parameters. Thus, the maximum peaks at 87, 93, and 117 C
were used for rough estimation of the apparent denaturation
temperature (Tm) of apo-, Mn-, and Os-TM1459, respectively
(Figure 3B, arrows). The observed peak shifts toward higher
temperature indicated that the protein folding was thermally
stabilized upon the metal binding to the apo-form. The value
of  Tm (apparent denaturation temperature difference be-
tween the metal-binding form and the apo-form) of Mn-
TM1459 and Os-TM1459 are 6 and 30 C, respectively. These
 Tm values reflect the strength of the metal binding as is
reported in the case of transferrin and cupredoxin.21,38 The 
Tm value of Os-TM1459 (30 C) is much higher than that of
Mn-TM1459 (6 C), suggesting that the binding of Os ion to
the protein is much stronger than that of Mn ion. Indeed, Os-
N (imidazole) bonds (average 2.11 Å) in Os-TM1459 are fairly
shorter than Mn-N (imidazole) bonds (average 2.21 Å) in
Mn-TM1459.
such ill-suited conditions for “protein” (50 % organic solvent
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and acidic pH, Figure S8B). Most of enzymes are hard to be
used in such a harsh conditions. Additionally, the enantiose-
lectivity is one of the most important issues of enzymatic
reactions. Unfortunately, however, enantioselectivity was
hardly observed in the present cis-dihydroxylation reaction. In
a
docking simulation experiment using 2-methoxy-6-
vinylnaphthalene as a substrate, we found two low-energy
conformations with nearly the same energy (Figure S9AB).
This indicates that the wild type Os-TM1459 may not be able
to distinguish the Si and Re faces of the olefin substrates due
to the relatively large cavity size of this protein.
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Taking the proposed reaction mechanism of Os-TPA for di-
hydroxylation of alkene into account,30 a possible catalytic
mechanism by Os-TM1459 is shown in Scheme 2. First, 1 (Os-
TM1459) is oxidized with 1 equiv of H2O2 generating the ac-
tive species, oxo-hydroxo-Os(V) complex 2, which reacts with
alkene via a concerted [3+2]-cycloaddition mechanism to
produce a five-membered glycolato-Os(III) intermediate 3.
Finally, the hydrolyzation of 3 provides the starting complex 1
as well as the corresponding diol product, completing the
catalytic cycle.
For native metalloproteins, a representative  Tm value has
been reported as 8 C for ZnII-carbonic anhydrase.39 Even
though higher values around 20 C have been known for CuII-
cupredoxin40 and FeIII-transferrin,38 the  Tm values were fur-
ther increased upon replacing by xenobiotic HgII and CdII (29
and 31C, respectively).21 The result could be explained by the
fact that soft donor atoms such as sulfur of Cys and Met bind
to softer metal ions such as HgII and CdII stronger than harder
metal ions such as CuII and FeIII. On the other hand, the large
 Tm value of Os-TM1459 (30 C) can be attributed to the
strong Os–N (imidazole) bonding due to strong back electron
donation from the Os center to the imidazole -orbital as in
the case of OsIII-TPA complex.41
Catalytic cis-1,2-Dihydroxylation of Alkenes in a Preparative
Scale. With intent to explore the catalytic performance of Os-
TM1459 for organic synthesis, the reactions were examined
in a preparative scale using various substrates (Table 1).
Yields and stereochemistry of the diol products were deter-
1
mined by H NMR by comparing that of authentic samples.
Styrene and -methylstyrene were efficiently converted to
the corresponding diols in good yields (entries 1 and 2). The
product yields somewhat decreased in the reaction of -
methylstyrene (trans and cis) and 2-methyl-1-phenylpropene
(entries 3–5). It should be noted that only the threo and
erythro products were obtained from the trans- and cis-
styrene derivatives, respectively, suggesting that the reac-
tions proceeded completely through syn-addition. When a
styrene with even electron-withdrawing substituent (methyl
cinnamate) was employed, the yield still retained a good level
(entry 6).
Besides the high thermal stability, chemical stability of Os-
TM1459 was also improved as confirmed by the CD meas-
urements under various conditions. Namely, the contents of
secondary structure of Os-TM1459 hardly changed under
both acidic and alkaline conditions (pH 2–10) even in the
presence of high concentration of organic solvents (50 % v/v
t-butylalchol (t-BuOH), Figure S8).
Peroxygenase Activity of TM1459. The catalytic activity of
Os-TM1459 was examined for the alkene dihydroxylation
using 2-methoxy-6-vinylnaphtalene (final, 10 mM) as a sub-
strate. The reaction condition was optimized by detecting the
diol product with reverse-phase HPLC (Table S7). The dihy-
droxylation reaction proceeded very efficiently under an acid-
ic conditions (pH 2.0, at 70 °C for 5 h, Table S7) to give the
corresponding diol (LC yield, 92 %) together with a small
amount of over-oxidation product, aldehyde. By decreasing
the catalytic amount of Os-TM1459 to 0.01 mol % and in-
creasing the substrate concentration to 50 mM, a high TON of
9100 was obtained after 12 h (Table S8), indicating the ro-
bustness of the catalyst (LC yield, 91 %). The TON are compa-
rable or even higher than those of the cis-dihydroxylation
reaction catalyzed by the Os-complexes; the Os-TPA complex
system30 and the Os-N4 complex sysetm (N4 = N,N’-dimethyl-
2,11-diaza[3.3](2,6)pyridinophane)42 showed maximum TON
of 2500 and 5500 in cyclohexene dihydroxylation, respective-
ly. Thus, the results clearly illustrate that Os-TM1459 acts as a
peroxygenase with the high catalytic performance even under
Scheme 2. Proposed Reaction Mechanism for the Catalytic
Dihydroxylation of Alkenes by Os-TM1459
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However, much sharper substrate selectivity was observed in
1
the case of Os-TM1459. Namely, the diol yield drastically de-
creased as the position of C=C double bond changed from 1-
alkene to 4-alkene (7524 %, Table 2, entries 1–4). Further-
more, such a simple tendency of substrate reactivity (1-
octene > 2-octene > 3-octence > 4-octene) was not observed
even in the case of Os-TPA system (1-octene  2-octene > 3-
octence  4-octene, Table S9). Thus, Os-TM1459 system
(7524 % (3.1 fold) from 1- to 4-alkene) exhibited the sharp-
er preference than Os-TPA system (9055 %, 1.6 fold), sug-
gesting that the steric effect is predominant in the case of Os-
TM1459. In support of this notion, Os-TM1459 system pre-
ferred less sterically hindered cis-derivative to trans-
derivative (58 vs 24 % (2.4 fold) for cis vs trans-4-octene)
more prominently than Os-TPA system (74 vs 55 % (1.3 fold)).
These results clearly demonstrated that the protein matrix
magnifies the substrate selectivity in the Os-catalyzed cis-
dihydroxylation reaction. This has been also confirmed by the
docking simulation (Figure S9CD).
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Table1. Catalytic cis-Dihydroxylation of Styrene Derivatives
Table2. Catalytic cis-Dihydroxylation of n-Octene (n = 1, 2, 3, 4)
1H NMR
1H NMR
entry
1b
substrate
product
yield (%)a
entry
1b
substrate
product
yield (%)a
78
90
59
68
53
55
75
2
3
4
5
6
2
3
4
5
6
52
33
24
79
58
a 1H NMR yield based on the substrate. b Reaction conditions: Os-
TM1459 (2 mol%), substrate (25 mM), and H2O2 (25 mM) in 40
mM Britton-Robinson buffer /t-BuOH = 1/1 (v/v) at 70 C for 5 h.
a 1H NMR yield based on the substrate. b Reaction conditions: Os-
TM1459 (2 mol%), substrate (25 mM) and H2O2 (25 mM) in 40
mM Britton-Robinson buffer / t-BuOH= 1/1 (v/v) at 70 C for 5 h.
The yields of diol products were diminished by the substitu-
tion on -position of styrene (Table 1). These results suggest
that the steric effect induced by the protein matrix appears to
govern the reaction efficiency rather than electronic effect.
To confirm this, the reactions were conducted by using oc-
tene derivatives as substrate. In this case as well, only the
threo and erythro products were obtained from the trans-
and cis-octene derivatives, respectively (Table 2). It has been
reported that simple OsO4/H2O2 system exhibited the reac-
tion yields that could be controlled via electronic effect (18
and 34 % for trans-2-octene and trans-4-octene, respectively,
Table S9),43 whereas Os-TPA showed the opposite tendency
(83 and 55 % for trans-2-octene and trans-4-octene, respec-
tively) due to the steric effects of the TPA ligand (Table S9).30
It has been reported that the diol yield decreased drastical-
ly, if H2O2 is added to the reaction solution at once in the case
of Fe-complex44 as well as Os-TPA.30 Thus, to obtain higher
product yields, H2O2 should be added slowly using a syringe
pump or dropwise iteratively. This is presumably due to a
catalase type disproportionation of H2O2 to O2 and H2O cata-
lyzed by the metal center involved in the catalytic cycle.
However, such a yield decrement was not observed in the Os-
TM1459 system. Namely, the steric effect of protein scaffold
and/or the interaction with the second coordination sphere
of the protein matrix are probably responsible for the sup-
pression of catalase-like activity by preventing the inter-
molecular decomposition of active species with H2O2 (Figure
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S10). Such a hydrogen-bonding network in the second coor-
Page 6 of 8
CONCLUSION
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dination sphere of protein matrix (Figure 4A), on the other
hand, may prohibit substrate access to the Os-center, sup-
pressing the intrinsic reactivity of the reactive oxidant. There-
fore, the site-directed mutagenesis into the surrounding ami-
no acid should lead to the improvement of catalytic activity.
In this work, TM1459 cupin superfamily protein from T.
maritima has been demonstrated to be a suitable scaffold for
developing an artificial metalloenzyme. It should be noted
that cupins belong to one of the most functionally diverse
protein superfamily,47 interestingly, in which many metallo-
proteins have occurred and the various sorts of first coordina-
tion spheres have been identified.48 Therefore, many cupins
will be highlighted as hopefully versatile ligands. In addition,
TM1459 has no additional histidines except metal-
coordinating four histidines. As a result, any non-specific
binding of Os ion to this protein was not seen in ICP-AES and
X-ray crystallographic analysis, and Os-TM1459 is able to
catalyze cis-dihydroxylation of alkenes with the high TON.
Constructing a well-defined complex of Os with protein led to
the enhancement of thermal stability of protein folding and
allowed us structure-based improvement of the catalytic ac-
tivity retaining the substrate selectivity. Such enzyme repur-
posing strategy (metal substitution based on the metal bind-
ing promiscuity) can be confidently expanded as a promising
means to explicit the power of platinum group elements as
well as to acquire the robust enzyme scaffold. The compatibil-
ity with the improvement of catalytic activity based on the
site-directed mutagenesis suggests that directed evolution
strategies might be used to further optimize its performance.
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Figure 4. (A) Amino acid residues and hydrogen bonds around Os
center in Os-TM1459. The protein main chain is displayed as
white ribbon and key amino acid residues are highlighted as
sticks. Hydrogen bonds are indicated in dotted green lines. (B)
Relative catalytic activity (turnover frequencies (TOF)) of R39A,
F41A, C106A, and I108A mutants. (C) Comparison of relative
activity among C106X mutants.
Improvement of Peroxygenase Activity of Os-TM1459 by
Site-targeted Mutagenesis. In order to examine the involve-
ment of the neighboring amino acids in the catalysis, brief
alanine-scanning mutagenesis was conducted toward the
amino acids surrounding the Os center (Arg39, Phe41,
Cys106, and Ile108, Figure 4AB, and Figure S11 and S12). The
kinetic analysis on the dihydroxylation of 2-methoxy-6-
vinylnaphtalene catalyzed by the wild type (WT) and these
four mutants were performed by following the fluorescence
of 2-methoxy-6-naphthalaldehyde derived by the post-
treatment of the diol product with NaIO4. Such property will
provide us the simple method in screening the catalytic activi-
ty of TM1459 variants.45,46. The amount of the product in-
creased linearly during the initial several hours and then
gradually slowed down to reach a plateau after 24 h (Figure
S13). Then, the relative catalytic activity was evaluated using
TOF determined from the initial straight line portion of the
plot. As shown in Figure 4B, F41A and I108A mutants exhibit-
ed nearly the same reactivity, whereas R39A showed about
50 % activity when compared with WT. These results are in-
dicative of the positive influence of the hydrogen bond net-
work (Figure 4A). Concerning Cys106, C106A mutant showed
1.5-fold higher activity, probably because oxidized thiol group
(sulfinate/sulfonate) cause a negative influence (see Figure S4
and S5). Thus, this position 106 was selected as a point for
further site-directed mutagenesis investigation. The results
are summarized in Figure 4C. Except the two mutants, C106R
and C106P, which formed inclusion bodies, C106X mutants
showed comparable to higher catalytic activity as compared
to WT. Especially, the TOF values of C106E, C106L, and C106S
were larger than that of WT by 2.5-, 2.5-, and 3-fold, respec-
tively. Furthermore, C106S mutant showed similar substrate
selectivity to that of WT in the preparative scale reaction (Ta-
ble S9). This supports that enhancement of the intrinsic reac-
tivity of the Os center could be possible without the influence
on the interaction with olefin substrate by site-directed mu-
tagenesis.
ASSOCIATED CONTENT
Supporting Information
Experimental section; supporting figures and tables (PDF). The
Supporting Information is available free of charge via the Inter-
net at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
fujieda@mls.eng.osaka-u.ac.jp (NF) and shinobu@mls.eng.osaka-
u.ac.jp (SI)
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manu-
script.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
N.F. thanks JSPS and MEXT, Japan (JSPS KAKENHI Grant Number
JP15K13743 and MEXT KAKENHI Grant Number JP15H01066 in
Hadean Bioscience, JP16H01025 in Precisely Designed Catalysts
with Customized Scaffolding). S.I. thanks JST (the CREST program,
161052502). We thank Dr. K. Oohora and Dr. T. Hayashi for their
kind assistance with ESI-MS, and Dr. E. Yamashita, Dr. A. Hi-
gashiura, and Dr. A. Nakagawa of SPring-8 BL-44XU for their sup-
port crystallographic data collection. This work was performed in
part using a synchrotron beamline BL44XU at SPring-8 under the
Cooperative Research Program of Institute for Protein Research,
Osaka University. Diffraction data were collected at the Osaka
University beamline BL44XU at SPring-8 (Harima, Japan) under
proposal numbers 2014A6500, 2014B6500, 2015A6500, and
2015B6500.
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(27) Jaroszewski, L.; Schwarzenbacher, R.; von Delft, F.; McMullan, D.;
ABBREVIATIONS
Brinen, L. S.; Canaves, J. M.; Dai, X.; Deacon, A. M.; DiDonato, M.;
Elsliger, M.-A.; Eshagi, S.; Floyd, R.; Godzik, A.; Grittini, C.;
Grzechnik, S. K.; Hampton, E.; Levin, I.; Karlak, C.; Klock, H. E.;
Koesema, E.; Kovarik, J. S.; Kreusch, A.; Kuhn, P.; Lesley, S. A.;
McPhillips, T. M.; Miller, M. D.; Morse, A.; Moy, K.; Ouyang, J.;
Page, R.; Quijano, K.; Reyes, R.; Rezezadeh, F.; Robb, A.; Sims, E.;
Spraggon, G.; Stevens, R. C.; van den Bedem, H.; Velasquez, J.;
Vincent, J.; Wang, X.; West, B.; Wolf, G.; Xu, Q.; Hodgson, K. O.;
Wooley, J.; Wilson, I. A. Proteins Struct. Funct. Bioinforma. 2004,
56, 611.
1
2
3
4
5
6
7
8
T. maritima, Thermotoga maritima; E. coli, Esherichia coli; t-
BuOH, tertiary-butylalcohol; TPA, tris(2-pyridylmethyl)amine;
DSC, differential scanning calorimetry; CD, circular dichroism,
HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; ICP-
AES, Inductively Coupled Plasma-Atomic Emission Spectrometry;
WT, wild type.
REFERENCES
9
(1)
Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.;
Moore, J. C.; Robins, K. Nature 2012, 485, 185.
(28) Hajnal, I.; Faber, K.; Schwab, H.; Hall, M.; Steiner, K. Adv. Synth.
Catal. 2015, 357, 3309.
(29) Que, L.; Tolman, W. B. Nature 2008, 455, 333.
(30) Sugimoto, H.; Kitayama, K.; Mori, S.; Itoh, S. J. Am. Chem. Soc.
2012, 134, 19270.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(2)
(3)
Ward, T. R. Chem. Eur. J. 2005, 11, 3798.
Boersma, A. J.; Megens, R. P.; Feringa, B. L.; Roelfes, G. Chem. Soc.
Rev. 2010, 39, 2083.
(4)
(5)
Park, S.; Sugiyama, H. Angew. Chemie Int. Ed. 2010, 49, 3870.
Köhler, V.; Wilson, Y. M.; Lo, C.; Sardo, A.; Ward, T. R. Curr. Opin.
Biotechnol. 2010, 21, 744.
(31) Kokubo, T.; Sugimoto, T.; Uchida, T.; Tanimoto, S.; Okano, M. J.
Chem. Soc., Chem. Commun. 1983, 769.
(32) Köhler, V.; Mao, J.; Heinisch, T.; Pordea, A.; Sardo, A.; Wilson, Y.
M.; Knörr, L.; Creus, M.; Prost, J.; Schirmer, T.; Ward, T. R. Angew.
Chemie Int. Ed. 2011, 50, 10863.
(33) Konieczny, S.; Leurs, M.; Tiller, J. C. ChemBioChem 2015, 16, 83.
(34) Meeuwissen, J.; Reek, J. N. H. Nat. Chem. 2010, 2, 615.
(35) Prier, C. K.; Arnold, F. H. J. Am. Chem. Soc. 2015, 137, 13992.
(36) Mayboroda, A.; Rheinwald, G.; Lang, H. Inorganica Chim. Acta
2003, 355, 69.
(6)
(7)
Heinisch, T.; Ward, T. R. Curr. Opin. Chem. Biol. 2010, 14, 184.
Yu, F.; Cangelosi, V. M.; Zastrow, M. L.; Tegoni, M.; Plegaria, J. S.;
Tebo, A. G.; Mocny, C. S.; Ruckthong, L.; Qayyum, H.; Pecoraro, V.
L. Chem. Rev. 2014, 114, 3495.
(8)
(9)
Bos, J.; Roelfes, G. Curr. Opin. Chem. Biol. 2014, 19, 135.
Matsuo, T.; Hirota, S. Bioorg. Med. Chem. 2014, 22, 5638.
(10) Hoarau, M.; Hureau, C.; Gras, E.; Faller, P. Coord. Chem. Rev. 2016,
308, 445.
(37) Dobson, J. C.; Takeuchi, K. J.; Pipes, D. W.; Geselowitz, D. A.;
(11) Pàmies, O.; Diéguez, M.; Bäckvall, J.-E. Adv. Synth. Catal. 2015,
357, 1567.
(12) Jeschek, M.; Reuter, R.; Heinisch, T.; Trindler, C.; Klehr, J.; Panke,
S.; Ward, T. R. Nature 2016, 537, 661.
(13) Key, H. M.; Dydio, P.; Clark, D. S.; Hartwig, J. F. Nature 2016, 534,
534.
(14) Dydio, P.; Key, H. M.; Nazarenko, A.; Rha, J. Y.-E.; Seyedkazemi, V.;
Clark, D. S.; Hartwig, J. F. Science 2016, 354, 102.
(15) Ueno, T.; Tabe, H.; Tanaka, Y. Chem. Asian J. 2013, 8, 1646.
(16) Lewis, J. C. Curr. Opin. Chem. Biol. 2015, 25, 27.
(17) Song, W. J.; Tezcan, F. A. Science 2014, 346, 1525.
(18) Fujieda, N.; Schätti, J.; Stuttfeld, E.; Ohkubo, K.; Maier, T.;
Fukuzumi, S.; Ward, T. R. Chem. Sci. 2015, 6, 4060.
(19) Bornscheuer, U. T.; Kazlauskas, R. J. Angew. Chemie Int. Ed. 2004,
43, 6032.
(20) Pordea, A. Curr. Opin. Chem. Biol. 2015, 25, 124.
(21) Engeseth, H. R.; McMillin, D. R. Biochemistry 1986, 25, 2448.
(22) Okrasa, K.; Kazlauskas, R. J. Chemistry 2006, 12, 1587.
(23) Fernández-Gacio, A.; Codina, A.; Fastrez, J.; Riant, O.; Soumillion, P.
Chembiochem 2006, 7, 1013.
Meyer, T. J. Inorg. Chem. 1986, 25, 2357.
(38) Mizutani, K.; Mikami, B.; Aibara, S.; Hirose, M. Acta Crystallogr.
Sect. D Biol. Crystallogr. 2005, 61, 1636.
(39) Lisi, G. P.; Hughes, R. P.; Wilcox, D. E. JBIC J. Biol. Inorg. Chem.
2016, 21, 659.
(40) Ma, J. K.; Bishop, G. R.; Davidson, V. L. Arch. Biochem. Biophys.
2005, 444, 27.
(41) Sugimoto, H.; Matsunami, C.; Koshi, C.; Yamasaki, M.; Umakoshi,
K.; Sasaki, Y. Bull. Chem. Soc. Jpn. 2001, 74, 2091.
(42) Sugimoto, H.; Ashikari, K.; Itoh, S. Chem. Asian J. 2013, 8, 2154.
(43) Bergstad, K.; Jonsson, S. Y.; Bäckvall, J.-E. J. Am. Chem. Soc. 1999,
121, 10424.
(44) Chen, M. S.; White, M. C. Science 2007, 318, 783.
(45) List, B.; Barbas, C. F.; Lerner, R. a. Proc. Natl. Acad. Sci. 1998, 95,
15351.
(46) Wahler, D.; Badalassi, F.; Crotti, P.; Reymond, J. Chem. Eur. J. 2002,
8, 3211.
(47) Dunwell, J. M.; Purvis, A.; Khuri, S. Phytochemistry 2004, 65, 7.
(48) Chavez, F.; Banerjee, A.; Sljivic, B. Modeling the metal binding site
in cupin proteins, On Biomimetics; Pramatarova, L., Ed.; InTech:
Rijeka, 2011.
(24) Jing, Q.; Okrasa, K.; Kazlauskas, R. J. Chem. Eur. J. 2009, 15, 1370.
(25) Jing, Q.; Kazlauskas, R. J. ChemCatChem 2010, 2, 953.
(26) Key, H. M.; Clark, D. S.; Hartwig, J. F. J. Am. Chem. Soc. 2015, 137,
8261.
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