Comparison of acyl-CoA synthetic activities and enantioselectivity toward 2-arylpropanoic acids in firefly luciferases

Article abstract of DOI:10.1271/bbb.110299

Measurement of thioesterification activities for dodecanoic acid (C12) and ketoprofen was done using five firefly luciferases, from Pyrocoelia miyako (PmL), Photinus pyralis (PpL), Luciola cruciata (LcL), Hotaria parvura (HpL), and Luciola mingrelica (LmL). Among these, PmL, PpL, and LcL showed the expected thioesterification activities toward both substrates. All the enzymes exhibited (R)-enantioselectivity toward ketoprofen, which had same tendency as firefly luciferase from Luciola lateralis (LUC-H). HpL and LmL, however, did not accept ketoprofen, although they had thioesterification activity toward C12. These results indicate that the substrate acceptance of luciferases for the thioesterification reaction varies dramatically relying on the origin of firefly. Hence we focused primarily on PmL and investigated the effect of pH on enzymatic activity. In addition, by determining the kinetic parameters at various pH values, we verified that the kcat parameter contributed to the preferential enantioselectivity of this enzyme.

Full text of DOI:10.1271/bbb.110299

Biosci. Biotechnol. Biochem., 75 (9), 1758–1762, 2011
Comparison of Acyl-CoA Synthetic Activities and Enantioselectivity
toward 2-Arylpropanoic Acids in Firefly Luciferases
y
Dai-ichiro KATO, Keisuke YOKOYAMA, Yoshihiro HIRAISHI, Masahiro TAKEO, and Seiji NEGORO
Department of Materials Science and Chemistry, Graduate School of Engineering, University of Hyogo,
2
167 Shosha, Himeji, Hyogo 671-2280, Japan
Received April 14, 2011; Accepted May 27, 2011; Online Publication, September 7, 2011
[doi:10.1271/bbb.110299]
Measurement of thioesterification activities for
whether other firefly luciferases can catalyze the
enantioselective thioesterification reaction. Here we
report the enantioselectivity of the acyl-CoA synthesis
of ketoprofen in other five firefly luciferases, from
Pyrocoelia miyako (PmL), Photinus pyralis (PpL),
Luciola cruciata (LcL), Hotaria parvura (HpL), and
Luciola mingrelica (LmL). In addition, we report on the
effect of pH on enzymatic activity and kinetic param-
eters determined using PmL.
dodecanoic acid (C12) and ketoprofen was done using
five firefly luciferases, from Pyrocoelia miyako (PmL),
Photinus pyralis (PpL), Luciola cruciata (LcL), Hotaria
parvura (HpL), and Luciola mingrelica (LmL). Among
these, PmL, PpL, and LcL showed the expected
thioesterification activities toward both substrates. All
the enzymes exhibited (R)-enantioselectivity toward
ketoprofen, which had same tendency as firefly lucifer-
ase from Luciola lateralis (LUC-H). HpL and LmL,
however, did not accept ketoprofen, although they had
thioesterification activity toward C12. These results
indicate that the substrate acceptance of luciferases for
the thioesterification reaction varies dramatically rely-
ing on the origin of firefly. Hence we focused primarily
on PmL and investigated the effect of pH on enzymatic
activity. In addition, by determining the kinetic param-
eters at various pH values, we verified that the kcat
parameter contributed to the preferential enantioselec-
tivity of this enzyme.
Materials and Methods
The pTrc-PmL and the pTrc-HpL plasmids, which code the cDNA
of PmL and HpL, were kindly provided by Professor Yoshihiro
Ohmiya of the National Institute of Advanced Industrial Science and
Technology (AIST).11) LmL and PpL were purchased from Sigma-
Aldrich (USA). LcL was from Wako Pure Chemicals (Japan). (R)-
12)
Ketoprofen was prepared according to a previously reported method.
Other chemicals were purchased from reputable vendors and were used
without further purification, unless otherwise noted. Protein concen-
trations were measured by Bradford Assay with bovine serum albumin
as standard.13)
Key words: firefly luciferase; enantioselective thioester-
ification; 2-arylpropanoic acid; kinetic res-
olution; Pyrocoelia miyako
Plasmid construction and expression of PmL and HpL. The
expression vectors for PmL and HpL were designed to produce N-
terminal His-Tag fused proteins (MNHKVHHHHHHIGGRH). The
PmL gene was amplified from pTrc-PmL by PCR using forward
(TTTGACATATGGAAGATGATAGTAAACATATTATGC) and re-
verse (GTTAGTCTAGAGTAAACCGAAGAAATTACA) primers. The
cDNA of the HpL gene was also amplified using forward (TTTGA-
CATATGGAAATGGAAAAGGAGGA) and reverse (TAGCATCTA-
GACCGATTTACATCTTG) primers. These primers were designed to
contain artificially introduced Nde I and Xba I cleavage sites (italic
Firefly luciferase is a bioluminescence enzyme that
catalyzes the oxidation of D-luciferin using molecular
oxygen in the presence of ATP and Mg2
þ
1–3)
.
Its amino
acid sequence exhibits high similarity to that of long-
chain acyl-CoA synthetase (LACS), and the firefly
luciferase reaction mechanism shares some features in
0
0
form) in the 5 and 3 ends of the PmL and the HpL gene respectively.
The underlined ATG represents the start codon. The amplified 1.7-kbp
fragment was subcloned into TA vector, which was prepared from
pXCmkn12 digested with Xcm I (National Institute of Genetics, Japan).
First the subcloned vectors were digested with Nde I and Xba I. Then
they were purified and inserted into a pCold I expression vector
4
)
common with the LACS mechanism. Based on this
similarity, Oba et al. have suggested that firefly
luciferases from Luciola cruciata (LcL) and Photinus
pyralis (PpL) have the same catalytic ability as for acyl-
CoA synthesis toward long-chain fatty acids such as
(Takara, Japan). The resulting plasmids, pColdI-PmL and pColdI-HpL,
were used to express the recombinant proteins.
5
)
dodecanoic acid (C12), and this has been verified. On
the other hand, LACS is also known to be involved in a
deracemization reaction and to catalyze the enantiose-
lective thioesterification reaction toward 2-arylpropanoic
E. coli BL21 (DE3) cells were transformed with pColdI-PmL or
pColdI-HpL, cultivated, and treated to express proteins following the
manufacturer’s instructions. After production of the recombinant
enzymes, the cells were collected, lysed by sonication (20 kHz, 30 s ꢀ
acids.6,7)
In this way, we found that Japanese firefly
luciferases from Luciola lateralis (LUC-H) had (R)-
1
0 times) in 5 mL of 50 mM potassium phosphate buffer (PPB)
(
pH 7.0) containing 300 mM NaCl, and centrifuged (14;500 ꢀ g for
enantioselective thioesterification ability when the sub-
8
ꢁ
1
0 min, 4 C). The enzymes were purified from the supernatant using a
,9)
strates were a series of 2-arylpropanoic acids (Fig. 1).
In nature, there are many kinds of firefly lucifer-
ases.10) Hence we were interested in determining
TALON Metal Affinity Resin (2 mL) (Clontech) following the
manufacturer’s instructions. Following purification, the enzymes were
identified by SDS–PAGE, and protein bands were visualized by
y
To whom correspondence should be addressed. Tel/Fax: +81-79-267-4969; E-mail: kato@eng.u-hyogo.ac.jp
Abbreviations: C12, dodecanoic acid; CoASH, coenzyme A; HpL, firefly luciferase from Hotaria parvula; LcL, firefly luciferase from Luciola
cruciata; LmL, firefly luciferase from Luciola mingrelica; LUC-H, firefly luciferase from Luciola lateralis; PPB, potassium phosphate buffer; PmL,
firefly luciferase from pyrocoelia miyako; PpL, firefly luciferase from Photinus pyralis

Enantioselective Thioesterification Activity in Luciferases
1759
O
O
CH3
CO H
O
CH3
COSCoA
Fast
2
LUC-H
ATP, Mg2+ , CoASH
(
R)-acid
(R)-thioester
CH3
CO H
O
CH3
COSCoA
Slow
2
(
S)-acid
(S)-thioester
Fig. 1. LUC-H-Catalyzed Enantioselective Thioesterification toward Ketoprofen.
staining with Coomassie Blue. The fractions containing luciferase were
combined and dialyzed overnight against a 100 mM Tris–HCl buffer
Table 1. Comparison of Properties of the Firefly Luciferases Cata-
lyzed Thioesterification Reaction toward Dodecanoic Acid and (ꢂ)-
Ketoprofen
(pH 7.5).
Relative activity of
thioesterification (%)
Spectrum measurement. Spectrum measurement was performed
using an AB-1850 LumiFL-Spectrocapture (Atto) (slit width, 1 mm;
Identity
%)c
ꢀmax
Luciferase
d
(
(nm)
spectral resolution, 0.5 nm) for 5–30 s immediately after mixing 2 mL
Dodecanoic
acid (C12)a
Ketoprofenb
of luciferase enzymes and 100 mL of substrate solution containing
.
0
.5 mM D-luciferin, 3 mM ATP-2Na, 8 mM MgSO4 7H2O, and 0.1 M
(552)e
LUC-H
LcL
70
74
100
98
100
93
64
66
60
60
Tris–HCl (pH 8.0) in a 0.2-mL PCR tube. All spectra were corrected
for the spectral sensitivity of the equipment and normalized.
563
552
558
568
568
PmL
PpL
100
95
63
58
Thioesterification activity assays. The activities of the thioester-
ification reaction were measured using three assay systems: a simple
extraction assay,8) a HPLC assay,8) and a spectrophotometric assay,9)
following published procedures.
HpL
LmL
22
20
N.D.
N.D.
N.D., not detected. Relative activity could not be calculated because the
specific activity was below the limit of detection (<0:01 nmol/min/mg).
aThe relative activities shown are relative to the value of PmL (64.0 nmol/
min/mg), set at 100.
bThe relative activities shown are relative to the value of LUC-H
(20.5 nmol/min/mg), set at 100.8)
cIdentity was determined using the amino acid sequence of LUC-H as
standard.
dMeasurement was performed in 100 mM Tris–HCl (pH 8.0).
eThis value is quoted from the literature.24)
Characterization of the PmL and the HpL catalyzed thioesterifica-
tion reactions. The substrate specificities of PmL and HpL were
analyzed by spectrophotometric assay.9) The final concentration of the
substrate was 0.25 mM.
The effect of pH on the thioesterification activity was determined
using 0.25 mM (ꢂ)-ketoprofen in the following 100-mM buffers: citric
acid-sodium citrate buffer (pH 3.0–4.0), succinate-KOH buffer
(pH 4.0–5.5), MES-KOH buffer (pH 5.5–6.0), PPB (pH 6.0–8.5),
Tris–HCl buffer (pH 8.0–10.0), and sodium carbonate-sodium hydro-
gen carbonate buffer (pH 10.0–11.0). Each assay was done 3 times,
and the initial velocity was determined using the spectrophotometric
assay at each pH.9) Relative activity was calculated from a comparison
of these velocities.
enantiodifferentiation activity in LUC-H when ketopro-
9
)
fen was subjected to this enzyme. Hence in this study,
to determine whether other firefly luciferases have
thioesterification activity toward C12 and ketoprofen,
activities were measured using the other five firefly
luciferases, PmL, PpL, LcL, HpL, and LmL (Table 1).
The thioesterification activity toward C12 was detected
in all luciferases. As for ketoprofen, however, HpL and
LmL did not recognize. The other three firefly lucifer-
ases, PmL, PpL, and LcL, showed the expected
activities, and the enantioselectivity of these enzymes
was found to be R. These results are identical to the
case of LUC-H. The specific activities of each of the
luciferases were different; in comparison to LcL and
LUC-H, the specific activities of PmL and PpL toward
ketoprofen were relatively low (about 60%). These
results are interesting because the substrate accessibil-
ities for the thioesterification reaction of luciferases are
quite different depending on the species of firefly.
Because firefly luciferase is thought to have evolved
To compare thioesterification activity at different pH values, the
initial velocities obtained using 0.25 mM (R)- or (S)-ketoprofen were
measured by spectrophotometric assay9) in three pH buffers: 100 mM
PPB (pH 7.0 and pH 7.9) and 100 mM Tris–HCl buffer (pH 9.5).
Kinetic analysis. The kinetic parameters of PmL were calculated by
the spectrophotometric method. The kcat and Km values were
determined by Michaelis-Menten kinetics analysis using GraphPad
prism version 5.01 (GraphPad, San Diego, CA). The kcat values were
expressed as the turnover number per subunit (Mr of the subunit,
6
1
2,841). To determine the effects of pH on thioesterification activity,
00 mM PPB (pH 7.0 or pH 7.9) or 100 mM Tris–HCl buffer (pH 9.5)
was used. The kinetics studies were done with various concentrations
of (R)-ketoprofen (0.01–0.5 mM). The concentration of the other
cofactors, ATP and CoASH, were fixed at 10 mM and 2 mM
respectively. Each assay was repeated 5 times.
Results and Discussion
1
4)
from a long-chain acyl-CoA synthetase,
it is not
Thioesterification activities of the other five firefly
luciferases toward dodecanoic acid (C12) and ketopro-
fen
surprising that HpL and LmL did not accept an artificial
substrate such as 2-arylpropanoic acid. During the
evolution process, other luciferases, LUC-H, PmL,
PpL, and LcL, might have obtained substrate accept-
ability to 2-arylpropanoic acid.
Firefly luciferase is a bifunctional enzyme that can
catalyze the thioesterification reaction toward long-
chain fatty acid such as C12 as well as the bio-
To make clear how these differences originated, the
amino acid residues in the active site were compared
5
)
luminescence reaction. In addition, we identified

1760
D. KATO et al.
using the 15 important positions for the bioluminescence
reaction in PpL, which were discovered by Branchini
et al.: R218, H245, G246, F247, F250, T251, G315,
PmL exhibited thioesterification activity toward a
series of (ꢂ)-2-arylpropanoic acids. Among them,
ketoprofen was the best substrate. When fatty acids of
various chain lengths were subjected to the reaction, we
found that PmL exhibited thioesterification activity
toward chains 8 to 18 carbons in length, and the highest
activity was found in C12. These results are similar to
the case of LUC-H. Anthraquinone-2-carboxylic acid
was also converted to the corresponding thioester with
moderate efficiency. In contrast, catalytic activities
decreased to below the limit of detection with 2-
anthracenecarboxylic acid as substrate. The catalytic
activity toward anthraquinone-2-carboxylic acid was
vastly different from that of 2-anthracenecarboxylic
acid, in spite of the fact that the steric bulkiness of these
two compounds is almost the same. It is also remarkable
that the activity of ketoprofen was higher than that of
the other 2-arylpropanoic acids. The compounds that
exhibited high activity might have done so as a result of
the presence of a carbonyl group in the compound, one
by which these compounds made hydrogen bond with
some residues in the active site and bound securely with
the enzyme.
G316, G341, L342, T343, S347, A348, I351 and
K529.1
5,16)
All these residues were, however, completely
consistent with the six firefly luciferases. Hence other
residues were thought to be involved in the thioester-
ification reaction. The maximum wavelengths of the
bioluminescence spectra are also listed in Table 1. The
wavelength of bioluminescence is affected by small
differences in the active site of the enzyme, and the
environment around the catalytic cavity is influenced by
the overall structure of the protein. Because the total
sequence homologies of the six firefly luciferases varied
widely, from 60 to 93%, these differences cause the
structural changes in the catalytic cavities. These dif-
ferences might also affect the thioesterification activity
as well as the maximum wavelengths of the biolumi-
9
)
1
7–19)
nescence spectra.
Substrate specificities of PmL and HpL
Based on specific activity measurements of the six
firefly luciferases toward C12 and ketoprofen, we
confirmed that the magnitudes of their thioesterification
abilities were different from one another. In the case of
LUC-H, detailed experiments had already been per-
In the case of HpL, we did not detect catalytic activity
toward a series of 2-arylpropanoic acids. This enzyme,
however, did show thioesterification activity toward
long-chain fatty acids (C10–C16). Thus we confirmed
that HpL possesses the acyl-CoA synthetic activity itself
but did not recognize 2-arylpropanoic acid as substrate.
Thus these luciferases are suitable for investigating
differences in recognition of substrate binding toward
2-arylpropanoic acid.
8
,9)
formed (Table 2).
This enzyme exhibits acyl-CoA
synthetic activity toward long-chain fatty acids, from 8
to 18 carbons (C8–C18), and toward 2-arylpropanoic
acids such as flurbiprofen, ibuprofen, ketoprofen, and
naproxen. Thus, in this study, the substrate specificity of
PmL, whose thioesterification activity toward ketopro-
fen was moderate against LUC-H, and HpL, which
lacked thioesterification activity toward ketoprofen,
were investigated.
Effects of pH on thioesterification activity and the
enantiodifferentiation of PmL
The activity-pH profile of the PmL-catalyzed thio-
esterification reaction toward ketoprofen is shown in
Fig. 2. The enzyme was highly active under basic
conditions, with maximum activity at pH 9–10. At pH
over 10, the apparent enzymatic activities fell dramat-
ically. Using the bioluminescence reaction for compar-
ison, the optimal pH for thioesterification activity was
found to be in the basic region. These results are in good
Table 2. Substrate Specificities of the LUC-H, PmL, and HpL
Catalyzed Thioesterification Reaction
b
Relative activity (%)
Carboxylic acida
LUC-Hc
PmL
HpL
(
(
(
(
ꢂ)-Flurbiprofen
ꢂ)-Ibuprofen
ꢂ)-Ketoprofen
ꢂ)-Naproxen
10
8
8
6
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
95
46
15
21
11
100
Acetic acid (C2)
N.D.
N.D.
N.D.
N.D.
7
N.D.
N.D.
N.D.
N.D.
11
Propanoic acid (C3)
Butanoic acid (C4)
Hexanoic acid (C6)
Octanoic acid (C8)
Decanoic acid (C10)
Dodecanoic acid (C12)
80
60
93
96
d
100
64
100
87
100
76
40
d
Butyldecanoic acid (C14)
Hexadecanoic acid (C16)
d
3
7
21
20
d
Octadecanoic acid (C18)
Anthraquinone-2-carboxylic acid
-Anthracenecarboxylic acid
3
49
3
23
N.D.
N.D.
N.D.
2
12
N.D.
0
2
3
4
5
6
7
8
9
10 11 12
N.D., not detected. Relative activity could not be calculated because the
specific activity was below the limit of detection (<0:01 nmol/min/mg).
aFinal concentration of carboxylic acid was 0.25 mM.
bRelative activity was calculated by a comparison of specific activities, with
pH
Fig. 2. Effects of pH on PmL-Catalyzed Thioester Formation of (ꢂ)-
Ketoprofen.
the value for dodecanoic acid (C12) (LUC-H, 44.6 nmol/min/mg; PmL,
6
4.0 nmol/min/mg; HpL, 13.8 nmol/min/mg) as standard.
Relative activity was calculated by comparing specific activities,
using the value obtained in 100 mM Tris–HCl (pH 9.5) as standard.
The specific activity of the thioester formation was determined at a
final concentration of 0.25 mM (ꢂ)-ketoprofen.
cThe values for this column are quoted from the previous report.9)
dTo dissolve the substrate, 0.2% Triton X-100 was added to the reaction
mixture, and the activity was measured under these conditions.

Enantioselective Thioesterification Activity in Luciferases
1761
Table 3. Comparison of the Specific Activities of PmL-Catalyzed
Thioester Formation toward Ketoprofen Enantiomers at Various pH
Values
Table 4. Determination of the Kinetic Parameters of PmL-Catalyzed
Thioester Formation for (R)-Ketoprofen at Various pH Values
pHa
pHb
Specific activity
nmol/min/mg)a
7
.0
7.9
9.5
(
7
.0
7.9
9.5
Km (mM)
0:15 ꢂ 0:03
0:12 ꢂ 0:01
0.80
0:15 ꢂ 0:03
0:25 ꢂ 0:03
1.66
0:15 ꢂ 0:03
0:57 ꢂ 0:12
3.73
ꢃ1
VR
VS
22.5
1.1
32.6
1.7
63.2
3.2
kcat (s
)
ꢃ1
ꢃ1
)
kcat/Km (s ꢄmM
Ratio (VR/VS)
19.7
19.0
20.0
Relative activity
1
2.1
4.7
aThe specific activity of the thioester formation was determined at a final
concentration of 0.25 mM (R)- or (S)-ketoprofen.
bThe pH of the reaction mixture was controlled using 100 mM PPB (pH 7.0
or pH 7.9) or 100 mM Tris–HCl (pH 9.5).
aThe pH of the reaction mixture was controlled using 100 mM PPB (pH 7.0
or pH 7.9) or 100 mM Tris–HCl (pH 9.5).
enzyme toward D-luciferin was strikingly different, and
the value at pH 6 was about 4 times larger than the value
2
3)
agreement with the results for the LUC-H catalyzed
)
at pH 8. This indicates that the mode of substrate
recognition is quite different between the thioesterifica-
tion and the bioluminescence reaction and that the
catalytic residues participating in these reactions are
dissimilar. Further investigation using 3D structure and/
or homology modeling should be effective in identifying
important residues for thioester formation toward keto-
profen.
8
thioester formation toward ketoprofen. This tendency
might explain the pKa value of the mercapto group of
2
0)
CoASH. This value was calculated to be 10.35.
Because the nucleophilic ability of CoASH increases
gradually as the pH of the reaction mixture approaches
to this value, the possibility of attacking the reactant
improves and in total the reaction rate is thought to
increase in the basic pH region.
In this study, we examined enantioselective thioester-
ification activity toward ketoprofen using other five
firefly luciferases, PmL, PpL, LcL, HpL, and LmL.
Among these, we verified that PmL, PpL, and LcL
showed R-enantioselective thioesterification activities,
whose results are identical to the case of LUC-H. HpL
and LmL, however, did not accept 2-arylpropanoic acid,
although these two firefly luciferases exhibited thioes-
terification activity toward long-chain fatty acid suchs as
C12. These results are interesting because the substrate
accessibilities of luciferases for the thioester formation
differ depending on the species of firefly. Hence we
focused primarily on PmL, and compared the initial
velocities of the thioesterification reaction toward two
enantiomers of ketoprofen at various pH values. It
became clear that the enantiodifferentiation ability of
this enzyme does not depend on pH. Moreover, based on
the kinetic parameters, we confirmed that kcat contrib-
utes to the differences in reactivities of different pH
values. These results are in contrast to the original
catalytic activity of firefly luciferase, the biolumines-
cence reaction.
It is known that bioluminescence activities of PmL
have influence the pH of the reaction mixture, and due to
its pH sensitivity, bioluminescent properties such as the
maximum wavelength and the quantum yield were
2
1,22)
dramatically changed.
Thus we inquired whether
pH also affects thioesterification activity, especially as
to enantioselectivity toward ketoprofen. We measured
the specific activities toward two enantiomers of
ketoprofen at various pH value (Table 3). Although
the values for each enantiomer changed depending on
pH, the ratio of the initial velocity for (R)-ketoprofen
versus (S)-ketoprofen was almost the same at each pH
value. Thus we confirmed that the pH of the reaction
mixture was not affected by the enantioselectivity of
the PmL-catalyzed thioesterification reaction. We have
determined the enantiodifferentiational ratio of LUC-H
in pH 7, and its value is almost the same as in this
experiment.8 The enantioselectivities of these lucifer-
ases are equivalent although the specific activities
toward ketoprofen are different from each other. These
results indicate that the enantiodifferentiation mecha-
nism toward ketoprofen is identical for these two
luciferases.
)
Acknowledgment
Determination of the kinetic parameters for (R)-
ketoprofen
We are deeply grateful to Dr. Hiromichi Ohta
(University of Nagasaki) for his comments and for
discussion this manuscript. We extend special thanks to
Dr. Kazuki Niwa of the National Institute of Advanced
Industrial Science and Technology (AIST) for support
and for allowing us to use LumiFL-Spectrocapture. This
work was partially supported by a Grant-in-Aid for
Young Scientists (B) (23750197) from the Japan Society
for the Promotion of Science (JSPS), and by the Hyogo
Science and Technology Association.
From the results for activity-pH profile, it was clear
that the thioesterification activity of PmL increased from
pH 7 to 9. Thus, to make clear the factor of activity
change through different pH values, the kinetic param-
eters for (R)-ketoprofen were determined at three pH
values, 7.0, 7.9, and 9.5. The results are listed in
Table 4. The Km values at each pH were same at
0
.15 mM, indicating that there was no difference in
binding affinities at these pH values. In contrast, the kcat
values showed an obvious difference, in that the value
increased with pH. Thus it became clear that the total
reaction efficiency was determined by the effect of the
kcat parameter. This is in marked contrast to the
bioluminescent reaction of a pH-sensitive firefly lucifer-
ase such as PpL. The bioluminescence Km value of this
References
1
)
McElroy WD and Green A, Arch. Biochem. Biophys., 64, 257–
71 (1956).
2
2)
Rhodes WC and McElroy WD, Science, 128, 253–254 (1958).
Rhodes WC and McElroy WD, J. Biol. Chem., 233, 1528–1537
(1958).
3
)

1
762
D. KATO et al.
Suzuki H, Kawarabayashi Y, Kondo J, Abe T, Nishikawa K,
Kimura S, Hashimoto T, and Yamamoto T, J. Biol. Chem., 256,
681–8685 (1990).
Oba Y, Ojika M, and Inouye S, FEBS Lett., 540, 251–254
2003).
4
)
15) Branchini BR, Southworth TL, Murtiashaw MH, Boije H, and
Fleet SE, Biochemistry, 42, 10429–10436 (2003).
8
16) Branchini BR, Southworth TL, Murtiashaw MH, Magyar RA,
Gonzalez SA, Ruggiero MC, and Stroh JG, J. Am. Chem. Soc.,
43, 7255–7262 (2004).
5)
6)
7)
8)
9)
(
Knihinicki RD, Williams KM, and Day RO, Biochem. Pharma-
col., 38, 4389–4395 (1989).
Hall SD and Xiaotao Q, Chem. Biol. Interact., 90, 235–251
17) Viviani VR, Arnoldi FGC, Neto AJS, Oehlmeyer TL, Bechara
EJH, and Ohmiya Y, Photochem. Photobiol. Sci., 7, 159–169
(2008).
(1994).
18) Hirano T, Hasumi Y, Ohtsuka K, Maki S, Niwa H, Yamaji M,
and Hashizume D, J. Am. Chem. Soc., 131, 2385–2396 (2009).
19) Naumov P, Ozawa Y, Ohkubo K, and Fukuzumi S, J. Am.
Chem. Soc., 131, 11590–11605 (2009).
Kato D, Teruya K, Yoshida H, Takeo M, Negoro S, and Ohta H,
FEBS J., 274, 3877–3885 (2007).
Kato D, Tatsumi T, Bansho A, Teruya K, Takeo M, and Negoro
S, J. Mol. Cat. B: Enzymatic, 69, 140–146 (2011).
0) Lloyd JE, ‘‘Bioluminescence in Action,’’ ed. Herring PJ,
20) Pitman IH and Morris IJ, Aust. J. Chem., 33, 1625–1630 (1980).
21) Niwa K, Ichino Y, Kumata S, Nakajima Y, Hiraishi Y, Kato D,
Viviani VR, and Ohmiya Y, Photochem. Photobiol., 86, 1046–
1049 (2010).
1
Academic Press, New York, pp. 241–272 (1978).
1
1) Ohmiya Y, Ohba N, Toh H, and Tsuji FI, Photochem.
Photobiol., 62, 309–313 (1995).
22) Ando Y, Niwa K, Yamada N, Enomoto T, Irie T, Kubota H,
Ohmiya Y, and Akiyama H, Nat. Photon., 2, 44–47 (2007).
23) Viviani VR, Oehlmeyer TL, Arnoldi FGC, and Brochetto-Braga
MR, Photochem. Photobiol., 81, 843–848 (2005).
1
2) Kato D, Miura A, Takeo M, and Negoro S, Rep. Grad. Sch.
Eng., Univ. Hyogo, 63, 24–28 (2010).
1
1
3) Bradford MM, Anal. Biochem., 78, 248–254 (1976).
4) Conti E, Franks NP, and Brick P, Structure, 4, 287–298
24) Kajiyama N, Masuda T, Tatsumi H, and Nakano E, Biochim.
Biophys. Acta, 1120, 228–232 (1992).
(1996).