AbstractFirefly luciferase can catalyze the formation of fatty acyl-CoA via fatty acyl-adenylate from fatty acid in the presence of ATP, Mg2+ and coenzyme A (CoA). A long chain fatty acyl-CoA (C16–C20), produced by luciferase from a North American firefly (Photinus pyralis) and a Japanese firefly (Luciola cruciata), was isolated and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis. Of a number of substrates tested, linolenic acid (C18:3) and arachidonic acid (C20:4) appear to be suitable for acyl-CoA synthesis. This evidence suggests that firefly luciferase within peroxisomes of the cells in the photogenic organ may be a bifunctional enzyme, catalyzing not only the bioluminescence reaction but also the fatty acyl-CoA synthetic reaction

Inouye, Satoshi

Oba, Yuichi

Ojika, Makoto

Elsevier - Publisher Connector 

Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase 

Fire£y luciferase is a bifunctional enzyme: ATP-dependent
monooxygenase and a long chain fatty acyl-CoA synthetase
Yuichi Obaa, Makoto Ojikaa, Satoshi Inouyeb;
aGraduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
bYokohama Research Center, Chisso Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan
Received 11 February 2003; revised 10 March 2003; accepted 12 March 2003
First published online 21 March 2003
Edited by Judit Ova¤di
Abstract Fire£y luciferase can catalyze the formation of fatty
acyl-CoA via fatty acyl-adenylate from fatty acid in the pres-
ence of ATP, Mg2+ and coenzyme A (CoA). A long chain fatty
acyl-CoA (C16^C20), produced by luciferase from a North
American ¢re£y (Photinus pyralis) and a Japanese ¢re£y (Lu-
ciola cruciata), was isolated and identi¢ed by matrix-assisted
laser desorption/ionization time-of-£ight mass spectrometry
analysis. Of a number of substrates tested, linolenic acid
(C18:3) and arachidonic acid (C20:4) appear to be suitable for
acyl-CoA synthesis. This evidence suggests that ¢re£y luciferase
within peroxisomes of the cells in the photogenic organ may be a
bifunctional enzyme, catalyzing not only the bioluminescence
reaction but also the fatty acyl-CoA synthetic reaction.
3 2003 Published by Elsevier Science B.V. on behalf of the
Federation of European Biochemical Societies.
Key words: Bioluminescence; Luciferin; CoA ligase;
Adenylation; Intermediate; L-Oxidation
1. Introduction
Fire£y luciferase (EC 1.13.12.7) is a well-characterized en-
zyme that is responsible for the bioluminescence reaction. It
catalyzes the oxidation of ¢re£y luciferin with molecular oxy-
gen in the presence of ATP and Mg2þ to emit yellow-green
light [1^4]. The initial reaction catalyzed by ¢re£y luciferase is
the formation of luciferyl adenylate with the release of inor-
ganic pyrophosphate. The luciferase-bound luciferyl adenylate
reacts rapidly with molecular oxygen to give light, CO2, AMP
and oxyluciferin (Fig. 1A). When excess luciferin and ATP are
added to the reaction mixture of luciferase, a £ash of light is
observed and then a rapid inhibition of luminescence occurs.
The addition of coenzyme A (CoA) to the reaction mixtures
prevents this rapid inhibition [5]. In 1967, McElroy et al. [6]
suggested that a luciferase from North American ¢re£y, Pho-
tinus pyralis, is remarkably similar in catalytic mechanism to
amino acyl-tRNA synthetase and fatty acyl-CoA synthetase.
After two decades, the primary structure of ¢re£y luciferase
was determined by cDNA cloning [7] and signi¢cant sequence
homology of P. pyralis luciferase to plant p-coumarate:CoA
ligase (EC 6.2.1.12) [8] and rat long chain acyl-CoA synthe-
tase (EC 6.2.1.3) [9] was reported. Further, conserved se-
quence motifs characteristic of adenylate formation were pro-
posed in ¢re£y luciferase [10], based on comparison with
several adenylate-forming enzymes including p-coumarate:
CoA ligase, fungal acetyl-CoA synthetase and rat long chain
acyl-CoA synthetase. At the present time, over 12 cDNAs
encoding luciferase from various ¢re£y species have been ob-
tained [11] and the crystal structure of P. pyralis luciferase has
been determined by X-ray analysis [12]. However, the struc-
ture^function relationship for the luminescence reaction has
not been well resolved [13,14]. Recently, it was reported that
¢re£y luciferase can synthesize the adenosine(5P)tetraphos-
pho(5P)adenosine (AppppA) from dehydroluciferyl adenylate,
ATP and Mg2þ, and the formation of AppppA was inhibited
by addition of CoA [15]. Furthermore, Ueda and Suzuki [16]
reported that the long chain fatty acids (C12^C20) inhibited the
light production in competition with ¢re£y luciferin in the
luciferase reaction.
With this background information, we have predicted the
ability of ¢re£y luciferase to synthesize fatty acyl-CoA via
fatty acyl-adenylate as an intermediate from fatty acid in
the presence of ATP, Mg2þ and CoA (Fig. 1B), and have
now shown long chain fatty acyl-CoA synthetic activity in
¢re£y luciferase.
2. Materials and methods
2.1. Materials
All chemicals were obtained from commercial sources: CoA triso-
dium salt, GTP, UTP, sodium acetate, linoleic acid and ferulic acid
(Wako Pure Chemicals, Osaka, Japan); propionic anhydride (Tokyo
Kasei Kogyo, Tokyo, Japan); oleoyl-CoA, arachidonic acid sodium
salt, p-coumaric acid, ca¡eic acid, 3-hydroxypicolinic acid, CTP, TTP,
and ITP (Sigma, St. Louis, MO, USA); oleic acid sodium salt (Al-
drich, Milwaukee, WI, USA); ¢re£y luciferin sodium salt (Nacalai
Tesque, Kyoto, Japan); palmitic acid sodium salt (Kanto Chemical,
Tokyo, Japan); linolenic acid (NOF, Tokyo, Japan); ATP and AMP
(Oriental Yeast, Osaka, Japan); recombinant ¢re£y luciferases from
P. pyralis (Promega, Madison, WI, USA) and Luciola cruciata
(Wako); recombinant aequorin (Chisso, Tokyo, Japan); acyl-CoA
synthetase from Pseudomonas sp. (Funakoshi, Tokyo, Japan); [K-
32P]ATP (3000 Ci/mmol) and [1-14C]oleic acid (56.0 mCi/mmol)
from NEN Life Science Products (Boston, MA, USA) and Amersham
Pharmacia Biotech (Piscataway, NY, USA), respectively.
2.2. Assay for acyl adenylation activity using [K-32P]ATP by TLC
analysis
The reaction mixture (20 Wl) contained [K-32P]ATP (0.33 WCi= 5.6
nM), CoA (250 WM), MgCl2 (5 mM), substrate (10 WM) and recombi-
nant P. pyralis ¢re£y luciferase (0.25 Wg= 0.2 WM) in 100 mM Tris^
HCl (pH 7.8). The reaction was started by adding luciferase and in-
0014-5793 / 03 / $22.00 M 2003 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
doi:10.1016/S0014-5793(03)00272-2
*Corresponding author. Fax: (81)-45-786-5512.
E-mail address: sinouye@chisso.co.jp (S. Inouye).
Abbreviations: CoA, coenzyme A; HPLC, high performance liquid
chromatography; MALDI-TOF-MS, matrix-assisted laser desorp-
tion/ionization time-of-£ight mass spectrometry; TLC, thin-layer
chromatography
FEBS 27131 26-3-03
FEBS 27131 FEBS Letters 540 (2003) 251^254
cubated at 30‡C for 30 min. After adding 20 Wl of ethanol to the
reaction mixture, 2 Wl of the reaction solution was spotted on thin-
layer chromatography (TLC) plates (Silica gel 60 F254, Merck). First
development was performed in dioxane/50 mM acetic acid (4:1), fol-
lowed by second development in dioxane/ammonium hydroxide/water
(6:1:5) at the same level as the ¢rst development. The Rf values of [K-
32P]ATP and [K-32P]AMP were identi¢ed to 0.17 and 0.51, respec-
tively, comparing with the Rf values for authentic samples of ATP
and AMP under a UV lamp at 254 nm. The radioactivity of [K-
32P]AMP was measured using an image analyzer (Fuji Film; a model
BAS 2500) for 30 min exposure and was obtained by subtracting the
background value. The intensity was calculated as a percentage of
that produced with ¢re£y luciferin as a substrate under the same
reaction conditions and the data were analyzed by Student’s t-test.
2.3. Requirements for oleoyl-CoA synthesis in ¢re£y luciferase reaction
The reaction mixture (20 Wl) contained [1-14C]oleic acid (19.2
nCi= 17.3 WM), ATP (250 WM), CoA (250 WM), MgCl2 (5 mM)
and recombinant P. pyralis ¢re£y luciferase (1.27 Wg= 1 WM) in 100
mM Tris^HCl (pH 7.8). The reaction was started by adding luciferase
and incubated at 30‡C for 60 min. After adding 20 Wl of ethanol to the
reaction mixture, 2 Wl of the reaction solution was spotted on TLC
plate (Silica gel 60 F254, Merck) and was developed in dioxane/am-
monium hydroxide/water (3:0.5:2). Oleoyl-CoA (Rf = 0.50) was iden-
ti¢ed using the authentic sample of oleoyl-CoA under a UV lamp at
254 nm. The radioactivity of [1-14C]oleoyl-CoA was measured in a
BAS 2500 (Fuji) and the relative intensity was obtained by subtracting
the background value.
2.4. Isolation of fatty acyl-CoA by reversed-phase HPLC
The reaction mixture (400 Wl) contained substrate (17.3 WM; pal-
mitic acid, oleic acid, linoleic acid, linolenic acid or arachidonic acid),
NTP (250 WM; ATP, GTP, CTP, TTP, UTP or ITP), CoA (250 WM),
MgCl2 (5 mM) and recombinant ¢re£y luciferase (25.4 Wg= 1 WM)
from P. pyralis or L. cruciata in 100 mM Tris^HCl (pH 7.8). The
enzyme reaction was started by addition of luciferase and incubated at
30‡C for 60 min. The reaction was terminated by adding 267 Wl of
acetonitrile and centrifuged at 10 000 rpm for 5 min. The resultant
supernatant was subjected to reversed-phase high performance liquid
chromatography (HPLC) using a Capcell Pak C18 (4.6U150 mm;
Shiseido, Japan) and with a linear gradient of 40^70% acetonitrile
in 25 mM KH2PO4 from 5 to 17 min, followed by 70% acetonitrile
for 8 min at a £ow rate of 0.8 ml/min. The fractions from the column
were monitored using a multi-wavelength detector (195^650 nm; MD-
2010 plus, Jasco). Elution times of palmitoyl-CoA, oleoyl-CoA, lino-
leoyl-CoA, linolenoyl-CoA and arachidonoyl-CoA were 18.4 min,
19.1 min, 17.1 min, 14.8 min and 16.2 min, respectively.
2.5. Identi¢cation of fatty acyl-CoA by MALDI-TOF-MS
The peak fraction isolated from HPLC analysis was subjected to
matrix-assisted laser desorption/ionization time-of-£ight mass spec-
trometry (MALDI-TOF-MS) with an AutoFLEX (Bruker Daltonics)
using 3-hydroxypicolinic acid as a matrix. The data were acquired in
the negative re£ector mode of operation.
2.6. Kinetic analyses
The reaction mixture (500 Wl) contained linolenic acid (0.1^100
WM), ATP (250 WM), CoA (250 WM), MgCl2 (5 mM) and recombi-
nant P. pyralis ¢re£y luciferase (6.32 Wg= 0.2 WM) in 100 mM Tris^
HCl (pH 7.8). The reaction was started by adding luciferase and in-
cubated at 30‡C for 10 min. The product, fatty acyl-CoA, was mea-
sured by reversed-phase HPLC as described in Section 2.4. Since a
strong inhibition for fatty acyl-CoA synthetic activity of ¢re£y lucif-
erase was observed at high concentrations (over 20 WM) of various
fatty acids, the Km, Vmax and Kcat values for linolenic acid were de-
termined at concentrations from 10 to 20 WM of fatty acids by the
method of Lineweaver^Burk plots.
2.7. Luminescence assay
The reaction mixture (100 Wl) contained substrate (10 WM; fatty
acid or ¢re£y luciferin), ATP (250 WM), CoA (250 WM) and MgCl2
(5 mM) in 100 mM Tris^HCl (pH 7.8). The reaction was started by
the addition of enzyme (1.27 Wg, recombinant P. pyralis ¢re£y lucif-
erase; 2 Wg, acyl-CoA synthetase from Pseudomonas sp.) at 25‡C. The
initial intensity of the luminescence was determined with an Atto
(Tokyo, Japan) model AB-2200 luminometer. The detection limit of
the luminometer corresponded to the light intensity from 1 pg of
recombinant aequorin (4.8U1015 photons/mg protein) as a light
source.
3. Results and discussion
In the ¢rst step, to screen a suitable substrate for acyl-CoA
formation by P. pyralis ¢re£y luciferase, the acyl-adenylate-
mediated reaction was monitored by the formation of [K-
32P]AMP from [K-32P]ATP using TLC analysis. In the pres-
ence of [K-32P]ATP, Mg2þ and CoA, the following substrates
were examined: acetic acid (C2), propionic acid (C3), palmitic
acid (C16:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic
acid (C18:3) and arachidonic acid (C20:4), p-coumaric acid
and its derivatives. The results suggest that long chain unsat-
urated fatty acids are the most suitable substrate for the ad-
enylation reaction (Fig. 2). Arachidonic acid, in particular,
has ca. 50% e⁄ciency of production of AMP in comparison
with that using ¢re£y luciferin as a substrate under the same
conditions. Other substrates of aromatic acids for plant
p-coumarate:CoA ligase and short chain acids were not uti-
lized e⁄ciently by ¢re£y luciferase. The identi¢cation of fatty
acyl-CoA formation via fatty acyl-adenylate was performed
using [1-14C]oleic acid as a substrate and oleoyl-CoA as an
authentic sample. Based on TLC analysis, [1-14C]oleoyl-CoA
was a product and ATP, Mg2þ and CoA were the essential
cofactors for the reaction (Table 1). Other nucleotides includ-
ing GTP, CTP, TTP, UTP and ITP did not stimulate the
formation of oleoyl-CoA signi¢cantly (data not shown). Fur-
thermore, the oleoyl-CoA product was also isolated by re-
versed-phase HPLC. A single major peak showing the identi-
cal retention time to the authentic oleoyl-CoA was separated
by HPLC (Fig. 3A). The peak fraction isolated was analyzed
Fig. 1. Schematic representation of bioluminescence reaction (A) and fatty acyl-CoA synthetic reaction (B), catalyzed by ¢re£y luciferase.
FEBS 27131 26-3-03
Y. Oba et al./FEBS Letters 540 (2003) 251^254252
with MALDI-TOF-MS. The mass value obtained from the
peak fraction was consistent with the predicted mass value
of oleoyl-CoA (Fig. 3B) and the mass pattern of the product
was also identical to that of authentic oleoyl-CoA. The CoA
products formed by ¢re£y luciferase with other fatty acids
were also identi¢ed by the same procedure (Table 2). The
conversion e⁄ciency to fatty acyl-CoA from its fatty acid
by P. pyralis luciferase was calculated from peak areas of
HPLC analysis : 19.3% from palmitic acid, 52.8% from oleic
acid, 63.3% from linoleic acid, over 98.0% from linolenic acid
and over 98.0% from arachidonic acid under these reaction
conditions. In the kinetic study on P. pyralis luciferase, the
Km and Vmax values for linolenic acid were 13.6 WM and 0.127
Wmol/min/mg protein, respectively. The Kcat value was calcu-
lated to 0.130/s. These values for linolenic acid were similar to
the Kcat and Km values for ¢re£y luciferin (Kcat = 0.125/s,
Km =15 WM) using recombinant P. pyralis luciferase [17].
Thus, ¢re£y luciferase acts as an enzyme that catalyzes the
fatty acyl-CoA synthesis. To determine whether fatty acyl-
CoA synthetic activity is a general property of ¢re£y lucifer-
ase, a Japanese ¢re£y L. cruciata luciferase (60.4% amino acid
sequence identity to P. pyralis luciferase) was also examined
and fatty acyl-CoA products were identi¢ed. As summarized
in Table 2, these results showed that ¢re£y luciferase has the
catalytic ability to synthesize a long chain fatty acyl-CoA
from various long chain fatty acids.
From the viewpoint of the luminescence reaction, ¢re£y
luciferase did not give any luminescence in the presence of
fatty acid, ATP, Mg2þ and CoA. On the other hands, an
acyl-CoA synthetase from Pseudomonas sp. (EC 6.2.1.3) hav-
ing fatty acyl-CoA synthetic activity did not show lumines-
cence activity in the presence of ¢re£y luciferin, ATP, Mg2þ
and CoA, and also did not produce luciferyl-CoA from lu-
ciferin (data not shown). The di¡erence between the lumines-
cence reaction and the fatty acyl-CoA synthetic reaction by
¢re£y luciferase would be explained as follows: luciferase cat-
alyzes the adenylation at the carboxyl group in both ¢re£y
luciferin and a long chain fatty acid with ATP. This adenyla-
tion step is presumably by an identical mechanism. The lumi-
nescence reaction will start by removing the proton at the C4
position in luciferyl adenylate, followed by addition of molec-
ular oxygen and then give light emission (Fig. 1A) [2^4]. In
Fig. 2. Detection of acyl-adenylate formation by ¢re£y luciferase.
Acyl-adenylate formation was detected by the formation of [K-
32P]AMP from [K-32P]ATP. The intensity of [K-32P]AMP was calcu-
lated as a percentage of that produced with ¢re£y luciferin as a sub-
strate under the same reaction conditions. Horizontal bars in the
shaded boxes represent the meanUS.E.M. (n=3). *P6 0.05 vs. ‘No
substrate’.
Table 1
Requirements of cofactors for fatty acyl-CoA synthesis by ¢re£y lu-
ciferase
Reaction conditions [1-14C]Oleoyl-CoA relative intensity (%)a
Controlb 764 (100)
^ Luciferase 0 (0)
^ ATP 11 (1)
^ CoA 9 (1)
^ Mg2þ 23 (3)
aRadioactivity was determined as relative intensity using an image
analyzer.
bControl contains the complete components of luciferase, ATP,
CoA and Mg2þ.
Fig. 3. Identi¢cation of oleoyl-CoA produced by ¢re£y luciferase. A: Isolation of oleoyl-CoA by reversed-phase HPLC. The peak fraction at
19.1 min was subjected to MALDI-TOF-MS. B: MALDI-TOF-MS analysis of oleoyl-CoA produced by ¢re£y luciferase. The signal at 1068.4
indicates the potassium adduct of oleoyl-CoA.
FEBS 27131 26-3-03
Y. Oba et al./FEBS Letters 540 (2003) 251^254 253
the case of fatty acyl-adenylate as an intermediate, fatty acyl-
CoA is only produced in the presence of CoA in the same
manner as other adenylate-mediated enzyme reactions (Fig.
1B) [9,10].
Considering the biological function of luciferase in the ¢re-
£y, the enzyme is localized within peroxisomes in the cells of
the photogenic organ [18,19], and the luciferase expressed in
mammalian cells is distributed into peroxisomes. The signal
sequence targeted into peroxisomes has also been identi¢ed at
the carboxy-terminus of luciferase [20]. Since the peroxisome
is the functional organelle for L-oxidation of long chain fatty
acids [21], ¢re£y luciferase could serve to synthesize a long
chain acyl-CoA in the activation step leading to degradation
of fatty acids. Our ¢ndings and the above evidence may sug-
gest that ¢re£y luciferase in peroxisomes keeps the catalytic
functions in bioluminescence and fatty acid metabolism.
Acknowledgements: This work was supported in part by a Grant-in-
Aid for Scienti¢c Research on Priority Areas (A) from the Ministry of
Education, Culture, Sports, Science and Technology.
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Table 2
Identi¢cation of fatty acyl-CoA produced by recombinant luciferase
from P. pyralis and L. cruciata with MALDI-TOF-MSa
Product Predicted [M3H]3 Observed [M3H]3
P. pyralis L. cruciata
Palmitoyl-CoA 1004.3 1004.6 N.D.
Oleoyl-CoA 1030.4 1030.4 1030.4
Linoleoyl-CoA 1028.3 1028.4 1028.6
Linolenoyl-CoA 1026.3 1026.4 1026.6
Arachidonoyl-CoA 1052.3 1052.5 1052.6
N.D.=not determined.
aSee Sections 2.4 and 2.5 for the details of reaction conditions and
the procedures for determination of fatty acyl-CoA, respectively.
FEBS 27131 26-3-03
Y. Oba et al./FEBS Letters 540 (2003) 251^254254


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Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase

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