Enzymatic and genetic characterization of firefly luciferase and Drosophila CG6178 as a fatty acyl-Coa synthetase

Article abstract of DOI:10.1271/bbb.69.819

Recently we found that firefly luciferase is a bifunctional enzyme, catalyzing not only the luminescence reaction but also long-chain fatty acyl-CoA synthesis. Further, the gene product of CG6178 (CG6178), an ortholog of firefly luciferase in Drosophila melanogaster, was found to be a long-chain fatty acyl-CoA synthetase and dose not function as a luciferase. We investigated the substrate specificities of firefly luciferase and CG6178 as an acyl-CoA synthetase utilizing a series of carboxylic acids. The results indicate that these enzymes synthesize acyl-CoA efficiently from various saturated medium-chain fatty acids. Lauric acid is the most suitable substrate for these enzymes, and the product of lauroyl CoA was identified with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Phylogenetic analysis indicated that firefly luciferase and CG6178 genes belong to the group of plant 4-coumarate:CoA ligases, and not to the group of medium- and long-chain fatty acyl-CoA synthetases in mammals. These results suggest that insects have a novel type of fatty acyl-CoA synthetase.

Full text of DOI:10.1271/bbb.69.819

Biosci. Biotechnol. Biochem., 69 (4), 819–828, 2005
Enzymatic and Genetic Characterization of Firefly Luciferase
and Drosophila CG6178 as a Fatty Acyl-CoA Synthetase
y
2
Yuichi OBA,1 Mitsunori SATO,1 Makoto OJIKA,1 and Satoshi INOUYE
1Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
2Yokohama Research Center, Chisso Co., 5-1 Okawa, Kanazawa-ku, Yokohama 236-8605, Japan
Received December 15, 2004; Accepted January 13, 2005
Recently we found that firefly luciferase is a bifunc-
tional enzyme, catalyzing not only the luminescence
reaction but also long-chain fatty acyl-CoA synthesis.
Further, the gene product of CG6178 (CG6178), an
ortholog of firefly luciferase in Drosophila melanogaster,
was found to be a long-chain fatty acyl-CoA synthetase
and dose not function as a luciferase. We investigated
the substrate specificities of firefly luciferase and
CG6178 as an acyl-CoA synthetase utilizing a series of
carboxylic acids. The results indicate that these enzymes
synthesize acyl-CoA efficiently from various saturated
medium-chain fatty acids. Lauric acid is the most
suitable substrate for these enzymes, and the product
of lauroyl CoA was identified with matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS). Phylogenetic analysis indicated
that firefly luciferase and CG6178 genes belong to the
group of plant 4-coumarate:CoA ligases, and not to the
group of medium- and long-chain fatty acyl-CoA
synthetases in mammals. These results suggest that
insects have a novel type of fatty acyl-CoA synthetase.
C18ꢁC20) in the presence of ATP, coenzyme A (CoA-
SH), and Mg2þ (Eq. 3 and 4).4)
R-COOH þ ATP ꢀ! R-CO-AMP þ PPi
R-CO-AMP þ CoA-SH
ð3Þ
ꢀ! R-CO-S-CoA þ AMP
ð4Þ
Thus firefly luciferase is a bifunctional enzyme, not only
monooxygenase but also long-chain fatty acyl-CoA
synthetase (ACSL). A suitable substrate for luciferase
from the North American firefly Photinus pyralis was
arachidonic acid (C20:4), which showed about 50%
catalytic efficiency with firefly luciferin.4)
Further, we demonstrated that the gene product of
CG6178 (CG6178), an ortholog of firefly luciferase in
Drosophila melanogaster, has an acyl-CoA synthetic
activity. Unsaturated long-chain fatty acids (C18ꢁC20)
were good substrates for CG6178, as in the case of
firefly luciferase. ꢀ-Linolenic acid (C18:3n-3) was the
best as far as we examined.5) But no luminescence
activity or acyl-CoA formation with firefly luciferin
were detected.
Various acyl-CoA synthetases (EC 6.2.1.-, Eq. 3 and
4) have been characterized, and were classified by
the substrate specificities of carboxylic acids: acetyl-
CoA synthetase (AceCS; C2), medium-chain fatty acyl-
CoA synthetase (ACSM; C4ꢁC14), ACSL (C10ꢁC20),
very-long-chain fatty acyl-CoA synthetase (VLCS; ꢂ
C22), 4-coumarate:CoA ligase (4CL), phenylacetyl-CoA
synthetase, etc. (http://www.chem.qmul.ac.uk/iubmb/
enzyme/).
Key words: acyl-CoA synthetase; bifunctional enzyme;
bioluminescence; lauric acid; ortholog gene
In firefly, the bioluminescence reaction is catalyzed
by luciferase (monooxygenase [EC 1.13.12.7]) with
firefly luciferin in the presence of ATP, Mg2þ, and
O2.1–3) The luminescence reaction consists of two steps,
the adenylation of luciferin (Eq. 1) and the oxygenation
of luciferyl adenylate (LH2-CO-AMP) (Eq. 2).
To identify the type of acyl-CoA synthetase for firefly
luciferases (from P. pyralis and the Japanese firefly
Luciola cruciata) and CG6178, the substrate specificities
of these enzymes were examined utilizing various
carboxylic acids. The results indicate that they have
similar specificity of ACSM or ACSL from mammals.
On the other hand, phylogenetic analysis of acyl-CoA
synthetases suggests that firefly luciferase and CG6178
LH2-COOH þ ATP ꢀ! LH2-CO-AMP þ PPi ð1Þ
LH2-CO-AMP þ O2
ꢀ! Oxyluciferin þ CO2 þ Light
ð2Þ
Recently, we found that firefly luciferase has a
catalytic function of acyl-CoA (R-CO-S-CoA) synthesis
from some unsaturated long-chain fatty acids (R-COOH,
y
To whom correspondence should be addressed. Tel/Fax: +81-52-789-4280; E-mail: oba@agr.nagoya-u.ac.jp
Abbreviations: AceCS, acetyl-CoA synthetase; ACSL, long-chain fatty acyl-CoA synthetase; ACSM, medium-chain fatty acyl-CoA synthetase;
4CL, 4-coumarate:CoA ligase; EPA, cis-5,8,11,14,17-eicosapentaenoic acid; HETE, hydroxyeicosatetraenoic acid; MALDI-TOF-MS, matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry; PTS, peroxisomal targeting signal; VLCS, very-long-chain fatty acyl-CoA synthetase

820
Y. OBA et al.
are not to be classified in the group of ACSM or ACSL.
From these results, we concluded that insects have a
novel type of fatty acyl-CoA synthetase.
radioactivity was obtained by subtracting the back-
ground value without substrate. Palmitoleic acid, p-
coumaric acid, caffeic acid, ferulic acid, linoleic acid, ꢀ-
linolenic acid, ꢁ-linolenic acid, 1-naphthylacetic acid, 2-
naphthylacetic acid, and lipoic acid were neutralized by
5 N NaOH before dissolving in water. n-Hexanoic acid
was dissolved in ethanol to a concentration of 100 mM
and then diluted with water for assay. The final
concentration of ethanol in the reaction mixture was
0.1%. The concentration of ethanol at 2% in the reaction
mixture did not affect the activity (data not shown).
Hydroxyeicosatetraenoic acids (HETEs) were dissolved
in 2% DMSO, and the assay was performed at 0.2%.
The concentration of DMSO at 3% in the reaction did
not affect the activity (data not shown). Other substrates
were dissolved in water. For sparingly soluble or
unstable compounds such as arachidonic acid and
EPA, concentrations and purity were analyzed using
HPLC before assay (data not shown).
Materials and Methods
Materials. The following materials were obtained
from commercial sources: [1-14C]lauric acid (60.0 mCi/
mmol, Amersham Biosciences, Buckinghamshire, UK);
lauric acid sodium salt, myristic acid sodium salt,
propionic anhydride, thiazolidine-2-carboxylic, acid and
n-hexanoic anhydride (Tokyo Kasei Kogyo, Tokyo); D-
proline, benzoic acid sodium salt, and 2-naphthylacetic
acid (Aldrich, Milwaukee, WI); linoleic acid, sodium
acetate, L-phenylalanine, ferulic acid, 1-naphthylacetic
acid, and 2-quinolinecarboxylic acid (Wako, Osaka,
Japan); octanoic acid sodium salt, decanoic acid sodium
salt, oleic acid sodium salt, palmitoleic acid, arachidonic
acid sodium salt, cis-5,8,11,14,17-eicosapentaenoic acid
(EPA) sodium salt, D-cysteine, thiaproline, p-coumaric
acid, caffeic acid, ꢁ-linolenic acid, and lauroyl CoA
lithium salt (Sigma, St. Louis, MO); L-proline, D-
luciferin (firefly luciferin) sodium salt, and DL-ꢀ-lipoic
acid (Nacalai Tesque, Kyoto, Japan); recombinant
P. pyralis luciferase (Promega, Madison, WI); [ꢀ-
32P]ATP (3000 Ci/mmol, NEN Life Science Products,
Boston, MA); palmitic acid sodium salt and L-cysteine
(Kanto Chemical, Tokyo); ꢀ-linolenic acid (NOF,
Tokyo); recombinant L. cruciata luciferase (Funakoshi,
Tokyo); (6E,8Z,11Z,14Z)-(ꢃ)-5-hydroxyeicosatetraeno-
ic acid (5-HETE), (5Z,9E,11Z,14Z)-(ꢃ)-8-hydroxyeico-
satetraenoic acid (8-HETE), (5E,7Z,11Z,14Z)-(ꢃ)-9-
hydroxyeicosatetraenoic acid (9-HETE), (5Z,8Z,12E,
14Z)-(ꢃ)-11-hydroxyeicosatetraenoic acid (11-HETE),
(5E,8Z,10Z,14Z)-(ꢃ)-12-hydroxyeicosatetraenoic acid
(12-HETE), and (5E,8Z,11Z,13Z)-(ꢃ)-15-hydroxyeico-
satetraenoic acid (15-HETE) (Cayman Chemical, Ann
Arbor, MI). His6-tagged CG6178 expressed in Esche-
richia coli was purified by Ni-chelating chromatogra-
phy, as previously described.5)
Detection of 14C-lauroyl CoA synthesis. The
reaction mixture (20 ml) containing [1-14C]lauric acid
(12.0 nCi = 10 mM), ATP (250 mM), CoA (250 mM),
MgCl2 (5 mM), and enzyme (50 nM) in 100 mM Tris–
HCl (pH 7.8) was incubated at 25 ꢄC. After 1 h, the
reaction was terminated by addition of 20 ml of ethanol,
and 2 ml was applied to TLC analysis (50 ꢅ 50 mm,
Silica gel 60 F254). The development was performed in
dioxane/ammonium hydroxide/water (3:0.5:2). The
radioactivity of 14C-lauroyl CoA was measured on
BAS 2500 after exposing for 17 h. Authentic lauroyl
CoA on TLC was detected using a 254 nm UV lamp.
Identification of lauroyl CoA by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS). The reaction mixture (500 ml) con-
tained lauric acid (10 mM), ATP (250 mM), CoA (250 mM),
MgCl2 (5 mM), and enzyme (50 nM) in 100 mM Tris–HCl
(pH 7.8). The reaction was started by addition of the
enzyme and incubated at 25 ꢄC for 2 h. It was terminated
by adding 333 ml of acetonitrile, and 750 ml was applied
to HPLC. Reversed-phase HPLC was performed using a
Capcell Pak C18 column (4:6 ꢅ 150 mm, Shiseido,
Japan) 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 flow rate of 0.8 ml/min. The
fractions were monitored using a multi-wavelength
detector (195–650 nm; MD-2010 plus, Jasco). The
elution time for lauroyl CoA was 11.3 min. MALDI-
TOF-MS analysis was performed with AutoFLEX
(Bruker Daltonics, Billerica, MA) using 3-hydroxypico-
linic acid as a matrix. The data were acquired in the
negative reflector mode of operation.
Assay for adenylation activity. Adenylation activity
for carboxylic acids was determined by 32P-AMP
generated from [ꢀ-32P]ATP using TLC analysis.4,5)
The reaction mixture (20 ml) containing [ꢀ-32P]ATP
(0.33 mCi), ATP (100 mM), CoA (250 mM), MgCl2
(5 mM), and enzyme (50 nM) in 100 mM Tris–HCl
(pH 7.8) was incubated at 25 ꢄC. After 30 min, the
reaction was terminated by addition of 20 ml of ethanol,
and 2 ml was spotted on a TLC plate (100 ꢅ 50 mm,
Silica gel 60 F254, Merck). First development was
performed in dioxane/50 mM acetic acid (4:1). After
drying, second development was in dioxane/ammonium
hydroxide/water (6:1:5).6) The Rf values for [ꢀ-32P]ATP
and 32P-AMP formed were identified as 0.17 and 0.51
respectively. The radioactivity of 32P-AMP was meas-
ured using an imaging analyzer (BAS 2500, Fuji Film,
Tokyo) after exposing for 2 h. Relative intensity of the
Effect of lauric acid concentration on acyl-CoA
synthesis. The reaction and separation conditions were
same as those described above section, except for the
concentrations of substrate (0 to 100 mM) and enzyme

Firefly Luciferase and CG6178 as a Fatty Acyl-CoA Synthetase
821
(5 nM) and the reaction time (10 min). The amount of
lauroyl CoA produced was determined by the peak area
of HPLC analysis at 260 nm. The linearity between peak
area and amount was verified from 0 to 1,000 pmol,
using authentic lauroyl CoA.
urated long-chain fatty acids. The profiles of specificity
were similar to each other (Fig. 2). The most suitable
substrate was lauric acid (C12:0), which showed about 4
times higher activity than firefly luciferin. Myristic acid
(C14:0) and decanoic acid (C10:0) were also good
substrates among saturated medium-chain fatty acids,
while octanoic acid (C8:0) and palmitic acid (C16:0)
were not used for firefly luciferase. With unsaturated
long-chain fatty acids, the longer and more highly
unsaturated fatty acids were better substrates for firefly
luciferase, except for palmitoleic acid (C16:1).
For the substrate specificity of CG6178, significant
activities were found only with saturated medium-chain
fatty acids and unsaturated long-chain fatty acids. As
with firefly luciferase, lauric acid (C12:0) was the most
suitable substrate for CG6178, and several fatty acids
(C8ꢁC20) were used broadly as substrates. HETEs are
monohydroxyl fatty acids, produced enzymatically and
non-enzymatically from arachidonic acid (C20:4).11,12)
They contain a hydroxyl group in their structure, like
firefly luciferin, but they were poor substrates (0 to 30%
activity with arachidonic acid) for firefly luciferases. In
contrast, some HETEs (5-HETE, 12-HETE, 15-HETE)
were good substrates (150 to 180% activity with
arachidonic acid) for CG6178 (Fig. 3). The catalytic
site of CG6178 for adenylation may have interactive
residues with the hydroxyl group of HETE.
Phylogenetic analysis. Amino acid sequences were
acquired from NCBI Database (http://www.ncbi.nlm.
nih.gov/) and FlyBase (http://flybase.bio.indiana.edu/).
The phenylalanine-activating subunit of gramicidin
synthetase 1 in Brevibacillus brevis (PheA)7,8) and the
A domain of ꢂ-(L-ꢀ-aminoadipyl)-L-cysteinyl-D-valine
synthetase in Penicillium chrysogenum (acvA)7,9) were
designated as outgroups. Multiple alignment was
achieved with the CLUSTAL X program.10) The Gonnet
series was selected for the protein weight matrix. All
sites containing gap were excluded, and 295 characters
were included for analysis. Neighbor joining tree was
constructed using PAUP* 4.0beta10 (http://paup.csit.
fsu.edu/).
Results
Substrate specificity
The substrate specificity of enzymes for acyl-CoA
synthesis was determined using a series of carboxylic
acids (Fig. 1). The results showed that firefly luciferases
of P. pyralis and L. cruciata exhibited significant
activity only with saturated medium-chain and unsat-
All other carboxylic acids were poor substrates for
firefly luciferase of L. cruciata and P. pyralis and
Fig. 1. Structure of Carboxylic Compounds for Substrate Specificity Analysis in Firefly Luciferases and CG6178.

822
Y. OBA et al.
Fig. 2. Substrate Specificity of Firefly Luciferases and CG6178 for Fatty Acids.
Fatty acyl-CoA synthetic activity was determined by the formation of acyl-adenylate from a series of fatty acids. Fatty acyl-adenylate
formation was monitored by detection of released 32P-AMP from [ꢀ-32P]ATP with TLC analysis. The data represent the means ꢃ SEM for
triplicate determinations. Relative activity for each enzyme is expressed as a percentage with respect to lauric acid. Abbreviations are as follows:
C8:0, octanoic acid; C10:0, decanoic acid; C12:0, lauric acid; C14:0, myristic acid; C16:0, palmitic acid; C16:1, palmitoleic acid; C18:1, oleic
acid; C18:2, linoleic acid; C18:3n-3, ꢀ-linolenic acid; C18:3n-6, ꢁ-linolenic acid; C20:4, arachidonic acid; C20:5, EPA; luciferin, firefly
luciferin.
Fig. 3. Substrate Specificity of Firefly Luciferases and CG6178 for Other Carboxylic Acids.
The assay method was same as that described in Fig. 2. Relative activity is expressed as a percentage with respect to arachidonic acid. Each
point represents the value of independent experiments (n ¼ 2 or 3). Closed triangle, P. pyralis luciferase; open diamond, L. cruciata luciferase;
closed circle, D. melanogaster CG6178. 2-Thz, thiazolidine-2-carboxylic acid; 2-QA, 2-quinolinecarboxylic acid.
CG6178 (Fig. 3). Very weak activities (0 to 10%
activity with arachidonic acid) were observed for amino
and imino acids (L-proline, D-proline, L-cysteine, D-
cysteine, L-phenylalanine, L-thiaproline, and thiazoli-
dine-2-carboxylic acid). These results suggest that the
structural similarity to the thiazole moiety of firefly
luciferin is not essential for substrate recognition in
acyl-CoA synthesis. No significant activity was ob-
served (less than 5% activity with arachidonic acid) for
short chain acids (acetic acid, propionic acid, n-hexanoic
acid, and lipoic acid) or aromatic acids (p-coumaric
acid, caffeic acid, ferulic acid, 1-naphtylacetic acid, 2-

Firefly Luciferase and CG6178 as a Fatty Acyl-CoA Synthetase
823
naphtylacetic acid, 2-quinolinecarboxylic acid, and
benzoic acid) in firefly luciferase or CG6178.
½M ꢀ Hꢆꢀ (m=z ¼ 948:3) and ½M þ Li ꢀ 2Hꢆꢀ (m=z ¼
954:3). Thus the reaction product of lauric acid
with firefly luciferase and CG6178 was lauroyl CoA
(Table 1).
Lauroyl CoA production by enzyme reaction
The reaction product of lauric acid with firefly
luciferases and CG6178 was identified as lauroyl CoA
by TLC analysis using [1-14C]lauric acid and MALDI-
TOF-MS analysis. TLC analysis showed that the Rf
value (¼ 0:38) of the radioactive spot generated from the
enzymatic reaction was identical to that of authentic
lauroyl CoA (Fig. 4). Also, the product from the
enzymatic reaction with lauric acid was isolated by
HPLC and applied to MALDI-TOF-MS analysis. The
mass values of the product corresponding to ½M ꢀ Hꢆꢀ
(calculated mass 948.3) and ½M þ K ꢀ 2Hꢆꢀ (calculated
mass 986.3) of lauroyl CoA were detected, and the
authentic sample of lauroyl CoA exhibited values of
Enzymatic properties of firefly luciferases and
CG6178 for lauric acid
The effects of the concentration of lauric acid on acyl-
CoA synthetic activity in firefly luciferase and CG6178
were investigated (Fig. 5). With P. pyralis luciferase
and CG6178, strong inhibition of acyl-CoA synthetic
activity was observed at high concentrations (over
20 mM) of long-chain fatty acids (C18ꢁC20), as report-
ed.4,5) However, no inhibition by lauric acid was
observed at a concentration of 40 mM. A weak inhibitory
effect was detected only over 80 mM of lauric acid in
CG6178. The Km, Vmax, and kcat values of these enzymes
for lauric acid at a concentration of 5–50 mM were
determined from Lineweaver–Burk plots (Table 2). The
Km values of P. pyralis luciferase, L. cruciata lucifer-
ase, and CG6178 were 7.41, 16.3, and 1.68 mM respec-
tively. The affinity of lauric acid for P. pyralis luciferase
was about 2 times higher than that for L. cruciata
luciferase. The affinity of lauric acid was about 2 times
Fig. 4. Autoradiography of 14C-Lauroyl CoA Produced by Enzymat-
ic Reaction.
½1-14C]Lauric acid was incubated with enzyme in the presence of
ATP, CoA, and Mg2þ. The product was separated by TLC and the
radioactivity was measured with an imaging analyzer. The arrow
indicates the position corresponding to authentic lauroyl CoA. Pp,
P. pyralis luciferase; Lc, L. cruciata luciferase; Dm, D. melanogast-
er CG6178; NE, no enzyme.
Table 1. MALDI-TOF-MS Analyses of Lauroyl CoA Produced by
Enzymatic Reaction
Lauroyl
CoA
Lauroyl
CoA
Fig. 5. Effect of Lauric Acid Concentration on Fatty Acyl-CoA
Synthesis.
P. pyralis L. cruciata CG6178
m=z
Calcd.
The formation of lauroyl CoA was determined by HPLC
procedures, as described in ‘‘Materials and Methods’’. The vertical
axis represents the initial velocity (v) of the reaction (mmol/min/mg
of enzyme). Closed triangle, P. pyralis luciferase; open diamond,
L. cruciata luciferase; closed circle, D. melanogaster CG6178.
948.2
—
986.1
948.3
—
986.4
948.5
—
986.3
948.3 948.3 ½M ꢀ Hꢆꢀ
954.3 954.3 ½M þ Li ꢀ 2Hꢆꢀ
—
986.3 ½M þ K ꢀ 2Hꢆꢀ
Table 2. Comparison of Kinetic Parameters
Km
Vmax
kcat
(sꢀ1
kcat=Km
ꢀ1
Enzyme
Substrate
(mM)
(mmol/min/mg)
)
(mM ꢇsꢀ1
)
P. pyralis luciferase
Lauric acid
ꢀ-Linolenic acida
Lauric acid
7.41
13.6
16.3
1.68
1.88
0.273
0.127
0.640
0.373
0.490
0.278
0.130
0.651
0.402
0.530
0.0375
0.0096
0.0399
0.2393
0.2819
L. cruciata luciferase
CG6178
Lauric acid
ꢀ-Linolenic acida
aRef. 5

824
Y. OBA et al.
higher than that of ꢀ-linolenic acid in P. pyralis
luciferase. The Km value of lauric acid for CG6178
was almost same as that of ꢀ-linolenic acid.
Furthermore, rat ACSL activity was observed in
peroxisomes,22,23) as in firefly luciferase.24) Firefly
luciferases and CG6178 possess a peroxisomal targeting
signal sequence at the C-terminus.25,26) Thus the sub-
strate specificity and the cell localization of firefly
luciferase are similar to that of mammalian ACSL rather
than ACSM.
Studies of the X-ray structure analysis of firefly
luciferase27) and the mutagenesis analysis28) have been
reported, and the binding residues of firefly luciferin in
luciferase have been proposed by molecular modeling.
Recently, the crystal structures of ACSL from Thermus
thermophilus, including complexes with ATP analog
and myristoyl-AMP, were first resolved.29) This report
suggests that firefly luciferase and ACSL are similar in
structure, consisting of a large N-terminal domain and a
small C-terminal domain. The catalytic site was formed
at the junction between the two domains. Furthermore,
three motifs specific for ACSLs were also conserved in
firefly luciferase.29) The structural similarities and the
substrate specificities support the conclusion that both
firefly luciferase and CG6178 are to be classified as
ACSL.
Phylogenetic analysis of acyl-CoA synthetase
(Table 3) suggests that these genes are mainly classified
into four different families: AceCS, ACSM, ACSL, and
VLCS.16) In this study, we constructed a phylogenetic
tree of acyl-CoA synthetase genes including beetle
luciferases, CG6178, plant 4CLs, unknown genes of
D. melanogaster, and fungi (Fig. 6). It indicates that
beetle luciferases and CG6178 form a clade with
unknown genes of fungi and plant 4CLs. This group is
independent of the AceCS, ACSM, ACSL, and VLCS
families, suggesting that beetle luciferases (and
CG6178) are phylogenetically different from the type
of ACSL in mammals and yeast. Interestingly, the gene
products possessing the peroxisomal targeting signal
sequence 1 (PTS1, marked by asterisks in Fig. 6) were
all located in this clade (referred to as the PTS1 family).
Furthermore, no gene grouped in the PTS1 family was
found in vertebrate genomes (including Fugu, zebrafish,
human, and mouse genomes, searched by genomic
BLAST: http://www.ncbi.nlm.nih.gov/). This suggests
that the ortholog of the PTS1 family has been lost in the
vertebrate lineage. We postulate that the PTS1 family
originates from ACSL (not AceCS), because a gene
product of FadD in E. coli (see Fig. 6) has been
identified as ACSL.30)
Discussion
In the present study, we found that P. pyralis
luciferase, L. cruciata luciferase, and D. melanogaster
CG6178 showed significant activity of fatty acyl-CoA
synthesis from saturated medium-chain fatty acids
(C8ꢁC14) and unsaturated long-chain fatty acids
(C16ꢁC20) (Fig. 2). Among the fatty acids examined,
lauric acid (C12:0) was the best substrate for acyl-CoA
synthesis in firefly luciferases and CG6178. At the
present time, the relationship between the carbon length
of lauric acid and the structure of firefly luciferin has not
been explained clearly. Other carboxylic acids, short-
chain acids (< C6), aromatic acids, amino acids, and
imino acids were not utilized as the substrate for the
adenylate formation in acyl-CoA synthesis. Based on
these results, firefly luciferase and CG6178 will be
classified into ACSM or ACSL.
Previously, Ueda and Suzuki13) and Matsuki et al.14)
reported that long-chain fatty acids (C14:0, C16:0,
C18:0, and C20:4) strongly inhibited the luminescence
activity of firefly luciferase (IC50 ¼ 0:62{0:68 mM), that
fatty acids of C10:0 and C12:0 were weaker inhibitors
(IC50 ¼ 13:2 and 1.2 mM, respectively), and that carbox-
ylic acids of C4:0ꢁC8:0 showed low inhibition (IC50
3{14 mM). Lipoic acid is also a strong inhibitor (IC50
0:05 mM),15) but it was not used for adenylation (Fig. 3).
Thus the carboxylic acid, which strongly inhibits the
luminescent activity of firefly luciferase, is not always a
good substrate for acyl-CoA synthesis.
¼
¼
Several genes having significant homology with those
coding for ACSM and ACSL were found in the insect
genome databases of D. melanogaster, Apis mellifera,
and Anopheles gambiae, but their gene products have
not been characterized enzymatically. In addition, no
genes for ACSM and ACSL were identified in insects. In
mammals, ACSM gene products have been studied.16)
These enzymes are localized in mitochondria and
exhibited substrate specificity for C6ꢁC12 saturated
acids. The best substrates are n-hexanoic acid (C6:0) and
octanoic acid (C8:0). Some aromatic acids, such as
benzoic acid and naphthylacetic acid, showed significant
activity for purified bovine ACSM.17) These character-
istics of ACSM in mammals are different from that of
firefly luciferases and CG6178 (Figs. 2 and 3). On the
other hand, ACSL gene products have been well
characterized from various organisms. Five genes were
cloned from rat, mouse, and human,18) and the substrate
specificities of these enzymes are different from each
other.19–21) Interestingly, the substrate specificity of rat
Acsl3 (rAcsl3 in Table 3 and Fig. 6) is similar to that of
firefly luciferases and CG6178. The efficient substrates
for rat Acsl3 are lauric acid (C12:0), myristic acid
(C14:0), arachidonic acid (C20:4), and EPA (C20:5).19)
In insects, studies of the content of medium-chain
fatty acids (C8ꢁC14) were not characterized in detail. In
D. melanogaster, C12:0 is not abundant, as compared to
long-chain fatty acids.31) In Coleoptera, including
Lampyridae, the amounts of fatty acids of C14:0 and
C14:1 are trace or not detected.32) On the contrary, the
content of long-chain fatty acids (C16ꢁC20) in insects
has been well determined, and the main components are
C16 and C18 (reviewed in ref. 32). Nor Aliza et al.33)
reported that the proportions of arachidonic acid (C20:4)

Firefly Luciferase and CG6178 as a Fatty Acyl-CoA Synthetase
825
Table 3. The Members of Acyl-CoA Synthetase
Name
Mammal
mAcsm
Access. No.
Organism
Function of gene product
PTS1 signala
Reference
NP 473435
NM 016870
AAB87982
NP 036952
NP 476448
NP 446075
NP 446059
NP 570095
NM 005622
NM 052956
NP 003636
BAA91273
AAF75064
Q9NUB1
Mus musculus
Mus musculus
Mus musculus
ACSM
ACSM
VLCS
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
16
16
35
19
19
20
21
36
mSA protein
mVLCS
rAcsl1
Rattus norvegicus
Rattus norvegicus
Rattus norvegicus
Rattus norvegicus
Rattus norvegicus
Homo sapiens
Rat ACSL
Rat ACSL
Rat ACSL
Rat ACSL
Rat ACSL
Human SA gene
Putative ACSM
VLCS
rAcsl3
rAcsl4
rAcsl5
rAcsl6
hSA protein
hACSM
hVLCS
Homo sapiens
Homo sapiens
37
38
BAA91273
hAceCS1
hAceCS2
Homo sapiens
Homo sapiens
Homo sapiens
Unknown
AceCS
Putative AceCS
Insect
P. pyralis
L. cruciata
Railroad worm
Click beetle
CG3961
AAA29795
M26194
Photinus pyralis
Luciola cruciata
Phrixothrix hirtus
Luciferase and ACSL
Luciferase and ACSL
Railroad worm luciferase
Jamaican click beetle luciferase
Unknown
þ
þ
þ
þ
ꢀ
ꢀ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢃ
ꢀ
ꢀ
4, 39
4, 40
41
AF139645
AAQ11720
NP 649067
NM 141903
NM 142964
NM 079984
NP 724696
NM 136935
S52154
Pyrophorus plagiophthalamus
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
Drosophila melanogaster
42
CG4830
CG6178
Unknown
ACSL
5
CG7400
CG8732
Putative fatty acid transporter
Unknown
Unknown
CG8834
CG9390
CG9993
Putative AceCS
Unknown
Unknown
NP 611518
NM 142574
NM 139736
CG11407
CG18586
Nematode
F11A3.1
Unknown
CAA94751
Caenorhabditis elegans
Unknown
þ
Plant
At4CL1
At4CL2
At4CL3
U18675
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis 4CL
Arabidopsis 4CL
Arabidopsis 4CL
ꢀ
ꢀ
ꢀ
þ
þ
þ
43
43
43
AF106085
AF106087
AY250835
AY250839
AY250834
At5g63380
At4g05160
At4g19010
Fungi
Arabidopsis 4CL-like protein
Arabidopsis 4CL-like protein
Arabidopsis 4CL-like protein
Ustilago 1
Ustilago 2
Aspergillus 1
Aspergillus 2
yAceCS1
yAceCS2
yVLCS
EAK81955
EAK85592
EAA57739
EAA61507
AAC04979
P52910
Ustilago maydis
Ustilago maydis
Unknown
Unknown
ꢀ
þ
þ
þ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Aspergillus nidulans
Aspergillus nidulans
Unknown
Unknown
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Yeast AceCS
Yeast AceCS
44
44
45
46
46
NP 009597
NP 014962
NP 010931
NP 013974
Yeast VLCS (Fat1p)
Yeast ACSL (Faa1p)
Yeast peroxisomal ACSL (Faa2p)
Yeast putative ACSL (FAA4)
yACSL (FAA1)
yACSL (FAA2)
yACSL (FAA4)
Bacteria
PheA
acvA
eAceCS
1AMUA
P19787
Brevibacillus brevis
Penicillium chrysogenum
Escherichia coli K12
(See in Materials and methods)
(See in Materials and methods)
Putative AceCS
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
NP 418493
AB126656
P29212
ttLC-FACS
FadD
ScCCL
Thermus thermophilus HB8
Escherichia coli K12
Streptomyces coelicolor A3(2)
ACSL
ACSL
Cinnamate:CoA ligase
29
30
47
NP 628552
aThe PTS1 signal was searched by the PTS1 predictor (http://mendel.imp.univie.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp). These genes were classified as
‘‘targeted’’ which possess the PTS1 signal (þ), ‘‘not targeted’’ which do not possess the PTS1 signal (ꢀ), or ‘‘twilight zone’’ which the predictor could not classify (ꢃ).
and EPA (C20:5) are high in tissue lipids of adult
P. pyralis, especially in the light organ. These facts are
of interest in connection with our present results that
arachidonic acid and EPA are good substrates for
P. pyralis and L. cruciata luciferases. Palmitoleic acid
(C16:1) is found in small amounts in a Lampyridae,32,33)
but is abundant in D. melanogaster.31,32) There is much
evidence that the ꢃ-oxidation pathway of fatty acids is

826
Y. OBA et al.
Fig. 6. Phylogenetic Tree of Acyl-CoA Synthetase Genes.
The tree was constructed by the neighbor-joining method using amino acid sequences. Abbreviated names of the genes are listed in Table 3.
Numbers indicate bootstrap values from 1,000 replicates. Only the bootstrap values over 50% are indicated on the nodes. Horizontal branch
lengths indicate genetic distances. The genes marked by an asterisk possess the PTS1 signal at the C-terminus. A shaded clade shows the PTS1
family.
present in insects, and ACSL activity in peroxisomes has
also been studied in several insect species.34) Firefly
luciferase might have another physiological role, such as
the ꢃ-oxidation of fatty acids.
In conclusion, our results suggest that beetle lucifer-
ase is evolved from a novel type of fatty acyl-CoA
synthetase, which differs from ACSM and ACSL in
mammals and yeast.
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