Chemical synthesis and firefly luciferase produced dehydroluciferyl- coenzyme A

Article abstract of DOI:10.1016/j.tetlet.2004.01.050

Dehydroluciferyl-coenzyme A (L-CoA) was chemically synthesized and characterized by MS, UV-vis spectrometry and RP-HPLC. The identity of the chemically synthesized compound with the one that was produced by firefly luciferase was confirmed. Moreover, the reversibility of the enzymatic conversion of dehydroluciferin?dehydroluciferyl-adenylate?L-CoA was also confirmed. The chemical synthesis of L-CoA, described here, may help the clarification of the activator effect of CoA on luciferase bioluminescent assays, in which the enzyme catalyzed formation of L-CoA and the consequent destruction of L-AMP is one of the possible explanations for that effect.

Full text of DOI:10.1016/j.tetlet.2004.01.050

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 2117–2120
Chemical synthesis and firefly luciferase produced
dehydroluciferyl-coenzyme A
a,b
Hugo Fraga, Joaquim C. G. Esteves da Silva and Rui Fontes
a,
b
*
a
LAQUIPAI, Chemistry Department, Faculdade de Ci ^e ncias do Porto, R. Campo Alegre 687, 4169-007 Porto, Portugal
Biochemistry Department (U38-FCT), Faculdade de Medicina do Porto, 4200-319 Porto, Portugal
b
Received 18 December 2003; revised 8 January 2004; accepted 13 January 2004
Abstract—Dehydroluciferyl-coenzyme A (L-CoA) was chemically synthesized and characterized by MS, UV–vis spectrometry and
RP-HPLC. The identity of the chemically synthesized compound with the one that was produced by firefly luciferase was confirmed.
Moreover, the reversibility of the enzymatic conversion of dehydroluciferin ꢀ dehydroluciferyl-adenylate ꢀ L-CoA was also
confirmed. The chemical synthesis of L-CoA, described here, may help the clarification of the activator effect of CoA on luciferase
bioluminescent assays, in which the enzyme catalyzed formation of L-CoA and the consequent destruction of L-AMP is one of the
possible explanations for that effect.
ꢀ
2004 Elsevier Ltd. All rights reserved.
Firefly luciferase is an enzyme that catalyzes the oxida-
0
E þ LH
2
þ ATP ! EÁLH
2
-AMP þ PPi
ð1Þ
0
tion of luciferin (LH ){(S)-2-[6 -hydroxy-2 -benzothia-
2
zolyl]-2-thiazoline-4-carboxylic acid} (Fig. 1) in the
presence of ATP and magnesium ion giving rise to
EÁLH -AMP þ O
2
2
! E þ AMP þ CO
2
1
þ oxyluciferin þ photon ð2Þ
light. The bioluminescent reaction involves the forma-
tion, from LH and ATP, of an enzyme bound adenylyl
2
The supplementation of reaction media with coenzyme
A (CoA) results in an enhancement in the light pro-
duction and in a delay in the characteristic inhibition
profile. In the course of the bioluminescent reaction
L-AMP and L are also produced, and the activating
effect of CoA on firefly light emission has been inter-
preted as a consequence of its reaction with the L moiety
of the EÆL-AMP complex yielding L-CoA and liberating
enzyme, allowing it to recycle with further light pro-
2
;3
intermediate (LH
sequent oxidation with release of AMP, pyrophosphate
0 0
PPi), CO2 and oxyluciferin {2-[6 -hydroxy-2 -benzo-
thiazolyl]-4-hydroxythiazole} (Fig. 1) (reaction 2), the
2
-AMP) (reaction 1) and its sub-
(
4
presumed light emitter. Apart from oxyluciferin,
0
0
dehydroluciferin (L) {2-[6 -hydroxy-2 -benzothiazolyl]-
thiazole-4-carboxylic acid} is also an oxidative product
of LH
2
. Its formation from the luciferin moiety of the
EÆLH -AMP was previously reported and its role on the
2
2;5–7
duction.
characteristic decay of the firefly light production has
5
been supported.
8
As pointed by McElroy et al. in 1968, the mechanism of
synthesis of L-CoA by luciferase (reactions 3 and 4) may
be similar to that of fatty acid activation by acyl-CoA
synthetases.
E þ L þ ATP ꢀ EÁL-AMP þ PPi
ð3Þ
ð4Þ
Figure 1. Chemical structures of luciferin and oxyluciferin.
EÁL-AMP þ CoA ꢀ E þ L-CoA þ AMP
The suspected structural relationship between firefly
luciferase and acyl-CoA synthetases was confirmed by
molecular biology studies in the last decade; these and
other enzymes are now grouped under the denomina-
tion of acyl-adenylate/thioester-forming enzyme family.
Keywords: Dehydroluciferyl-coenzyme A; Firefly luciferase; Co-
enzyme A; Bioluminescence.
1
*
Corresponding author. Tel.: +351-22-608-2869; fax: +351-22-608-
959; e-mail: jcsilva@fc.up.pt
2
0
040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.01.050

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H. Fraga et al. / Tetrahedron Letters 45 (2004) 2117–2120
Despite the relevance of the enzymatic process of syn-
thesis of L-CoA, the unequivocal assignment of L-CoA
was never done mainly because the chemical synthesis of
this compound has never been achieved.
With the objective of unequivocally identify the luci-
ferase synthesized L-CoA the chemical synthesis of this
compound was undergone by modifying a procedure
9
employed for the synthesis of acyl-CoA derivatives,
(
Fig. 2). The synthesis started with the dissolution of
chemically synthesized L in tetrahydrofuran (THF)
and left to react overnight with carbonyldiimidazole at
room temperature. The activated dehydroluciferin was
left to react with CoA in an alkaline medium for one
day. Purification was performed by RP-HPLC. The
purified compound was characterized by MALDI mass
spectroscopy (Fig. 3, panel A) and RP-HPLC-DAD
10
11
11
12
(
UV–vis, Fig. 3, panel B). The mass spectra confirmed
the expected mass for the compound formed from a
thioester bound between L and CoA (calculated
M
w
¼ 1027:83 g/mol).
The identification of the luciferase synthesized L-CoA
was obtained by RP-HPLC-DAD, using methodologies
developed for the separation of compounds involved in
Figure 3. MALDI mass (A) and UV–vis (B) spectra of the chemically
synthesized L-CoA.
3
;4;7
the firefly luciferase catalyzed reactions.
luciferase is also able to catalyze the synthesis of L-CoA
when, starting from LH and ATP, the intermediate
Although
2
LH -AMP is oxidized to L-AMP, which then transfers
2
3
the L moiety to CoA, in this work chemically synthe-
sized L-AMP was used as L donor (reaction 4). That is,
the enzymatic synthesis was performed by incubating
13
L-AMP and CoA in the presence of luciferase. The
observed chromatographic retention times and UV–vis
spectrum for both chemically and enzyme synthesized
L-CoA coincided (Fig. 4).
6
Airth et al. were able to verify the reversibility of
reactions 3 and 4 by observing the disappearance of L in
reaction mixtures containing ATP, L, CoA and lucifer-
ase and its reformation when the assay media was sup-
Figure 4. Enzyme and chemically synthesized L-CoA. Control corre-
sponds to an assay similar to Lase-10 min where Lase was omitted.
plemented with AMP and pyrophosphate (PPi). The
entire process was monitored by fluorescence, taking
advantage of the fact that both L-CoA and L-AMP are
nonfluorescent in contrast with L that exhibits strong
fluorescence (excitation 353 nm; emission 540 nm). It
should be noted that some confusion exists in luciferase
related literature because, by that time, L and L-CoA
were wrongly named as oxyluciferin (the light emitter)
2;5;6
Figure 2. Scheme of chemical synthesis of L-CoA.
and oxyluciferyl-CoA, respectively.

H. Fraga et al. / Tetrahedron Letters 45 (2004) 2117–2120
2119
Figure 6. Effect of the pH on the luciferase catalyzed synthesis of
L-CoA from L, ATP and CoA (d; continuous line) or from L-AMP
and CoA (ꢀ; broken line).
and test the conflicting theories on the mechanism of the
stimulating effect of CoA on light emission.
5;6;16;17
Acknowledgements
Figure 5. Luciferase catalyzed synthesis of L from L-CoA, AMP and
PPi. Panel A––fluorescence profiles: curves 1, 2and 3 correspond to
different concentrations of luciferase; 0.011, 0.023 and 0.033 mg pro-
tein/mL, respectively. Panel B––chromatograms of the reaction mix-
ture stopped at 5 and 60 min of incubation; control corresponds to an
assay where luciferase was omitted stopped at 60 min.
Financial support from FCT (FSE-FEDER) (project
POCTI/QUI/37768/2001) is acknowledged.
References and notes
1
2
. Wood, K. V. Photochem. Photobiol. 1995, 62, 662–673.
. Rhodes, W. C.; McElroy, W. D. J. Biol. Chem. 1958, 233,
We have checked the same reversibility using the
chemically synthesized and purified L-CoA mixed with
1
528–1537.
14
AMP, PPi and luciferase; the reaction was monitored
by fluorescence and also by RP-HPLC (Fig. 5). The
chromatographic results showed that, in those condi-
tions, L-CoA was converted into L (Fig. 5, panel B).
When the reaction was monitored by fluorescence we
confirmed the expected increase of fluorescence along
the incubation time (Fig. 5, panel A) and the effect of
luciferase concentration on the speed of the reaction.
3
4
. Fraga, H.; Esteves da Silva, J. C. G.; Fontes, R.
ChemBioChem. 2004, 5, 110–115.
. Esteves da Silva, J. C. G.; Magalh ~a es, J. M. C. S.; Fontes,
R. Tetrahedron Lett. 2001, 42, 8173–8176.
5. Fontes, R.; Dukhovich, A.; de Diego, A.; Sillero, A.;
G u€ nther Sillero, M. A. Biochem. Biophys. Res. Commun.
1
997, 237, 445–450.
. Airth, R. L.; Rhodes, W. C.; McElroy, W. D. Biochim.
Biophys. Acta 1958, 27, 519–532.
. Fontes, R.; Ortiz, B.; de Diego, A.; Sillero, A.; G u€ nther
Sillero, M. A. FEBS Lett. 1998, 438, 190–194.
. McElroy, W. D.; DeLuca, M.; Travis, J. Science 1967,
57, 150–160.
. Kawaguchi, A.; Yoshimura, T.; Okuda, S. J. Biochem.
981, 89, 337–391.
6
7
8
9
The formation of L from L-CoA could not be attributed
to hydrolysis and was catalyzed by luciferase: the reac-
tion depended on the simultaneous presence of AMP,
PPi and luciferase (reactions 4 and 3).
1
1
15
Using the same chromatographic methodologies the
pH dependence of the luciferase catalyzed synthesis of
L-CoA either from ATP, L and CoA or from L-AMP
and CoA was also studied. The obtained pH profiles
10. Dehydroluciferin was synthesized from 2-cyano-6-meth-
oxibenzothiazole as described by White, E. H.; McCapra,
F.; Field, G. F. J. Am. Chem. Soc. 1963, 85, 337–343.
1
1. L (380 lmol) was dissolved in THF until total solubiliza-
tion (about 100 mL). To this solution 800 lmol of
carbonyldiimidazole in THF were added and the reaction
mixture was left overnight. The solvent was evaporated off
(Fig. 6) showed that, in both cases, the enzyme activity
had a maximum at about 7.5.
and the residue was dissolved in THF–H
solution was supplemented with 250 lmol of CoA dis-
solved in 25 mL of THF–H 0 (2:1); the pH of the reaction
was adjusted to 7.0–7.5 with 0.5 M NaOH and the reaction
mixture was left for about one day. THF was evaporated
2
0 (2:1). This
This is the first report of the chemical synthesis of
L-CoA. The observed stability of this compound may
allow extended purification processes that will permit the
study of its effect on luciferase bioluminescent reaction
2

2
120
H. Fraga et al. / Tetrahedron Letters 45 (2004) 2117–2120
off and the residual aqueous solution was acidified to
pH 3–4 with the addition of small amounts of a cation
purified L-CoA (estimated final concentrations of L-CoA
2.5 lM, L 0.13 lM in 2mM phosphate buffer pH 7.0),
200 lM AMP, 200 lM PPi and luciferase (0.011; 0.022;
0.033 mg protein/mL). The reactions were initiated with
the addition of luciferase and were performed in quartz
cell in a Perkin–Elmer luminescence spectrometer LS-50
(excitation 340 nm; emission 550 nm). In the assays studied
by RP-HPLC the reaction mixtures contained in the final
volume of 100 lL: 10 lL of partially purified L-CoA
(estimated concentration of 140 lM L-CoA and 7 lM L);
1 mM AMP, 1 mM PPi, 100 mM Hepes (pH 7.8) and
luciferase (0.13 mg protein/mL). At different times of
incubation (0–60 min) 20 lL aliquots were withdrawn
and added to 40 lL of 10 mM EDTA in 66% methanol.
The elution conditions were the same as referred above.
15. The reaction for the enzymatic synthesis of L-CoA from
ATP, L and CoA in different pH media contained the
following in a total volume of 60 lL: 30 lM L, 500 lM
þ
exchange resin (Amberlite IR-120-H ). The resin was
removed by filtration, and the filtrate was extracted with
diethyl ether (2 · 20 mL) to remove unreacted L. The
aqueous phase and the dissolved residue of the organic
phase (after evaporation of the ether) were analyzed by
RP-HPLC. The chromatographic system was constituted
by a HP-1100 isocratic pump, a rheodyne manual injection
valve, silica-based octadecyl columns and a Unicam
Cristal 250 ultraviolet (UV–vis) diode array detector.
The eluent was a solution of 29% methanol, 2 mM
phosphate buffer (pH 7.0), and the flow rate was set to
1
2. The purification of L-CoA present in the aqueous phase
mL/min. The aqueous phase was lyophilized.
1
1
was performed by HPLC using a Supelco semi-preparative
chromatographic column, the eluent was a solution of 35%
methanol, phosphate buffer pH 7.0, with a flow rate of
2mL/min.
ATP, 2mM MgCl
2
, 100 mM buffer (MES pH ¼ 6.3 or
3. The reaction mixture for the enzymatic synthesis of
L-CoA contained in the final volume of 200 lL: 0.5 mM
CoA, 60 lM L-AMP, 100 mM Hepes buffer (pH 7.8) and
luciferase (0.08 mg protein/mL). At different times of
incubation (0–10 min) 35 lL aliquots were withdrawn
from the reaction mixtures and added to 35 lL of
HEPES pH ¼ 7.0, 7.5 or 8.3), PPase 1 U/mL and luciferase
(0.035 mg protein/mL). In the case of the synthesis of
L-CoA from L-AMP (30 lM) and CoA the reactions
2
mixtures did not contain ATP, L, MgCl and PPase. After
5 min (synthesis from ATP, L and CoA) or 30 s (synthesis
from L-AMP and CoA) of incubation, 15 lL aliquots
were withdrawn from the reaction mixtures, added to
66% methanol and analyzed by RP-HPLC as referred
above.
1
0 mM EDTA in 66% methanol. Aliquots (50 lL) were
injected, the flow rate was 0.6 mL/min and the eluent was a
solution of 29% methanol (v/v) and 1 mM phosphate
buffer (pH 7). These elution conditions were also used with
chemically synthesized L-CoA (see Fig. 4).
16. Ford, R. F.; Hall, M. S.; Leach, F. R. Anal. Biochem.
1992, 204, 283–291.
1
4. In the assays studied by fluorescence the reaction mixtures
contained in the final volume of 600 lL: 50 lL of partially
17. Ford, R. F.; Buck, L. M.; Leach, F. R. Biochim. Biophys.
Acta 1995, 180–184.