- Email: [email protected]
Stereoisomeric bio-inversion key to biosynthesis of firefly d -luciferin
FEBS Letters 580 (2006) 5283–5287
Stereoisomeric bio-inversion key to biosynthesis of firefly D -luciferin Kazuki Niwaa,b, Mitsuhiro Nakamurac, Yoshihiro Ohmiyaa,* a b
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Japan National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (AIST), Japan c Integrated Research Institute, Tokyo Institute of Technology, Japan Received 6 June 2006; revised 7 August 2006; accepted 30 August 2006 Available online 11 September 2006 Edited by Judit Ova´di
Abstract The chirality of the luciferin substrate is critical to the luciferin–luciferase reaction producing bioluminescence. In firefly, the biosynthetic pathway of D -luciferin is still unclear, although it can be synthesized in vitro from D -cysteine. Here, we show that the firefly produces both D - and L -luciferin, and that the amount of active D -luciferin increases gradually with maturation stage. Studies of firefly body extracts indicate the possible conversion of L -cysteine via L -luciferin into D -luciferin, suggesting that the biosynthesis is enzymatically regulated by stereoisomeric bio-inversion of L -luciferin. We conclude that the selection of chirality in living organisms is not as rigid as previously thought. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Bioluminescence; Biosynthesis; Enantiomer; Firefly; Luciferase; Luciferin
1. Introduction Biologically active natural products are usually homochiral: proteins are composed of mainly L -amino acids and L -menthol produces a mint-like smell whereas D -menthol is a stronger stimulant than its L -counterpart. Thus, the determination and selection of chirality in natural products is considered as a rigid system. Bioluminescence is a typical example of a substrate-enzyme reaction [1]. Most such reactions are efficient and most luciferins are chiral and are biosynthesized from peptides or L -amino acid. For instance, only Cypridina L -luciferin exhibits bioluminescence potential although in chemiluminescence, the D -, L -, and DL -forms produce light of the same intensity [2]. These results indicate that Cypridina luciferase recognizes only one of the enantiomers and this luciferin is biosynthesized from L amino acids or tripeptides. Similarly, only firefly D -luciferin contributes to bioluminescence, whereas L -luciferin does not but strongly inhibits [3,4]. How does firefly biosynthesize D luciferin?
* Corresponding author. Address: 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan. Fax: +81 72 751 9628. E-mail address: [email protected] (Y. Ohmiya).
Abbreviations: ABD-F, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; BSA, Bovine serum albumin; CHBT, 2-cyano-6-hydroxybenzothiazole; CoA, coenzyme A; TBP, tri-n-butylphosphine
Firefly D -luciferin can be chemically synthesized from 2-cyano-6-hydroxybenzothiazole (CHBT) and D -cysteine [5] whose stereochemistry corresponds to that of D -luciferin (Fig. 1A). The putative biosynthetic precursors of D -luciferin are also cysteine and CHBT [6]. Incorporations of isotope-labeled DL -cystine and CHBT into D -luciferin in fireflies have been reported [7–9]. However, the detail biosynthetic mechanism of firefly D -luciferin has remained ambiguous for almost half a century since the first report of its chemical structure [5]. Especially, CHBT is so active that even in the absence of enzyme, it can react non-enzymatically with L - as well as D -cysteine, producing L -luciferin [6]. Although some D -amino acids were reported to have functional roles and to be widely distributed in living organisms [10], there are no reports to date of the presence of free D -cysteine and no biosynthetic studies of it in any eukaryotes [11]. From these findings, we supposed that L -luciferin could be the biosynthetic intermediate despite the fact that it is the enantiomer of a biologically active natural product (Fig. 1B). On the other hand, Lembert and we have previously reported the in vitro enzymatic formation of D luciferin from L -luciferin [4,12]. In this study, we propose a new biosynthetic pathway of firefly D -luciferin, focussing on its chirality, which has remained ambiguous since its chemical structure was first reported 45 years ago. We show that the firefly contains a considerable amount of L -luciferin, an enantiomer of active D -luciferin, and that the enantiomer is enzymatically inverted to D -luciferin. Furthermore, the chirality of cysteine, the putative biosynthetic precursor of D -luciferin, is almost L -form. As we have already reported, luciferase could be responsible for the stereoisomeric inversion of L -luciferin to D -luciferin [12]. These facts suggest that firefly D -luciferin is synthesized in vivo from L -cysteine and the biosynthetic intermediate is L -luciferin.
2. Materials and methods 2.1. Materials The Japanese firefly Luciola lateralis (Coleoptera) was raised in our laboratory with a commercially available incubation system (Environmental Engineering Laboratory Co., Ltd., Maebashi, Japan). Authentic D - and L -luciferin were prepared from CHBT as previously described [13]. 2.2. Preparation and chiral analysis of firefly luciferin Twelve larvae, three larvae in cocoon, twelve pupae, six adults in cocoon, and eight adults of L. lateralis were analyzed as described below. Luciferin in a firefly body was eluted with 200 ll of ethanol containing 0.5% tri-n-butylphosphine (TBP) at 70 C, and homogenized. The
0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.08.073
5284
K. Niwa et al. / FEBS Letters 580 (2006) 5283–5287
N
A
CN
+
S
HO
H2N * COOH HS
/ buffer
HO
N * COOH
S
S
D-luciferin
D-cysteine
CHBT
N
H2N * COOH
B
CHBT
HS
?
H2N * COOH
D-cysteine
HS L-cysteine
N
N * COOH
S
S
CHBT HO
HO ?
N
N * COOH
S
S
D-luciferin
L-luciferin Fig. 1. Synthesis of firefly D -luciferin. (A) Chemical synthesis of D -luciferin. The asymmetric carbon of cysteine corresponds to that of luciferin. (B) Hypothetical biosynthetic pathways of D -luciferin from L -cysteine.
sample was centrifuged (13 000 rpm, 20 min) and filtered with 0.45 lm membrane filter. The filtrate was injected into a high-performance liquid chromatography (HPLC) system (Alliance HPLC System with 2695 Separation Module, 2475 Multi k Fluorescence Detector, and 2996 Photodiode Array Detector, Waters). Linear gradient elution (15–40% acetonitrile/water with 0.1% TFA, 20 min, 1.0 ml/min) was adopted for the separation with a chiral fused silica column, CHIRALCEL OD-RH (4.6 · 150 mm; Daicel Chemical Industry, Tokyo, Japan). D - and L -Luciferin were detected with a fluorescence detector (excitation k = 330 nm, emission k = 530 nm, see Fig. 2A).
Intensity (fluorescence signal)
A 200 D-luciferin
100
L-luciferin
0 0
5
10
15
25
20
Retention time (min)
Intensity (fluorescence signal)
B
40
D-Cys-ABD L-Cys-ABD
20
Authentic (5 nmol)
0
2.3. Preparation and chiral analysis of firefly cysteine To extract cysteine, four adult firefly bodies suspended in 400 ll of 1 N HCl and 10 ll of 10% TBP/acetonitrile were homogenized and incubated at 100 C for 5 min. The sample was centrifuged (13 000 rpm, 20 min) and filtered with 0.45 lm membrane filter. Cysteine from 100 ll of the filtrate was purified with an ODS column (OD-SR-5, Daicel), and dried. To label fluorescence, the cysteine fraction was dissolved in 125 ll of 0.1 M sodium borate buffer (pH 8.0) containing 1 mM EDTA, 50 ll of 1.0 mM 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F, from Dojindo Laboratories, Kumamoto, Japan) in borate buffer, and 5 ll of 10% TBP/acetonitrile. The mixture was kept at 50 C for 5 min, and then placed on ice and mixed with 60 ll of 0.1 N HCl. The fluorescence-labeled cysteine was injected into the HPLC system equipped with a chiral crown ether fused column, CROWNPAK CR (+) (4.6 · 150 mm; Daicel Chemical Industry, Tokyo, Japan). Isocratic elution with 0.1% TFA in water (1.2 ml/min) was adopted for the separation. Fluorescence-labeled D - and L -cysteine were detected with a fluorescence detector (excitation k = 380 nm, emission k = 530 nm, see Fig. 2B, upper chart). 2.4. Stereoisomeric inversion assay of firefly extract To extract proteins, the light organ of an adult firefly was homogenized in 200 ll of 0.1 M Tris–HCl buffer (pH 8.0) containing 0.1 M NaCl, and this was followed by centrifugation (13 000 rpm, 20 min) to remove debris. The extract was dialyzed to remove contamination by small molecules. An aliquot (200 ll) of 0.1 mM L -luciferin, 1 mM coenzyme A (CoA), 3 mM ATP, and 8 mM MgSO4 in 0.1 M Tris– HCl buffer (pH 8.0) was mixed with 40 ll of the dialyzed protein extract and incubated at room temperature. Luciferin chirality was analyzed with the same procedure as that described above, except that the eluent was changed to isocratic 27% acetonitrile/water containing 0.1% TFA (Fig. 4A). To test the enzymatic reaction, an aliquot (2 ll) of 1 mM L -luciferin was mixed with 40 ll of various protein solutions and 198 ll of various cofactor solutions (Fig. 4C). Protein extracts from Luciola cruciata (sampled from Tsushima Island, Nagasaki) and Photinus pyralis (from Sigma) were also tested.
20
Extract
3. Results and discussion
10 0 0
5
10
15
20
25
Retention time (min) Fig. 2. Chiral analyses of in vivo luciferin and cysteine. (A) Chromatogram of luciferin in an adult firefly, obtained with a chiral column. (B) Chromatogram of ABD-labeled cysteine from four adult fireflies, obtained with a chiral column.
In order to gain an insight into the biosynthesis of active luciferin, we analyzed bioluminescence-related compounds during the life cycle of L. lateralis (Fig. 3). The adult of this firefly produces flashing light signals to communicate and attract a mate [14]. On the other hand, the larva and pupa occasionally produce a continuous dim glow, the biological function of which is not known. The larva, an aquatic glowworm, feeds on snails but the other stages do not eat anything.
K. Niwa et al. / FEBS Letters 580 (2006) 5283–5287
5285
Larva
Pupa
Aquatic
In cocoon of soil
Mating
1.2 1.0 0.8 0.6 0.4 0.2 0
100 80 60 40 20 0
L
LC
P
AC
Enantiomeric excess of D-luciferin (% ee)
Fasting
Feeding Total DL-luciferin (nmol / body)
Adult
A
Fig. 3. Life stage, luciferin content, and D -luciferin chirality. Illustrated are the five distinctive life stages of the Japanese firefly, L. lateralis. L, larva; LC, larva in cocoon; P, pupa; AC, adult in cocoon; A, adult. Graphs show the total amounts of D - and L -luciferin in an individual body and the enantiomeric excess of D -luciferin (% ee). Error bars indicate standard deviations.
It thrives inside a cocoon made of soil during metamorphosis from larva through pupa to adult. We raised the firefly in our laboratory and collected them to analyze their luciferin by HPLC with a chiral column. Individual samples were divided into five groups depending on the morphologically distinctive life stages: larva, larva in cocoon, pupa, adult in cocoon, and adult. As shown in Fig. 3, the more mature the firefly was, the higher the total amount of luciferin was, demonstrating that the firefly produces and stores luciferin during pupa and adult in cocoon stages despite the fasting period. Both L and D -luciferin were identified by their retention times (Fig. 2A) and biological activities [15]. Both luciferins were detectable at all stages and the enantiomeric excess of D -luciferin was highest at the adult stage, while it was relatively low during larval to pupal stages and variable as individual differences (Fig. 3). Both luciferins were detected in the egg, although its amount and ratio could not be determined accurately (data not shown). We previously reported that L -luciferin is a substrate for the non-luminous enzymatic reaction with firefly luciferase in vitro [12,15] and Lembert have previously reported the light production from L -luciferin [4]. But these facts could not prove the presence and biological role of L -luciferin in vivo. Now we proved the existence of L -luciferin in vivo and the change in ratio of D - and L -luciferin with development, implying that L -luciferin is the biosynthetic intermediate. As regards to the hypothetical biosynthetic precursor for luciferin, we analyzed the chirality of cysteine in vivo (Fig. 2B, lower chart). Although our in vitro assay system enabled detection of D - or L -cysteine in amounts as low as 10 10 moles (data not shown), faint peaks around the retention time for D -cysteine were recognized only when the extracts from four individuals of living fireflies were combined. This result could prove the extreme dominance of L -cysteine over D -cysteine. As CHBT readily reacts with both L - and D -cysteine, it is difficult for the firefly to prevent from producing corre-
sponding luciferins, suggesting that L -luciferin in vivo could be derived from L -cysteine. In vivo, however, L -luciferin must be inverted to D -luciferin to induce the bioluminescence reaction because inactive L -luciferin strongly inhibits the luciferin–luciferase reaction [4]. The next question is what is responsible for the disproportionation of luciferin chirality? We previously reported that in the presence of ATP, Mg2+ and CoA, firefly luciferase exhibits coenzyme A ligase (CoA-ligase) activity on L -luciferin in vitro, but not on D -luciferin, to give luciferyl-CoA [15]. Modification of the carboxyl group in L -luciferin accelerates enolization to give racemic luciferyl-CoA. Hydrolysis of racemic luciferyl-CoA could yield both L - and D -luciferin. We recently reported that L -luciferin was stereoisomerically bioinverted to produce D -luciferin under enzymatic conditions in vitro [12]. We examined the enzymatic activity for the stereoisomeric inversion of L -luciferin in L. lateralis extract (Fig. 4). Soluble proteins extracted from the light organ and dialyzed with buffer to remove small molecules exhibited inversion activity on authentic L -luciferin in the presence of ATP, Mg2+, and CoA (Fig. 4A and B). This activity was suppressed when the extract was boiled and could not be substituted by known proteins including bovine serum albumin (BSA), indicating that the stereoisomeric inversion is an enzymatic reaction in the firefly bioluminescence system (Fig. 4C). The activity was also suppressed in the absence of ATP, Mg2+, or CoA (Fig. 4C), suggesting that the CoA ligation of L -luciferin is essential for the bio-inversion reaction and luciferyl-CoA could be the intermediate. Therefore, CoA-thioesterase could catalyze the hydrolysis of D -luciferyl-CoA to yield D -luciferin. Furthermore, the same potential to invert L -luciferin to D -luciferin was found in the protein extracts from another Japanese firefly, L. cruciata, as well as a North American firefly, P. pyralis (data not shown), suggesting that the inversion activity could be ubiquitous among luminous beetles.
5286
K. Niwa et al. / FEBS Letters 580 (2006) 5283–5287
A
Relative amount (%)
B
Intensity (fluorescence signal)
L-luciferin D-luciferin
2-min reaction 15-min reaction
0
1
2
3
4
5
6
7
8
9
100
D-luciferin L-luciferin 0 0
10
Retention time (min)
Relative amount (%)
C
100
30
60
90
Reaction time (min)
L-luciferin
D-luciferin
before
extract +
0
Firefly extract Boiled extract BSA (10 ug/ml) ATP (3 mM) MgSO4 (8 mM) CoA (1 mM)
boiled
BSA
- ATP +
- Mg2+ +
- CoA +
+ + + + +
+ + +
+ + +
+ + +
+ + +
+ +
+
Fig. 4. Stereoisomeric inversion of L -luciferin to D -luciferin in L. lateralis extract. (A) Chromatograms of a reaction mixture containing L -luciferin, the protein extract, ATP, Mg2+, and CoA. Dashed and solid lines indicate the reaction mixture incubated for 2 and 15 min, respectively. (B) Time course of the inversion reaction. Ratios of D - and L -luciferin to the initial amount of total luciferin are plotted. (C) Inversion activities under various reaction conditions. Ratios of D - and L -luciferin in the reaction mixtures incubated at room temperature for more than 2 h.
nescence [12]. Our new proposal is supported by the inversion metabolism of ibuprofen, in which stereoselective CoA ligation of (S)-ibuprofen is the key step [16]. The reaction mixture for the stereoisomeric inversion, which contained luciferase, ATP, and Mg2+, was sufficient for light production from D -luciferin.
Based on our results and our previous findings [12,15], we propose a new pathway for D -luciferin biosynthesis, as shown in Fig. 5. The most important process is the stereoisomeric bioinversion at the final steps. Luciferase could be responsible for stereoselective CoA ligation of L -luciferin as well as biolumiH2N * COOH HS
HO
L-cysteine
N
N * COOH
S
S
HO
L-luciferin Luciferyl-CoA synthase (Luciferase)
N * COOH
S
S
D-luciferin
ATP, Mg2+, CoA
O
OH
N * C S
CoA
N
C
O S
N * C S
CoA
S
S L-luciferyl-CoA
CoA
S
(enol)
D-luciferyl-CoA hydrolysis enzyme
hydrolysis enzyme
HO
N
N
N * COOH
S
S
L-luciferin
HO
N
N * COOH
S
S
D-luciferin
Fig. 5. Proposed biosynthetic pathway of D -luciferin. L -luciferin is produced from natural L -cysteine. L -Luciferin is converted into L -luciferyl-CoA that is easy to racemize by enolization. Hydrolysis of D -luciferyl-CoA gives the bioluminescent substrate, D -luciferin.
K. Niwa et al. / FEBS Letters 580 (2006) 5283–5287
The reason why D -luciferin remained in the reaction mixture despite being under bioluminescence reaction conditions was because the bioluminescence reaction rate was so low [15] that the consumption of D -luciferin could not be detected in our reaction conditions. Lembert [4] first reported the light production from L -luciferin and proposed that L -luciferin was racemized to give D -luciferin and it was effectively stimulated by the addition of pyrophosphate. This racemization is an in vitro reaction of luciferase in the absence of CoA. In the presence of CoA, L -luciferin is readily converted into luciferyl-CoA [15]. Luciferase in vivo is localized in peroxisome where metabolisms involved in CoA mainly take place [17,18]. Furthermore, biosynthetic pathway should produce homochiral D -luciferin. Therefore, we consider our proposal is reasonable as an in vivo reaction. Because the firefly is able to invert L -luciferin to D -luciferin efficiently, as shown in Fig. 4B, it is not necessary for the firefly to produce D -cysteine. Although there remains the possibility, the firefly does not have to keep mechanisms that CHBT selectively reacts with minimal amounts of D -cysteine to produce D -luciferin alone. On the other hand, in the luciferin recycling pathway [19], the luciferin regeneration enzyme produces CHBT, although the de novo biosynthesis of CHBT as well as any benzothiazole compounds in vivo is not clear. Recently, reports of the multiple functions of luciferase have emerged: CoA-ligase activity on fatty acids [20], and adenosine polyphosphate synthetase activity on ATP, ADP, and AMP [21]. Our results strongly suggest that luciferase also participates in the biosynthesis of D -luciferin by producing luciferylCoA from L -luciferin. However, further research is required to elucidate the detailed mechanism after luciferyl-CoA formation. In previous research, the amount of L -luciferin in vivo and the existence of enzymes for the inversion of L -luciferin to D -luciferin have not been considered. This report introduces a new aspect of the firefly luciferase system. What is the meaning of disproportionation of luciferin chirality depending on the developmental stage? The flashing light of the adult L. lateralis is highly regulated whereas the continuous dim glow before metamorphosis is not passively regulated. For the adult, the saving of active D -luciferin could be helpful for flashing light communication. The flexibility of luciferin chirality could be part of a physiological regulatory system. According to the lock and key theory of E. Fischer, substrate chirality is a rigid system. However, studies of intact organisms indicate that substrate chirality is more flexible than previously thought. The firefly bioluminescence system is a good example of chiral diversity and enzymatic flexibility. Acknowledgements: We thank N. Ohba (Yokosuka City Musium) for many suggestions about culturing L. lateralis and its life cycle. We also thank N. Fujii (Kyoto University) for discussion and suggestions about chiral analysis of cysteine and T. Goto (Tohoku University) about stereoisomeric bio-inversion metabolism. This work was supported by grants from the Ministry of Economy, Trade and Industry of Japan and Takeda Science Foundation.
5287
References [1] Wilson, T. and Hastings, J.W. (1998) Bioluminescence. Annu. Rev. Cell. Dev. Biol. 14, 197–230. [2] Wu, C., Kawasaki, K., Ohgiya, S. and Ohmiya, Y. (2006) Syntheses and evaluation of the bioluminescent activity of (S)-Cypridina luciferin and its analogs. Tetrahedron Lett. 47, 753–756. [3] Seliger, H.H., McElroy, W.D., White, E.H. and Field, G.F. (1961) Stereospecificity and firefly bioluminescence, a comparison of natural and synthetic luciferins. Proc. Natl. Acad. Sci. USA 47, 1129–1134. [4] Lembert, N. (1996) Firefly luciferase can use L -luciferin to produce light. Biochem. J. 317, 273–277. [5] White, E.H., McCapra, F. and Field, G.F. (1963) The structure and synthesis of firefly luciferin. J. Am. Chem. Soc. 85, 337–343. [6] Day, J.C., Tisi, L.C. and Bailey, M.J. (2004) Evolution of beetle bioluminescence: the origin of beetle luciferin. Luminescence 19, 8–20. [7] McCapra, F. and Razavi, Z. (1976) Biosynthesis of luciferin in Pyrophorus pellucens. J.C.S. Chem. Commun. 153–154. [8] Okada, K., Ito, H. and Goto, T. (1976) Biosynthesis of firefly luciferin. Probable formation of benzothiazole from p-benzoquinone and cysteine. J.C.S. Chem. Commun. 32. [9] Colepicolo, P., Pagni, D. and Bechara, E.J.H. (1988) Luciferin biosynthesis in larval Pyrearinus termitilluminans (Coleoptera: Elateridae). Comp. Biochem. Physiol. 91B, 143–147. [10] Friedman, M. (1999) Chemistry, nutrition, and microbiology of D -amino acids. J. Agric. Food Chem. 47, 3457–3479. [11] Riemenschneider, A., Wegele, R., Schmidt, A. and Papenbrock, J. (2005) Isolation and characterization of a D -cysteine desulfhydrase protein from Arabidopsis thaliana. FEBS J. 272, 1291– 1304. [12] Nakamura, M., Niwa, K., Maki, S., Hirano, T., Ohmiya, Y. and Niwa, H. (2006) Construction of a new firefly bioluminescence system using L -luciferin as substrate. Tetrahedron Lett. 47, 1197– 1200. [13] Toya, Y., Takagi, M., Nakata, H., Suzuki, N., Isobe, M. and Goto, T. (1992) A convenient synthetic method of 2-cyano-6methoxybenzothiazole, a key intermediate for the synthesis of firefly luciferin. Bull. Chem. Soc. Jpn. 65, 392–395. [14] Ohba, N. (2004) Flash communication systems of Japanese fireflies. Integr. Comp. Biol. 44, 225–233. [15] Nakamura, M., Maki, S., Amano, Y., Ohkita, Y., Niwa, K., Hirano, T., Ohmiya, Y. and Niwa, H. (2005) Firefly luciferase exhibits bimodal action depending on the luciferin chirality. Biochem. Biophys. Res. Commun. 331, 471–475. [16] Reichel, C., Brugger, R., Bang, H., Geisslinger, G. and Brune, K. (1997) Molecular cloning and expression of a 2-arylpropionylcoenzyme A epimerase: a key enzyme in the inversion metabolism of ibuprofen. Mol. Pharmacol. 51, 576–582. [17] Keller, G.A., Gould, S., DeLuca, M. and Subramani, S. (1987) Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc. Natl. Acad. Sci. USA 84, 3264–3268. [18] Hunt, M.C., Stefan, E.H. and Alexson, E.H. (2002) The role Acyl-CoA thioesterase play in mediating intracellular lipid metabolism. Prog. Lipid Res. 41, 99–130. [19] Gomi, K. and Kajiyama, N. (2001) Oxyluciferin, a luminescence product of firefly luciferase, is enzymatically regenerated into luciferin. J. Biol. Chem. 276, 36508–36518. [20] Oba, Y., Ojika, M. and Inouye, S. (2003) Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase. FEBS Lett. 540, 251–254. [21] Fontes, R., Dukhovich, A., Sillero, A. and Sillero, M.A.G. (1997) Synthesis of dehydroluciferin by firefly luciferase: effect of dehydroluciferin, coenzyme A and nucleoside triphosphates on the luminescent reaction. Biochem. Biophys. Res. Commun. 137, 445–450.
Stereoisomeric bio-inversion key to biosynthesis of firefly D -luciferin Kazuki Niwaa,b, Mitsuhiro Nakamurac, Yoshihiro Ohmiyaa,* a b
Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Japan National Metrology Institute of Japan, National Institute of Advanced Industrial Science and Technology (AIST), Japan c Integrated Research Institute, Tokyo Institute of Technology, Japan Received 6 June 2006; revised 7 August 2006; accepted 30 August 2006 Available online 11 September 2006 Edited by Judit Ova´di
Abstract The chirality of the luciferin substrate is critical to the luciferin–luciferase reaction producing bioluminescence. In firefly, the biosynthetic pathway of D -luciferin is still unclear, although it can be synthesized in vitro from D -cysteine. Here, we show that the firefly produces both D - and L -luciferin, and that the amount of active D -luciferin increases gradually with maturation stage. Studies of firefly body extracts indicate the possible conversion of L -cysteine via L -luciferin into D -luciferin, suggesting that the biosynthesis is enzymatically regulated by stereoisomeric bio-inversion of L -luciferin. We conclude that the selection of chirality in living organisms is not as rigid as previously thought. 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Bioluminescence; Biosynthesis; Enantiomer; Firefly; Luciferase; Luciferin
1. Introduction Biologically active natural products are usually homochiral: proteins are composed of mainly L -amino acids and L -menthol produces a mint-like smell whereas D -menthol is a stronger stimulant than its L -counterpart. Thus, the determination and selection of chirality in natural products is considered as a rigid system. Bioluminescence is a typical example of a substrate-enzyme reaction [1]. Most such reactions are efficient and most luciferins are chiral and are biosynthesized from peptides or L -amino acid. For instance, only Cypridina L -luciferin exhibits bioluminescence potential although in chemiluminescence, the D -, L -, and DL -forms produce light of the same intensity [2]. These results indicate that Cypridina luciferase recognizes only one of the enantiomers and this luciferin is biosynthesized from L amino acids or tripeptides. Similarly, only firefly D -luciferin contributes to bioluminescence, whereas L -luciferin does not but strongly inhibits [3,4]. How does firefly biosynthesize D luciferin?
* Corresponding author. Address: 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japan. Fax: +81 72 751 9628. E-mail address: [email protected] (Y. Ohmiya).
Abbreviations: ABD-F, 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; BSA, Bovine serum albumin; CHBT, 2-cyano-6-hydroxybenzothiazole; CoA, coenzyme A; TBP, tri-n-butylphosphine
Firefly D -luciferin can be chemically synthesized from 2-cyano-6-hydroxybenzothiazole (CHBT) and D -cysteine [5] whose stereochemistry corresponds to that of D -luciferin (Fig. 1A). The putative biosynthetic precursors of D -luciferin are also cysteine and CHBT [6]. Incorporations of isotope-labeled DL -cystine and CHBT into D -luciferin in fireflies have been reported [7–9]. However, the detail biosynthetic mechanism of firefly D -luciferin has remained ambiguous for almost half a century since the first report of its chemical structure [5]. Especially, CHBT is so active that even in the absence of enzyme, it can react non-enzymatically with L - as well as D -cysteine, producing L -luciferin [6]. Although some D -amino acids were reported to have functional roles and to be widely distributed in living organisms [10], there are no reports to date of the presence of free D -cysteine and no biosynthetic studies of it in any eukaryotes [11]. From these findings, we supposed that L -luciferin could be the biosynthetic intermediate despite the fact that it is the enantiomer of a biologically active natural product (Fig. 1B). On the other hand, Lembert and we have previously reported the in vitro enzymatic formation of D luciferin from L -luciferin [4,12]. In this study, we propose a new biosynthetic pathway of firefly D -luciferin, focussing on its chirality, which has remained ambiguous since its chemical structure was first reported 45 years ago. We show that the firefly contains a considerable amount of L -luciferin, an enantiomer of active D -luciferin, and that the enantiomer is enzymatically inverted to D -luciferin. Furthermore, the chirality of cysteine, the putative biosynthetic precursor of D -luciferin, is almost L -form. As we have already reported, luciferase could be responsible for the stereoisomeric inversion of L -luciferin to D -luciferin [12]. These facts suggest that firefly D -luciferin is synthesized in vivo from L -cysteine and the biosynthetic intermediate is L -luciferin.
2. Materials and methods 2.1. Materials The Japanese firefly Luciola lateralis (Coleoptera) was raised in our laboratory with a commercially available incubation system (Environmental Engineering Laboratory Co., Ltd., Maebashi, Japan). Authentic D - and L -luciferin were prepared from CHBT as previously described [13]. 2.2. Preparation and chiral analysis of firefly luciferin Twelve larvae, three larvae in cocoon, twelve pupae, six adults in cocoon, and eight adults of L. lateralis were analyzed as described below. Luciferin in a firefly body was eluted with 200 ll of ethanol containing 0.5% tri-n-butylphosphine (TBP) at 70 C, and homogenized. The
0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.08.073
5284
K. Niwa et al. / FEBS Letters 580 (2006) 5283–5287
N
A
CN
+
S
HO
H2N * COOH HS
/ buffer
HO
N * COOH
S
S
D-luciferin
D-cysteine
CHBT
N
H2N * COOH
B
CHBT
HS
?
H2N * COOH
D-cysteine
HS L-cysteine
N
N * COOH
S
S
CHBT HO
HO ?
N
N * COOH
S
S
D-luciferin
L-luciferin Fig. 1. Synthesis of firefly D -luciferin. (A) Chemical synthesis of D -luciferin. The asymmetric carbon of cysteine corresponds to that of luciferin. (B) Hypothetical biosynthetic pathways of D -luciferin from L -cysteine.
sample was centrifuged (13 000 rpm, 20 min) and filtered with 0.45 lm membrane filter. The filtrate was injected into a high-performance liquid chromatography (HPLC) system (Alliance HPLC System with 2695 Separation Module, 2475 Multi k Fluorescence Detector, and 2996 Photodiode Array Detector, Waters). Linear gradient elution (15–40% acetonitrile/water with 0.1% TFA, 20 min, 1.0 ml/min) was adopted for the separation with a chiral fused silica column, CHIRALCEL OD-RH (4.6 · 150 mm; Daicel Chemical Industry, Tokyo, Japan). D - and L -Luciferin were detected with a fluorescence detector (excitation k = 330 nm, emission k = 530 nm, see Fig. 2A).
Intensity (fluorescence signal)
A 200 D-luciferin
100
L-luciferin
0 0
5
10
15
25
20
Retention time (min)
Intensity (fluorescence signal)
B
40
D-Cys-ABD L-Cys-ABD
20
Authentic (5 nmol)
0
2.3. Preparation and chiral analysis of firefly cysteine To extract cysteine, four adult firefly bodies suspended in 400 ll of 1 N HCl and 10 ll of 10% TBP/acetonitrile were homogenized and incubated at 100 C for 5 min. The sample was centrifuged (13 000 rpm, 20 min) and filtered with 0.45 lm membrane filter. Cysteine from 100 ll of the filtrate was purified with an ODS column (OD-SR-5, Daicel), and dried. To label fluorescence, the cysteine fraction was dissolved in 125 ll of 0.1 M sodium borate buffer (pH 8.0) containing 1 mM EDTA, 50 ll of 1.0 mM 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F, from Dojindo Laboratories, Kumamoto, Japan) in borate buffer, and 5 ll of 10% TBP/acetonitrile. The mixture was kept at 50 C for 5 min, and then placed on ice and mixed with 60 ll of 0.1 N HCl. The fluorescence-labeled cysteine was injected into the HPLC system equipped with a chiral crown ether fused column, CROWNPAK CR (+) (4.6 · 150 mm; Daicel Chemical Industry, Tokyo, Japan). Isocratic elution with 0.1% TFA in water (1.2 ml/min) was adopted for the separation. Fluorescence-labeled D - and L -cysteine were detected with a fluorescence detector (excitation k = 380 nm, emission k = 530 nm, see Fig. 2B, upper chart). 2.4. Stereoisomeric inversion assay of firefly extract To extract proteins, the light organ of an adult firefly was homogenized in 200 ll of 0.1 M Tris–HCl buffer (pH 8.0) containing 0.1 M NaCl, and this was followed by centrifugation (13 000 rpm, 20 min) to remove debris. The extract was dialyzed to remove contamination by small molecules. An aliquot (200 ll) of 0.1 mM L -luciferin, 1 mM coenzyme A (CoA), 3 mM ATP, and 8 mM MgSO4 in 0.1 M Tris– HCl buffer (pH 8.0) was mixed with 40 ll of the dialyzed protein extract and incubated at room temperature. Luciferin chirality was analyzed with the same procedure as that described above, except that the eluent was changed to isocratic 27% acetonitrile/water containing 0.1% TFA (Fig. 4A). To test the enzymatic reaction, an aliquot (2 ll) of 1 mM L -luciferin was mixed with 40 ll of various protein solutions and 198 ll of various cofactor solutions (Fig. 4C). Protein extracts from Luciola cruciata (sampled from Tsushima Island, Nagasaki) and Photinus pyralis (from Sigma) were also tested.
20
Extract
3. Results and discussion
10 0 0
5
10
15
20
25
Retention time (min) Fig. 2. Chiral analyses of in vivo luciferin and cysteine. (A) Chromatogram of luciferin in an adult firefly, obtained with a chiral column. (B) Chromatogram of ABD-labeled cysteine from four adult fireflies, obtained with a chiral column.
In order to gain an insight into the biosynthesis of active luciferin, we analyzed bioluminescence-related compounds during the life cycle of L. lateralis (Fig. 3). The adult of this firefly produces flashing light signals to communicate and attract a mate [14]. On the other hand, the larva and pupa occasionally produce a continuous dim glow, the biological function of which is not known. The larva, an aquatic glowworm, feeds on snails but the other stages do not eat anything.
K. Niwa et al. / FEBS Letters 580 (2006) 5283–5287
5285
Larva
Pupa
Aquatic
In cocoon of soil
Mating
1.2 1.0 0.8 0.6 0.4 0.2 0
100 80 60 40 20 0
L
LC
P
AC
Enantiomeric excess of D-luciferin (% ee)
Fasting
Feeding Total DL-luciferin (nmol / body)
Adult
A
Fig. 3. Life stage, luciferin content, and D -luciferin chirality. Illustrated are the five distinctive life stages of the Japanese firefly, L. lateralis. L, larva; LC, larva in cocoon; P, pupa; AC, adult in cocoon; A, adult. Graphs show the total amounts of D - and L -luciferin in an individual body and the enantiomeric excess of D -luciferin (% ee). Error bars indicate standard deviations.
It thrives inside a cocoon made of soil during metamorphosis from larva through pupa to adult. We raised the firefly in our laboratory and collected them to analyze their luciferin by HPLC with a chiral column. Individual samples were divided into five groups depending on the morphologically distinctive life stages: larva, larva in cocoon, pupa, adult in cocoon, and adult. As shown in Fig. 3, the more mature the firefly was, the higher the total amount of luciferin was, demonstrating that the firefly produces and stores luciferin during pupa and adult in cocoon stages despite the fasting period. Both L and D -luciferin were identified by their retention times (Fig. 2A) and biological activities [15]. Both luciferins were detectable at all stages and the enantiomeric excess of D -luciferin was highest at the adult stage, while it was relatively low during larval to pupal stages and variable as individual differences (Fig. 3). Both luciferins were detected in the egg, although its amount and ratio could not be determined accurately (data not shown). We previously reported that L -luciferin is a substrate for the non-luminous enzymatic reaction with firefly luciferase in vitro [12,15] and Lembert have previously reported the light production from L -luciferin [4]. But these facts could not prove the presence and biological role of L -luciferin in vivo. Now we proved the existence of L -luciferin in vivo and the change in ratio of D - and L -luciferin with development, implying that L -luciferin is the biosynthetic intermediate. As regards to the hypothetical biosynthetic precursor for luciferin, we analyzed the chirality of cysteine in vivo (Fig. 2B, lower chart). Although our in vitro assay system enabled detection of D - or L -cysteine in amounts as low as 10 10 moles (data not shown), faint peaks around the retention time for D -cysteine were recognized only when the extracts from four individuals of living fireflies were combined. This result could prove the extreme dominance of L -cysteine over D -cysteine. As CHBT readily reacts with both L - and D -cysteine, it is difficult for the firefly to prevent from producing corre-
sponding luciferins, suggesting that L -luciferin in vivo could be derived from L -cysteine. In vivo, however, L -luciferin must be inverted to D -luciferin to induce the bioluminescence reaction because inactive L -luciferin strongly inhibits the luciferin–luciferase reaction [4]. The next question is what is responsible for the disproportionation of luciferin chirality? We previously reported that in the presence of ATP, Mg2+ and CoA, firefly luciferase exhibits coenzyme A ligase (CoA-ligase) activity on L -luciferin in vitro, but not on D -luciferin, to give luciferyl-CoA [15]. Modification of the carboxyl group in L -luciferin accelerates enolization to give racemic luciferyl-CoA. Hydrolysis of racemic luciferyl-CoA could yield both L - and D -luciferin. We recently reported that L -luciferin was stereoisomerically bioinverted to produce D -luciferin under enzymatic conditions in vitro [12]. We examined the enzymatic activity for the stereoisomeric inversion of L -luciferin in L. lateralis extract (Fig. 4). Soluble proteins extracted from the light organ and dialyzed with buffer to remove small molecules exhibited inversion activity on authentic L -luciferin in the presence of ATP, Mg2+, and CoA (Fig. 4A and B). This activity was suppressed when the extract was boiled and could not be substituted by known proteins including bovine serum albumin (BSA), indicating that the stereoisomeric inversion is an enzymatic reaction in the firefly bioluminescence system (Fig. 4C). The activity was also suppressed in the absence of ATP, Mg2+, or CoA (Fig. 4C), suggesting that the CoA ligation of L -luciferin is essential for the bio-inversion reaction and luciferyl-CoA could be the intermediate. Therefore, CoA-thioesterase could catalyze the hydrolysis of D -luciferyl-CoA to yield D -luciferin. Furthermore, the same potential to invert L -luciferin to D -luciferin was found in the protein extracts from another Japanese firefly, L. cruciata, as well as a North American firefly, P. pyralis (data not shown), suggesting that the inversion activity could be ubiquitous among luminous beetles.
5286
K. Niwa et al. / FEBS Letters 580 (2006) 5283–5287
A
Relative amount (%)
B
Intensity (fluorescence signal)
L-luciferin D-luciferin
2-min reaction 15-min reaction
0
1
2
3
4
5
6
7
8
9
100
D-luciferin L-luciferin 0 0
10
Retention time (min)
Relative amount (%)
C
100
30
60
90
Reaction time (min)
L-luciferin
D-luciferin
before
extract +
0
Firefly extract Boiled extract BSA (10 ug/ml) ATP (3 mM) MgSO4 (8 mM) CoA (1 mM)
boiled
BSA
- ATP +
- Mg2+ +
- CoA +
+ + + + +
+ + +
+ + +
+ + +
+ + +
+ +
+
Fig. 4. Stereoisomeric inversion of L -luciferin to D -luciferin in L. lateralis extract. (A) Chromatograms of a reaction mixture containing L -luciferin, the protein extract, ATP, Mg2+, and CoA. Dashed and solid lines indicate the reaction mixture incubated for 2 and 15 min, respectively. (B) Time course of the inversion reaction. Ratios of D - and L -luciferin to the initial amount of total luciferin are plotted. (C) Inversion activities under various reaction conditions. Ratios of D - and L -luciferin in the reaction mixtures incubated at room temperature for more than 2 h.
nescence [12]. Our new proposal is supported by the inversion metabolism of ibuprofen, in which stereoselective CoA ligation of (S)-ibuprofen is the key step [16]. The reaction mixture for the stereoisomeric inversion, which contained luciferase, ATP, and Mg2+, was sufficient for light production from D -luciferin.
Based on our results and our previous findings [12,15], we propose a new pathway for D -luciferin biosynthesis, as shown in Fig. 5. The most important process is the stereoisomeric bioinversion at the final steps. Luciferase could be responsible for stereoselective CoA ligation of L -luciferin as well as biolumiH2N * COOH HS
HO
L-cysteine
N
N * COOH
S
S
HO
L-luciferin Luciferyl-CoA synthase (Luciferase)
N * COOH
S
S
D-luciferin
ATP, Mg2+, CoA
O
OH
N * C S
CoA
N
C
O S
N * C S
CoA
S
S L-luciferyl-CoA
CoA
S
(enol)
D-luciferyl-CoA hydrolysis enzyme
hydrolysis enzyme
HO
N
N
N * COOH
S
S
L-luciferin
HO
N
N * COOH
S
S
D-luciferin
Fig. 5. Proposed biosynthetic pathway of D -luciferin. L -luciferin is produced from natural L -cysteine. L -Luciferin is converted into L -luciferyl-CoA that is easy to racemize by enolization. Hydrolysis of D -luciferyl-CoA gives the bioluminescent substrate, D -luciferin.
K. Niwa et al. / FEBS Letters 580 (2006) 5283–5287
The reason why D -luciferin remained in the reaction mixture despite being under bioluminescence reaction conditions was because the bioluminescence reaction rate was so low [15] that the consumption of D -luciferin could not be detected in our reaction conditions. Lembert [4] first reported the light production from L -luciferin and proposed that L -luciferin was racemized to give D -luciferin and it was effectively stimulated by the addition of pyrophosphate. This racemization is an in vitro reaction of luciferase in the absence of CoA. In the presence of CoA, L -luciferin is readily converted into luciferyl-CoA [15]. Luciferase in vivo is localized in peroxisome where metabolisms involved in CoA mainly take place [17,18]. Furthermore, biosynthetic pathway should produce homochiral D -luciferin. Therefore, we consider our proposal is reasonable as an in vivo reaction. Because the firefly is able to invert L -luciferin to D -luciferin efficiently, as shown in Fig. 4B, it is not necessary for the firefly to produce D -cysteine. Although there remains the possibility, the firefly does not have to keep mechanisms that CHBT selectively reacts with minimal amounts of D -cysteine to produce D -luciferin alone. On the other hand, in the luciferin recycling pathway [19], the luciferin regeneration enzyme produces CHBT, although the de novo biosynthesis of CHBT as well as any benzothiazole compounds in vivo is not clear. Recently, reports of the multiple functions of luciferase have emerged: CoA-ligase activity on fatty acids [20], and adenosine polyphosphate synthetase activity on ATP, ADP, and AMP [21]. Our results strongly suggest that luciferase also participates in the biosynthesis of D -luciferin by producing luciferylCoA from L -luciferin. However, further research is required to elucidate the detailed mechanism after luciferyl-CoA formation. In previous research, the amount of L -luciferin in vivo and the existence of enzymes for the inversion of L -luciferin to D -luciferin have not been considered. This report introduces a new aspect of the firefly luciferase system. What is the meaning of disproportionation of luciferin chirality depending on the developmental stage? The flashing light of the adult L. lateralis is highly regulated whereas the continuous dim glow before metamorphosis is not passively regulated. For the adult, the saving of active D -luciferin could be helpful for flashing light communication. The flexibility of luciferin chirality could be part of a physiological regulatory system. According to the lock and key theory of E. Fischer, substrate chirality is a rigid system. However, studies of intact organisms indicate that substrate chirality is more flexible than previously thought. The firefly bioluminescence system is a good example of chiral diversity and enzymatic flexibility. Acknowledgements: We thank N. Ohba (Yokosuka City Musium) for many suggestions about culturing L. lateralis and its life cycle. We also thank N. Fujii (Kyoto University) for discussion and suggestions about chiral analysis of cysteine and T. Goto (Tohoku University) about stereoisomeric bio-inversion metabolism. This work was supported by grants from the Ministry of Economy, Trade and Industry of Japan and Takeda Science Foundation.
5287
References [1] Wilson, T. and Hastings, J.W. (1998) Bioluminescence. Annu. Rev. Cell. Dev. Biol. 14, 197–230. [2] Wu, C., Kawasaki, K., Ohgiya, S. and Ohmiya, Y. (2006) Syntheses and evaluation of the bioluminescent activity of (S)-Cypridina luciferin and its analogs. Tetrahedron Lett. 47, 753–756. [3] Seliger, H.H., McElroy, W.D., White, E.H. and Field, G.F. (1961) Stereospecificity and firefly bioluminescence, a comparison of natural and synthetic luciferins. Proc. Natl. Acad. Sci. USA 47, 1129–1134. [4] Lembert, N. (1996) Firefly luciferase can use L -luciferin to produce light. Biochem. J. 317, 273–277. [5] White, E.H., McCapra, F. and Field, G.F. (1963) The structure and synthesis of firefly luciferin. J. Am. Chem. Soc. 85, 337–343. [6] Day, J.C., Tisi, L.C. and Bailey, M.J. (2004) Evolution of beetle bioluminescence: the origin of beetle luciferin. Luminescence 19, 8–20. [7] McCapra, F. and Razavi, Z. (1976) Biosynthesis of luciferin in Pyrophorus pellucens. J.C.S. Chem. Commun. 153–154. [8] Okada, K., Ito, H. and Goto, T. (1976) Biosynthesis of firefly luciferin. Probable formation of benzothiazole from p-benzoquinone and cysteine. J.C.S. Chem. Commun. 32. [9] Colepicolo, P., Pagni, D. and Bechara, E.J.H. (1988) Luciferin biosynthesis in larval Pyrearinus termitilluminans (Coleoptera: Elateridae). Comp. Biochem. Physiol. 91B, 143–147. [10] Friedman, M. (1999) Chemistry, nutrition, and microbiology of D -amino acids. J. Agric. Food Chem. 47, 3457–3479. [11] Riemenschneider, A., Wegele, R., Schmidt, A. and Papenbrock, J. (2005) Isolation and characterization of a D -cysteine desulfhydrase protein from Arabidopsis thaliana. FEBS J. 272, 1291– 1304. [12] Nakamura, M., Niwa, K., Maki, S., Hirano, T., Ohmiya, Y. and Niwa, H. (2006) Construction of a new firefly bioluminescence system using L -luciferin as substrate. Tetrahedron Lett. 47, 1197– 1200. [13] Toya, Y., Takagi, M., Nakata, H., Suzuki, N., Isobe, M. and Goto, T. (1992) A convenient synthetic method of 2-cyano-6methoxybenzothiazole, a key intermediate for the synthesis of firefly luciferin. Bull. Chem. Soc. Jpn. 65, 392–395. [14] Ohba, N. (2004) Flash communication systems of Japanese fireflies. Integr. Comp. Biol. 44, 225–233. [15] Nakamura, M., Maki, S., Amano, Y., Ohkita, Y., Niwa, K., Hirano, T., Ohmiya, Y. and Niwa, H. (2005) Firefly luciferase exhibits bimodal action depending on the luciferin chirality. Biochem. Biophys. Res. Commun. 331, 471–475. [16] Reichel, C., Brugger, R., Bang, H., Geisslinger, G. and Brune, K. (1997) Molecular cloning and expression of a 2-arylpropionylcoenzyme A epimerase: a key enzyme in the inversion metabolism of ibuprofen. Mol. Pharmacol. 51, 576–582. [17] Keller, G.A., Gould, S., DeLuca, M. and Subramani, S. (1987) Firefly luciferase is targeted to peroxisomes in mammalian cells. Proc. Natl. Acad. Sci. USA 84, 3264–3268. [18] Hunt, M.C., Stefan, E.H. and Alexson, E.H. (2002) The role Acyl-CoA thioesterase play in mediating intracellular lipid metabolism. Prog. Lipid Res. 41, 99–130. [19] Gomi, K. and Kajiyama, N. (2001) Oxyluciferin, a luminescence product of firefly luciferase, is enzymatically regenerated into luciferin. J. Biol. Chem. 276, 36508–36518. [20] Oba, Y., Ojika, M. and Inouye, S. (2003) Firefly luciferase is a bifunctional enzyme: ATP-dependent monooxygenase and a long chain fatty acyl-CoA synthetase. FEBS Lett. 540, 251–254. [21] Fontes, R., Dukhovich, A., Sillero, A. and Sillero, M.A.G. (1997) Synthesis of dehydroluciferin by firefly luciferase: effect of dehydroluciferin, coenzyme A and nucleoside triphosphates on the luminescent reaction. Biochem. Biophys. Res. Commun. 137, 445–450.