Biosynthesis of Firefly Luciferin in Adult Lantern: Decarboxylation of ʟ-Cysteine is a Key Step for Benzothiazole Ring Formation in Firefly Luciferin Synthesis

Background Bioluminescence in fireflies and click beetles is produced by a luciferase-luciferin reaction. The luminescence property and protein structure of firefly luciferase have been investigated, and its cDNA has been used for various assay systems. The chemical structure of firefly luciferin was identified as the ᴅ-form in 1963 and studies on the biosynthesis of firefly luciferin began early in the 1970’s. Incorporation experiments using 14C-labeled compounds were performed, and cysteine and benzoquinone/hydroquinone were proposed to be biosynthetic component for firefly luciferin. However, there have been no clear conclusions regarding the biosynthetic components of firefly luciferin over 30 years. Methodology/Principal Findings Incorporation studies were performed by injecting stable isotope-labeled compounds, including ʟ-[U-13C3]-cysteine, ʟ-[1-13C]-cysteine, ʟ-[3-13C]-cysteine, 1,4-[D6]-hydroquinone, and p-[2,3,5,6-D]-benzoquinone, into the adult lantern of the living Japanese firefly Luciola lateralis. After extracting firefly luciferin from the lantern, the incorporation of stable isotope-labeled compounds into firefly luciferin was identified by LC/ESI-TOF-MS. The positions of the stable isotope atoms in firefly luciferin were determined by the mass fragmentation of firefly luciferin. Conclusions We demonstrated for the first time that ᴅ- and ʟ-firefly luciferins are biosynthesized in the lantern of the adult firefly from two ʟ-cysteine molecules with p-benzoquinone/1,4-hydroquinone, accompanied by the decarboxylation of ʟ-cysteine.


Introduction
Bioluminescence is the emission of visible light produced by living organisms [1,2]. Among insects, the luminous species have been found in three Coleoptera families: Lampyridae (firefly), Elateridae (click beetle), and Phengodidae (railroad worm) [3]. Light emission in these insects is produced by an enzymatic reaction of a luciferase (enzyme) and a luciferin (substrate). The luminescence system is essentially the same with an identical luciferin, ATP, Mg 2+ , and a highly conserved luciferase [4]. The luciferin is referred to as ''firefly luciferin'' or ''beetle luciferin'', and the chemical structure has been identified as (S)-2-(69hydroxy-29-benzothiazolyl)-2-thiazoline-4-carboxylic acid (I, Dfirefly luciferin), which consists of two structural units, benzothiazole and thiazoline rings ( Figure 1A). The chirality of the carboxyl group in natural firefly luciferin was determined to be the S form by the chemical synthesis of D-firefly luciferin from 2-cyano-6hydroxybenzothiazole (III) and D-cysteine [5,6]. L-Firefly luciferin with the R form is not used for the luminescence reaction by firefly luciferase [7]. Thus, firefly luciferase oxidizes only D-firefly luciferin to emit light and produces oxyluciferin (II) and CO 2 ( Figure 1A).
On the other hand, in marine luminous organisms, Cypridina luciferin and coelenterazine are widely used in the luciferase reactions [2]. Coelenterazine is also used as the light-emitting substrate for the Ca 2+ -binding photoproteins such as aequorin [8]. Recently, we have been studying the biosyntheses of Cypridina luciferin and coelenterazine in living specimens by feeding experiments using stable isotope-labeled compounds. The incorporation of stable isotopes into the luciferin was determined by mass spectrometry [9][10][11][12]. In the luminous ostracods Cypridina (presently Vargula) hilgendorfii and Cypridina noctiluca, we concluded that Cypridina luciferin is biosynthesized from the natural amino acids of L-tryptophan, L-arginine, and L-isoleucine [9][10][11]13]. Further, we demonstrated that coelenterazine is biosynthesized from two L-tyrosines and L-phenylalanine in the deep-sea luminous copepod Metridia pacifica [12]. Thus, similar to the method using the radioisotope-labeled compounds, the method of mass spectral analysis accompanied by the incorporation of stable isotope-labeled compounds is useful for investigating the biosynthetic process. With regard to studies on the biosynthesis of luciferin in firefly and click beetle, four biochemical investigations involving incorporation experiments with 14 C-labeled compounds have been reported [14][15][16][17] and studies using biomimetic synthesis have also been reported [16,18].
The initial study on the biosynthesis of firefly luciferin was reported in 1974 [14]. Based on the chemical synthesis of D-firefly luciferin from 2-cyano-6-hydroxybenzothiazole and D-cysteine [6], [2][3][4][5][6][7][8][9][10][11][12][13][14] C]-oxyluciferin and 2-[cyano-14 C]-6-hydroxybenzothiazole were chemically synthesized and were injected into the adult lantern of the Japanese firefly Luciola cruciata. To determine the incorporation of 14 C-labeled compounds into firefly luciferin, 14 Clabeled firefly luciferin with an excess of cold D-firefly luciferin was converted to its diacetate derivative and crystallized, following which the radioactivity was determined [14]. From these results, 2cyano-6-hydroxybenzothiazole (III) was proposed to be a candidate for the biosynthetic precursor of firefly luciferin, and oxyluciferin (II) could be regenerated to luciferin through 2cyano-6-hydroxybenzothiazole in the firefly lantern. Furthermore, when cell-free extracts from the frozen lanterns were incubated with 14 C-oxyluciferin and cysteine in the presence of ATP, the incorporation of 14 C-oxyluciferin into firefly luciferin was increased. However, the following controversial points exist in this report: (i) The configuration of L-or D-luciferin has not been not mentioned in the report. (ii) oxyluciferin is degraded to 2cyano-6-hydroxybenzothiazole (III) under non-enzymatic conditions at pH 7-9. (iii) the condensation of 2-cyano-6-hydroxybenzothiazole (III) with D-and L-cysteine proceeds spontaneously in aqueous solutions (pH 8) at room temperature [6,14] and forms Dand L-firefly luciferin, respectively; and (iv) the presence of 2cyano-6-hydroxybenzothiazole (III) or an intermediate of its derivatives has not been identified in the firefly. Thus, it is still unclear whether oxyluciferin and 2-cyano-6-hydorxybenzothiazole (III) are not intermediates for luciferin biosynthesis and recycling intermediates from oxyluciferin to luciferin in a living firefly [19,20].
For studies on the biosynthesis of beetle luciferin, the luminous click beetle Pyrophorus pellucens was used in 1976 [16]. The biosynthesis of luciferin was examined by feeding experiments using the adult specimen of P. pellucens with a 10% sucrose solution containing D/L-[1-14 C]-cystine (a dimer of D-and/or L-cysteine). After the addition of an excess of D-luciferin into the extracts of photophores, the 14 C-labeled luciferin recovered by TLC was crystallized and the radioactivity was determined. The results suggested that D/L-cystine was reduced to D-and L-cysteine and they were incorporated into beetle luciferin and that D-and/or Lcysteine are a biosynthetic unit of luciferin. In this report, the important point was that the possibility of the decarboxylation from a cysteine was predicted during benzothiazole ring formation. However, the configuration of cysteine incorporated in beetle luciferin and the incorporation of cysteine into the benzothiazole ring with decarboxylation were not revealed. Further, in 1988, the injection experiment of [U-14 C 6 ]-cystine into the larvae of the luminous click beetle Pyrearinus termitilluminans was performed, and the 14 C-labeled luciferin was extracted and was determined by TLC without the addition of luciferin [17]. As a result, cysteine from [U-14 C 6 ]-cystine was incorporated into luciferin, similar to the case of adult P. pellucens [16]. Unfortunately, the configuration of [U-14 C 6 ]-cystine used was not described in the report and the number of [U-14 C 3 ]-cysteine incorporated into luciferin was not determined.
In this study, we incorporated stable isotope-labeled L-cysteine, p-benzoquinone, and 1,4-hydroquinone into firefly luciferin in the adult lantern of a living firefly, and identified the positions of the stable isotopes incorporated into firefly luciferin by LC/ESI-TOF-MS analysis. We revealed that D-and L-firefly luciferins (beetle luciferin) are biosynthesized from p-benzoquinone/1,4-hydorquinone with two L-cysteines, accompanied by the decarboxylation of L-cysteine.  Figure 1C). One microliter of each isotopic compounds dissolved in sterile H 2 O was injected into the body cavity of an adult lantern using a syringe. The injected specimens were kept in a moisture chamber for 24 h, and the survived specimen was used for mass spectral analysis. The amount of the compounds injected was 550 nmol per specimen, except for pbenzoquinone which was 55 nmol. Owing to the toxicity of pbenzoquinone, the specimens injected with 550 nmol of pbenzoquinone were hardly survived for 24 h.

LC/ESI-TOF-MS Analysis of Stable Isotope-labeled Firefly Luciferins in the Lantern
We have reported that a single specimen of the adult L. lateralis contains approximately 0.5 nmol of firefly luciferin [21] and this amount is enough for analysis by LC/ESI-TOF-MS under our experimental conditions. Chemically synthesized D-and L-firefly luciferins were used as authentic samples to obtain the standard mass spectrum by LC/ESI-TOF-MS ( Figure 2 and Table S1 and Figure S1-S4). As shown in Fig. 2, the parent ion of D-firefly luciferin was observed at m/z 281 ((a) in Figure 2A). The fragment ions were formed at m/z 235 ((b) in Figure 2A), m/z 194 and m/z 177 ((c) in Figure 2A) by increasing the voltage of nozzle potential up to 360 V. The isotopic fragment ions of (b) and (c) were used for determining the positions of the 13 C-labeled atom in firefly luciferin. In our injection experiments, the incorporation efficiencies of stable isotope-labeled compounds into luciferin were estimated to be between 7% and 48% by calculating the peak intensities of the isotopic ions. The incorporation experiments were repeated 2-3 times to confirm reproducibility.

Incorporation of L-Cys[U-13 C 3 ] into Firefly Luciferin in the Presence of Non-isotopic 1,4-hydroquinone or pbenzoquinone
To determine whether L-cysteine is a biosynthetic component for both 6-hydroxybenzothiazole and 2-thiazoline-4-carboxylate moieties in firefly luciferin ( Figure 1A), the incorporation experiments were performed with L-Cys[U-13 C 3 ] in the presence and absence of 1,4-hydroquinone or p-benzoquinone. Because pbenzoquinone shows high toxicity in living organisms, the concentration of p-benzoquinone injected was 10-fold lower than that of 1,4-hydroquinone. The results of ESI-TOF-MS analysis are summarized in Table 1.

Incorporation of [D 6 ]-hydroquinone or [D 4 ]benzoquinone into Firefly Luciferin in the Presence of Non-isotopic L-cysteine (L-Cys)
To confirm 1,4-hydroquinone and p-benzoquinone as biosynthetic components for firefly luciferin, the injection experiments of [D 6 ]-hydroquinone and [D 4 ]-benzoquinone with L-cysteine were performed as follows ( Table 2). Notably, the incorporation efficiency of [D 4 ]-benzoquinone into firefly luciferin ( Figure S9) was higher than that of [D 6 ]hydroquinone (Table 2, Figure S7), despite the fact that the amount of p-benzoquinone injected was 10 times lower than that of 1,4-hydroquinone. This result indicated that p-benzoquinone might be preferred over 1,4-hydroquinone for firefly luciferin synthesis, and that 1,4-hydroquinone may converted to pbenzoquinone and immediately used for the biosynthesis of firefly luciferin in the lantern.

Identification of Endogenous D-and L-firefly Luciferin in an Adult Lantern in L. lateralis and Incorporation of L-Cys[U-13 C 3 ] into D-and L-firefly Luciferin
To characterize the chirality of firefly luciferin, firefly luciferin was extracted from the adult lantern of L. lateralis without racemization between D-and L-luciferin (see experimental section), following which then D-and L-luciferins were separated by HPLC with a chiral column ( Figure 9A). The peak ratio of D-luciferin to Lluciferin was approximately 9:1 ( Figure 9A-c), indicating that Lluciferin was present in an adult lantern. Following this, an incorporation study of L-Cys[U-13 C 3 ] and 1,4-hydroquinone was performed and the peak ratio of D-luciferin to L-luciferin was changed to 7:3 with an increase in L-luciferin ( Figure 9A-d). These peak fractions were collected and subsequently subjected to LC/ ESI-TOF-MS analysis ( Figure 9B). Interestingly, L-cysteine was incorporated into not only L-luciferin but also D-luciferin, indicating that L-cysteine is a biosynthetic component of Dluciferin.

Identification of Free 1,4-hydroquinone and Arbutin in Firefly Lantern
As described above, 1,4-hydroquinone is a biosynthetic component of firefly luciferin. To examine the presence of free 1,4-hydroquinone or its storage forms such as arbutin in the lantern of an adult firefly, we analyzed the lantern extracts by HPLC. Under our analytical conditions, free 1,4-hydroquinone was not detected in the lantern extracts. However, we successfully detected arbutin in the extracts by HPLC analysis ( Figure 10A). After the arbutin fraction was hydrolyzed with HCl ( Figure 10C), the hydrolyzed sample was subjected to HPLC analysis and the fluorescence peak of 1,4-hydroquinone was detected ( Figure 10B). Furthermore, the structure of 1,4hydroquinone in the hydrolyzed sample was confirmed as an acetylated derivative by LC/ESI-TOF-MS ( Figure S12). The content of 1,4-hydroquinone after hydrolysis was estimated to be 144634 pmol per specimen using the standard curve of 1,4hydroquinone (data not shown). This result suggested that 1,4hydroquinone would be released from a glycoside derivative such as arbutin in the lantern and used for the biosynthesis of firefly luciferin.

Discussion
Studies on the biosynthesis of luciferin in firefly and click beetle were initiated in the early 1970's using 14 C-labeled compounds. A hypothesis that firefly luciferin (beetle luciferin) is biosynthesized from p-benzoquinone and two cysteines was proposed [14][15][16]18]. In this report, we have identified the biosynthetic components of firefly luciferin by mass spectroscopy with stable isotope-labeled compounds. The 13 C-labeled Lcysteine, p-[D 4 ]-benzoquinone and 1,4-[D 6 ]-hydroquinone were incorporated into firefly luciferin in an adult lantern of a firefly. The incorporation experiment with L-Cys[U-13 C 3 ] indicated that L-cysteine was incorporated into both the benzothiazole and thiazoline unit of firefly luciferin (Figures 1 and 3). This is the first demonstration that two L-cysteine molecules are the biosynthetic components of firefly luciferin (Figure 3 and Table 2). Furthermore, the incorporation of L-[1-13 C]-cysteine and L-[3-13 C]-cysteine into firefly luciferin revealed that the carboxyl group of L-[1-13 C]-cysteine was eliminated during the benzothiazole ring formation of firefly luciferin (Figure 3 and Table 1), followed by the thiazoline ring formation of firefly luciferin ( Figure 11). This result clearly explains the previous observation that the radioisotope activity of 14 C-labeled firefly luciferin was lost following acetylation at the carboxyl group of luciferin [16]. Previously, a biosynthetic pathway of firefly luciferin from p-benzoquinone and a dipeptide of cysteine was proposed [18]. This possibility was not acceptable from the evidence that the carboxyl group from L-cysteine was eliminated. However, it is unclear whether the carbon atoms at the C2' in the benzothiazole unit and the C2 in the thiazoline unit were derived from the carbon atom at the C2 or C3 position of Lcysteine in our experiments (Figure 7).
On the other hand, the results of incorporation studies with p-[D 4 ]-benzoquinone and 1,4-[D 6 ]-hydroquinone were in good agreement with those of a study with 14 C-lableled p-benzoquinone and 1,4-hydroquinone [15]. Thus, p-benzoquinone and 1,4hydroquinone are components of the benzothiazole unit of firefly luciferin.
It is known that quinones including p-benzoquinone and its derivatives are found in some beetles (Coleoptera) [22][23][24] and pbenzoquinone is a metabolite produced by the oxidation of 1,4hydroquinone [25]. Because p-benzoquinone shows high toxicity to living organisms, the concentration of p-benzoquinone was 10fold lower than that of 1,4-hydroquinone in our injection experiments. The incorporation efficiency of p-benzoquinone into firefly luciferin was higher than that of 1,4-hydroquinone, suggesting that p-benzoquinone may be a preferred substance for the biosynthesis of firefly luciferin in the adult lantern. We detect arbutin, but not 1,4-hydroquionone in the firefly. It is considered that 1,4-hydroquione with low toxicity is stored as a non-toxic form of glycoside such as arbutin and released by the digestive enzyme b-glucosidase [25] and possibly oxidized to p-benzoquinone immediately for luciferin synthesis. However, the possibility that 1,4-hydroquinone is a direct biosynthetic precursor still remains.
Recently, the conversion of L-luciferin to D-luciferin in firefly has been proposed by the racemization through L-luciferyl CoA to Dluciferyl CoA, followed by its hydrolysis with an esterase [20]. L-Luciferyl CoA was produced from L-luciferin by ''firefly luciferase'' in the presence of ATP, Mg 2+ and CoA, although L-luciferin is a potent inhibitor of firefly luciferase [26,27]. However, another possibility that the D-configuration in luciferin is formed during the thiazoline ring formation accompanied by the conversion of Lform to D-form of cysteine still remains.
In this report, we determined the absolute configuration of the isotope-labeled firefly luciferin by HPLC analysis with a chiral column ( Figure 9) and found that L-cysteine was incorporated into not only L-luciferin but also D-luciferin, indicating that L-cysteine is a biosynthetic component of D-luciferin. The mechanism by which D-firefly luciferin is biosynthesized from L-cysteine remains unclear.
In conclusion, we have demonstrated that the 6-hydroxybenzothiazole moiety in D-and L-firefly luciferins is biosynthesized from 1,4-hydroquinone/benzoquinone with L-cysteine, accompanied by the elimination of the carboxyl group of L-cysteine, and that the 2-thiazoline-4-carboxylate moiety is derived from the second L-cysteine in the adult lantern of the firefly.

Injection of Stable Isotope-labeled Compounds into the Adult Lantern of L. lateralis
The stock solutions of stable isotope-labeled and non-labeled compounds were prepared by dissolving compound in sterile H 2 O to be 550 mM, excepting for 55 mM of p-benzoquinone and [D 4 ]benzoquinone. For incorporation experiments, 1 mL of the stock solution was injected into the adult lantern of a female L. lateralis (within 4 days after adult emergence) using a microsyringe (701RN 10 mL SYR; Hamilton, Reno, NV) ( Figure S13). After keeping fireflies in a moisture chamber at 2462uC for 24 h, the living specimens injected were frozen in liquid nitrogen and stored at 280uC.

Extraction of the Labeled Firefly Luciferin from an Adult Lantern of L. lateralis
A single lantern was separated from a frozen specimen using a razor blade, and was homogenized in a tube with 70 mL of hot H 2 O using a plastic pestle in a heating block at 95uC for 5 min to    inactivate luciferase activity. The homogenate was centrifuged at 17,4006g for 3 min at 4uC, and the resultant supernatant was filtrated by an Ultrafree-MC centrifugal filter (0.45 mm; Millipore, Billerica, MA). The filtrate obtained was washed twice by nhexane (60 mL) and 5 mL of aqueous layer (ca. 30 mL) was subjected to LC/ESI-TOF-MS analysis. Under above extraction conditions at 95uC, the racemization of D/L-firefly luciferin could be partially occurred.

Identification of D-and L-luciferin in an Adult Lantern of L. lateralis by HPLC Analysis with a Chiral Column
To avoid racemization of D/L-firefly luciferin during the extraction from an adult lantern, a single lantern was homogenized in 70 mL of methanol at 4uC instead of hot H 2 O at 95uC. Under above conditions, the racemization of D-luciferin to Lluciferin was not occurred at 4uC for 60 min. In contrast, by incubating D-luciferin in methanol at 70uC for 60 min, 21% of Lfirefly luciferin was yielded by racemization (data not shown). Methanol extracts (18 mL) were analyzed by reversed-phase HPLC equipped with a chiral column, CHIRALCEL OD-RH (4.66150 mm; Daicel Chemical Industry, Tokyo, Japan) and a fluorescence detector (FP-1520, Jasco). HPLC conditions: mobile phase, 27% acetonitrile in H 2 O containing 0.1% formic acid; flow rate, 1.0 mL/min; excitation, 330 nm; emission, 530 nm. The eluted fractions containing D-or L-luciferin (1.2 mL) were collected, concentrated to ,20 mL using a rotary evaporator (N-N series, EYELA, Tokyo, Japan), and applied to LC/ESI-TOF-MS analysis.

LC/ESI-TOF-MS Analysis
LC/ESI-TOF-MS analysis was performed by electrospray ionization-ion trap-mass spectrometry (ESI-TOF-MS) on an Agilent 1100 HPLC system (Agilent Technologies, Santa Clara, CA) with a Mariner Biospectrometry Workstation (Applied Biosystems, Foster City, CA). HPLC conditions: column, Unison UK-C8 (7562 mm; Imtakt, Kyoto, Japan); mobile phase, a linear gradient of methanol in H 2 O containing 0.1% formic acid from 50% to 90% for 12 min; flow rate 0.1 mL/min; sprit ratio, 1:20 (5 mL/min); nozzle potential, 120-360 V; ion mode, positive. Under these conditions, the retention time of the mass ion peaks for D-and L-luciferin with its fragments was at approximately 5.5 min. The mass value was calibrated using angiotensin I (m/ z = 324.9272 and 432.9603) and neurotensin (m/z = 558.3111) as external standards.

Isolation and Identification of Arbutin from L. lateralis
To isolate arbutin from L. lateralis, two frozen female adults were homogenized in 400 mL of methanol on ice. After centrifugation at 17,4006g for 3 min, the supernatant was filtrated by a 0.45 mm centrifugal filter. The filtrate was dried down under N 2 and suspended in 100 mL of 80% methanol. After incubating for 60 min on ice, the precipitate was removed by centrifugation at 17,4006g for 30 min. The resultant supernatant was dried down and dissolved in 50 mL of H 2 O. The aqueous solution was washed three times with 50 mL of ethyl acetate, filtrated, and subjected to reversed-phase HPLC equipped with a Develosil ODS-UG-5 (4.66250 mm; Nomura Chemical, Aichi, Japan) and a fluorescence detector (FP-1520, Jasco). HPLC conditions: mobile phase, 5% methanol in H 2 O; flow rate, 1.0 mL/min; excitation, 280 nm; emission, 320 nm. Figure 10. Identification of arbutin in L. lateralis by HPLC analysis. A. HPLC analysis of the extracts from an adult L. lateralis by using a fluorescence detector. (a) authentic arbutin, (b) the extracts of L. lateralis lantern. The arbutin fraction between the vertical dashed lines is used for hydrolysis as in Fig. 10C. B. HPLC analysis of the hydrolyzed arbutin fraction in Fig. 10A-b. (a) authentic 1,4-hydroquinone (labeled peak 1) containing benzoquinone (labeled peak 2), (b) the hydrolyzed products of the peak fraction between the dashed lines in Fig. 10A  To identify arbutin, the peak fraction (1.2 mL) containing arbutin was concentrated to 10 mL, and 5 mL was used for acid hydrolysis to release 1,4-hydroquinone from arbutin. The total reaction mixture (200 mL) containing 1.1 N HCl was incubated at 95uC for 1 h. After adding 200 mL of H 2 O, the mixture was extracted three times with 400 mL of diethyl ether and the extracts were dried down under N 2 . The resultant solid was immediately dissolved in 30 mL of H 2 O and was analyzed by reversed-phase HPLC equipped with a Develosil ODS-UG-5 (4.66250 mm) and a fluorescence detector. HPLC conditions: mobile phase, 25% methanol in H 2 O; flow rate, 0.8 mL/min; excitation, 290 nm; emission, 338 nm.
To identify 1,4-hydroquinone in firefly, the hydrolyzed extracts obtained from 10 specimens were acetylated in 200 mL of acetic anhydride (Wako Pure Chemicals) and 1 mL of sulfuric acid (Wako Pure Chemicals) at room temperature for 5 min [30]. The acetylated products were subjected to LC/ESI-TOF-MS analysis as described above.