Figure 1.
Strategy to study on the biosynthetic pathway of firefly luciferin in an adult lantern of a living firefly by injecting the stable isotope-labeled compounds, and the bioluminescence reaction catalyzed by firefly/beetle luciferase.
A. Proposed biosynthetic pathway of firefly luciferin (I) from p-benzoquinone and two ʟ-cysteines in an adult lantern, and the luminescence reaction of luciferin with firefly luciferase, followed by the formation of 2-cyano-6-hydroxybenzothiazole (III) from oxyluciferin (II). B. Stable isotope-labeled ʟ-cysteines used in the experiments. Asterisk indicates the position of a 13C atom. C. Preparation of p-[D4]-benzoquinone from 1,4-[D6]-hydroquinone by the oxidation reaction using silver oxide with H2O2.
Figure 2.
Mass spectrum of ᴅ-firefly luciferin by ESI-TOF-MS analysis.
A. The structure of ᴅ-firefly luciferin and the predicted mass fragment ions. The parent ion of ᴅ-luciferin at m/z 281 (M+H)+ (a), and its fragment ions at m/z 235 (b) and 177 (c). B. ESI-TOF-MS analysis of synthetic ᴅ-firefly luciferin.
Table 1.
Relative isotopic peak intensities (%) of the parent and its fragment mass from firefly luciferin in the lantern extracts after injecting ʟ-Cys[U-13C3] with 1,4-hydroquinone or p-benzoquinone into the adult of L. lateralis.
Figure 3.
Incorporation of 1,4-hydroquinone and 13C-labeled ʟ-cysteines into firefly luciferin in an adult lantern of L. lateralis.
A. Predicted firefly luciferins incorporated from 1,4-hydroquinone and ʟ-Cys[U-13C3]. B. Predicted firefly luciferins incorporated from 1,4-hydroquinone and ʟ-Cys[1-13C3]. C. Predicted firefly luciferins incorporated from 1,4-hydroquinone and ʟ-Cys[3-13C3]. The number in parenthesis on the right indicates the number of 13C-atom incorporated into firefly luciferin. Asterisk indicates the position of a 13C-atom.
Figure 4.
Mass spectra of firefly luciferin labeled with ʟ-Cys[U-13C3] and 1,4-hydroquinone in the adult lantern of L. lateralis.
(a), the parent ion; (b) and (c), the fragments ions of firefly luciferin.
Table 2.
Relative isotopic peak intensities (%) of the parent and its fragment mass from firefly luciferin in the lantern extracts after injecting [D6]-hydroquinone or [D4]-benzoquinone into the adult of L. lateralis.
Figure 5.
Incorporation of p-[D4]-benzoquinone or 1,4-[D6]-hydroquinone with ʟ-cysteines into firefly luciferin in an adult lantern of L. lateralis.
A. Predicted firefly luciferins incorporated from p-[D4]-benzoquinone and ʟ-cysteine. B. Predicted firefly luciferins incorporated from 1,4-[D6]-hydroquinone and ʟ-cysteine. C. Predicted firefly luciferins incorporated from 1,4-[D6]-hydroquinone and ʟ-Cys[3-13C3]. The number in parenthesis on the right indicates the number of the stable isotope atoms incorporated into firefly luciferin. Asterisk indicates the position of a 13C-atom.
Figure 6.
Mass spectra of firefly luciferin labeled with ʟ-Cys[1-13C] and 1,4-hydroquinone in the adult lantern of L. lateralis.
(a), the parent ion; (b) and (c), the fragments ions of firefly luciferin.
Table 3.
Relative isotopic peak intensities (%) of the parent and its fragment mass from firefly luciferin in the lantern extracts after injecting ʟ-Cys[1-13C] or ʟ-Cys[3-13C] with 1,4-hydroquinone into the adult of L. lateralis.
Figure 7.
Mass spectra of firefly luciferin labeled with ʟ-Cys[3-13C] and 1,4-hydroquinone in the adult lantern of L. lateralis.
(a), the parent ion; (b) and (c), the fragments ions of firefly luciferin.
Figure 8.
Mass spectrum of firefly luciferin labeled with ʟ-Cys[U-13C] and 1,4-[D6]-hydroquinone into the adult lantern of L. lateralis.
Figure 9.
Identification of ʟ- and ᴅ-luciferin in an adult lantern of L. lateralis by HPLC analysis and the incorporation of ʟ-cysteine into ᴅ- and ʟ-luciferin.
A. Isolation of ʟ- and ᴅ-firefly luciferin by HPLC analysis with a chiral column. (a) authentic ᴅ-luciferin, (b) authentic ʟ-luciferin, (c) the extracts of L. lateralis lantern without injections, (d) the extracts of L. lateralis lantern after injection with ʟ-Cys[U-13C3] and 1,4-hydroquinone. The peak fractions of 1 and 2, corresponding to ʟ-luciferin and ᴅ-luciferin, respectively, are subjected to ESI-TOF-MS analysis, as shown in Fig. 9B. B. ESI-TOF-MS analyses of the HPLC fractions for ʟ-luciferin and ᴅ-luciferin, separated by HPLC analysis as in Fig.9A–d. (a) ʟ-luciferin separated from the lantern. (b) ᴅ-luciferin separated from the lantern.
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–b. Asterisk indicates 1,4-hydroquinone from arbutin. C. A scheme of acid hydrolysis of arbutin to 1,4-hydroquinone by acid treatment with HCl.
Figure 11.
Proposed biosynthetic pathway of firefly luciferin in the lantern of adult firefly.