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

Date Published: December 31, 2013

Publisher: Public Library of Science

Author(s): Yuichi Oba, Naoki Yoshida, Shusei Kanie, Makoto Ojika, Satoshi Inouye, Fanis Missirlis.

http://doi.org/10.1371/journal.pone.0084023

Abstract

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.

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.

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.

Partial Text

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, Mg2+, 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-(6′-hydroxy-2′-benzothiazolyl)-2-thiazoline-4-carboxylic acid (I, ᴅ-firefly 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 ᴅ-firefly luciferin from 2-cyano-6-hydroxybenzothiazole (III) and ᴅ-cysteine [5], [6]. ʟ-Firefly luciferin with the R form is not used for the luminescence reaction by firefly luciferase [7]. Thus, firefly luciferase oxidizes only ᴅ-firefly luciferin to emit light and produces oxyluciferin (II) and CO2 (Figure 1A).

Studies on the biosynthesis of luciferin in firefly and click beetle were initiated in the early 1970’s using 14C-labeled compounds. A hypothesis that firefly luciferin (beetle luciferin) is biosynthesized from p-benzoquinone and two cysteines was proposed [14]–[16], [18]. In this report, we have identified the biosynthetic components of firefly luciferin by mass spectroscopy with stable isotope-labeled compounds. The 13C-labeled ʟ-cysteine, p-[D4]-benzoquinone and 1,4-[D6]-hydroquinone were incorporated into firefly luciferin in an adult lantern of a firefly. The incorporation experiment with ʟ-Cys[U-13C3] indicated that ʟ-cysteine was incorporated into both the benzothiazole and thiazoline unit of firefly luciferin (Figures 1 and 3). This is the first demonstration that two ʟ-cysteine molecules are the biosynthetic components of firefly luciferin (Figure 3 and Table 2). Furthermore, the incorporation of ʟ-[1-13C]-cysteine and ʟ-[3-13C]-cysteine into firefly luciferin revealed that the carboxyl group of ʟ-[1-13C]-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 14C-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 ʟ-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 ʟ-cysteine in our experiments (Figure 7).

 

Source:

http://doi.org/10.1371/journal.pone.0084023