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

Abstract

BackgroundBioluminescence 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 FindingsIncorporation 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.ConclusionsWe 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.

Beyond D-luciferin: expanding the scope of bioluminescence imaging in vivo

“…In an amazing feat of in vivo chemistry that remains somewhat mysterious, the firefly synthesizes D-luciferin in a multi-step process from L-cysteine and benzoquinone [47]. In the laboratory, D-luciferin and aminoluciferin analogs are typically synthesized by the condensation of an electron-deficient nitrile with D-cysteine (Figure 3).…”

Termite soldiers contribute to social immunity by synthesizing potent oral secretions

“…darwiniensis soldiers, the identified chemical compounds are thought to be mixed in the labial glands (Sillam‐Dusses et al ., ) with p ‐benzoquinone being the dominant component found in secreted fluids (Moore, ) (Table ). The process of quinone production had been hypothesized in Eleodes longicollis and Tribolium castaneum (Happ, ), and also as a part of luciferin biosynthesis in the firefly Luciola lateralis (Oba et al ., ) but the pathways responsible for the production of these chemicals has remained largely unclear. In our study, we identified glucosylceramidase, oxidoreductases (in particular, glucose dehydrogenase [FAD‐quinone]) and laccase 2, which are likely to be involved in the of benzoquinone biosynthesis in soldier secretions.…”

Enzymatic promiscuity and the evolution of bioluminescence

“…The synthesis of d ‐luciferin in the laboratory typically employs condensation of 2‐cyano‐6‐hydroxybenzothiazole with d ‐cysteine as the final step . Although early work suggested this reaction can occur upon co‐injection of 2‐cyano‐6‐hydroxybenzothiazole and d ‐cysteine into firefly lantern organs , this is not likely the method that fireflies use to make their luciferin . First, amino acids are made in the l ‐form, and there is no evidence of excess d ‐cysteine in firefly lanterns.…”