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Bioluminescence, the conversion of chemical energy into light in living organisms, is dependent on two principal components, an enzyme luciferase and the substrate luciferin. In beetles, the enzyme luciferase has been extensively studied, with significant enzymological, sequence and structural data now available. Furthermore, the enzyme has been employed in a remarkable number of important applications, from microbial detection and medical imaging to GM gene expression studies. However, there is little information regarding the biosynthesis of beetle luciferin, and here we review the literature and speculate as to its evolutionary origins. Luciferin consists of a benzothiazole moiety attached to a thiazole carboxylic acid moiety, the former being rarely observed in nature but the latter being observed in a broad range of biologically derived molecules. Benzothiazoles are, however, observed in melanogenesis and we speculate as to whether this may be relevant to the understanding of luciferin biosynthesis in beetles. This review examines recent novel insights into beetle luciferin recycling and we assess a range of possible biosynthetic mechanisms.

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Firefly luciferase catalyzes the highly efficient emission of yellow-green light from the substrates luciferin, Mg-ATP, and oxygen in a two-step process. The enzyme first catalyzes the adenylation of the carboxylate substrate luciferin with Mg-ATP followed by the oxidation of the acyl-adenylate to the light-emitting oxyluciferin product. The beetle luciferases are members of a large family of nonbioluminescent proteins that catalyze reactions of ATP with carboxylate substrates to form acyl-adenylates. Formation of the luciferase-luciferyl-AMP complex is a specific example of the chemistry common to this enzyme family. Site-directed mutants at positions Lys529, Thr343, and His245 were studied to determine the effects of the amino acid changes at these positions on the individual luciferase-catalyzed adenylation and oxidation reactions. The results suggest that Lys529 is a critical residue for effective substrate orientation and that it provides favorable polar interactions important for transition state stabilization leading to efficient adenylate production. These findings as well as those with the Thr343 and His245 mutants are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

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The chemical structure of two luciferin precursors PS-A and PS-B, isolated from the luminous mushroom Panellus stipticus, were determined as 1-O-decanoylpanal (2) and 1-O-dodecanoylpanal (3), respectively. Both PS-A and PS-B were converted into chemiluminescent luciferins by treatment with 50 mmol/l methylamine in a pH 3.5 buffer solution containing an anionic surfactant Tergitol 4 at 25-35 degrees C. The luciferins emitted chemiluminescence in a pH 7-8 buffer solution containing a cationic surfactant in the presence of O2 and O2-.

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A 10-fold circadian variation in the amount of luciferin binding protein (LBP) in the marine dinoflagellate Gonyaulax polyedra is reported. This protein binds and stabilizes luciferin, the bioluminescence substrate. In early night phase, when bioluminescence is increasing and LBP levels are rising in the cell, pulse labeling experiments show that LBP is being rapidly synthesized in vivo. At other times, the rate of LBP synthesis is at least 50 times lower, while the rate of synthesis of most other proteins remains the same. The LBP mRNA levels, as determined by in vitro translations and by RNA (Northern) hybridizations, do not vary over the daily cycle, indicating that circadian control of bioluminescence in this species is mediated by translation.

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For the first time, luciferin from a bioluminescent earthworm has been purified, identified, and synthesized. This luciferin from the North American species, Diplocardia longa, is a simple aldehyde compound, N-isovaleryl-3-aminopropanal, with an amide functional group. It is a clear, odorless oil at room temperature. It is nonvolatile and has no near-uv-visible absorption or fluorescence. Derivatives of this compound were made to facilitate its identification: the luciferin 2,4-dinitrophenylhydrazone (mp 174 degrees C), a yellow crystalline solid; and the luciferin alcohol, a clear oil. Synthesis of Diplocardia luciferin yielded an oil of identical spectroscopic (proton nuclear magnetic resonance (NMR), 13C NMR, mass, and ir), chemical (dinitrophenylhydrazone and alcohol derivatives, bioluminescence activity), and physical (thin-layer chromatography, volatility) properties to those of the purified native Diplocardia luciferin.

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