This research will focus on the fundamental biochemical mechanisms of light emitting systems in marine dinoflagellates (responsible for the "phosphorescence of the sea") and terrestrial Diptera (flies), two major and evolutionarily independent groups of bioluminescent organisms. The biochemical components of the light-emitting reaction of the dinoflagellate Gonyaulax polyedra - the luciferase enzyme (LCF), the luciferin substrate (LH2), a unique tetrapyrrole, and a luciferin binding protein (LBP)- are sequestered in an organelle, the scintillon. LCF consists of three highly conserved and contiguous sequences, each of which is catalytically active. The minimum peptide size necessary for activity and the location of the active site will be determined. Comparison of LCF sequences from other bioluminescent dinoflagellates will allow residues conserved for enzyme activity and regulation to be identified, and our understanding of the evolution of the luciferase gene structure will be increased. Larvae of the web-building, carnivorous North American dipteran Platyura fultoni (<15 mm long) have two light organs (head and tail) and emit a blue luminescence with an emission peak at 460 nm, but the biochemistry of the bioluminescence reaction is not known. Oxygen is required, suggesting the involvement of a peroxidic intermediate, as in all bioluminescence reactions. A granular secretion ascribed to the mitochondria of giant cells located in these organs is reported to be associated with the light emission. The study will focus on these unusual cytological aspects and on the identification of the biochemical components. The web-weaving larvae of an Australian bioluminescent dipteran, Arachnocampa flava, present similarities with Platyura despite differences in light organ anatomy; the biochemistry of the light emitting reactions of these two diptera will be compared.
An understanding of the basic mechanisms in bioluminescence will advance the knowledge of energy metabolism, as well as our understanding of processes involving reactive oxygen species, now known to serve as cellular messengers as well as agents of cellular damage. Fundamental knowledge of the genes, enzymes and substrates responsible for bioluminescence has led to extensive applications, ranging from sensitive visualization of gene expression to analytical measurements of drugs and metabolites. The dinoflagellate studies proposed here promise to contribute significant advances in enzymology, as well as in molecular evolution. The biochemistry of the dipteran bioluminescence, which the investigator recently began studying, is entirely novel, hence rich in promises of new knowledge and applications.
Specific aims for this project are as follows: (a) examine luciferase genes from other bioluminescent dinoflagellates so as to gain insight into gene evolution and to aid in the determination of amino acid residues involved in catalysis; (b) define the structural regions required for luciferase activity within the individual domains; (c) characterize the biochemical components of the bioluminescence reactions of the dipterans Platyura and Arachnocampa, isolate and partially purify luciferin and luciferase; test for the ability of the beetle luciferase to react with the dipteran luciferin.
