Molecular evolution of dinoflagellate luciferases, enzymes with three catalytic domains in a single polypeptide.

Enzymes with multiple catalytic sites are rare, and their evolutionary significance remains to be established. This study of luciferases from seven dinoflagellate species examines the previously undescribed evolution of such proteins. All these enzymes have the same unique structure: three homologous domains, each with catalytic activity, preceded by an N-terminal region of unknown function. Both pairwise comparison and phylogenetic inference indicate that the similarity of the corresponding individual domains between species is greater than that between the three different domains of each polypeptide. Trees constructed from each of the three individual domains are congruent with the tree of the full-length coding sequence. Luciferase and ribosomal DNA trees both indicate that the Lingulodinium polyedrum luciferase diverged early from the other six. In all species, the amino acid sequence in the central regions of the three domains is strongly conserved, suggesting it as the catalytic site. Synonymous substitution rates also are greatly reduced in the central regions of two species but not in the other five. This lineage-specific difference in synonymous substitution rates in the central region of the domains correlates inversely with the content of GC3, which can be accounted for by the biased usage toward C-ending codons at the degenerate sites. RNA modeling of the central region of the L. polyedrum luciferase domain suggests a function of the constrained synonymous substitutions in the circadian-controlled protein synthesis.

Two bioluminescent diptera: the North American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different bioluminescence systems.

Orfelia fultoni is the only bioluminescent dipteran (Mycetophilidae) found in North America. Its larvae live on stream banks in the Appalachian Mountains. Like their Australasian relative Arachnocampa spp., they build sticky webs to which their bioluminescence attracts flying prey. They bear two translucent lanterns at the extremities of the body, histologically distinct from the single caudal lantern of Arachnocampa spp., and emit the bluest bioluminescence recorded for luminescent insects (lambda(max) = 460 nm versus 484 nm from Arachnocampa). A preliminary characterization of these two bioluminescent systems indicates that they are markedly different. In Orfelia a luciferin-luciferase reaction was demonstrated by mixing a hot extract prepared with dithiothreitol (DTT) under argon with a crude cold extract. Bioluminescence is not activated by adenosine triphosphate (ATP) but is strongly stimulated by DTT and ascorbic acid. Using gel filtration, we isolated a luciferase fraction of approximately 140 kDa and an additional high molecular weight fraction (possibly a luciferin-binding protein) that activated bioluminescence in the presence of luciferase and DTT. The Arachnocampa luciferin-luciferase system involves a 36 kDa luciferase and a luciferin soluble in ethyl acetate under acidic conditions; the bioluminescence is activated by ATP but not by DTT. The present findings indicate that the bioluminescence of O. fultoni constitutes a novel bioluminescent system unrelated to that of Arachnocampa.