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Identification of Luciferyl Adenylate and Luciferyl Coenzyme A Synthesized by Firefly Luciferase

The firefly luciferase reaction intermediate luciferyl adenylate was detected by RP‐HPLC analysis when the luciferase reaction was performed under a nitrogen atmosphere. Although this compound is always specified as an intermediate in the light‐production reaction, this is the first report of its identification by HPLC in a luciferase assay medium. Under a low‐oxygen atmosphere, luciferase can catalyze the synthesis of luciferyl coenzyme A from luciferin, ATP, and coenzyme A, but in air dehydroluciferyl coenzyme A was produced. The luciferase‐catalyzed synthesis of these coenzyme A derivatives may be a consequence of the postulated recent evolutionary origin of firefly luciferases from an ancestral acyl‐coenzyme A synthetase.