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

Abstract

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.

Firefly Luciferase Produces Hydrogen Peroxide as a Coproduct in Dehydroluciferyl Adenylate Formation

“…This proton removal is considered the limiting step in firefly bioluminescence, allowing carbanion formation, oxygen attack with subsequent dioxetanone ring formation, and light emission (Scheme 1). [1,29,30] As shown in Figure 4, when we incubated l-LH 2 with ATP and Luc we observed, as expected, minimal l-LH 2 consumption (the consumption observed can be explained by chemical racemization of the l-LH 2 -AMP formed [18,28] ) and minimal L formation, so we were able to conclude that, as in the light reaction, L-AMP formation from d-LH 2 -AMP is a stereospecific process, also involving proton removal from C4.…”

“…In previous publications from our group, H 2 O 2 has been pointed to as a possibility. [18,19] In this report we present results showing that H 2 O 2 is indeed the reduced coproduct formed when LH 2 -AMP is oxidized into L-AMP by molecular oxygen (Scheme 1). We were able to correlate L and H 2 O 2 production in firefly bioluminescence reactions directly with the aid of a selective and sensitive H 2 O 2 probe (horseradish peroxidase-catalyzed oxidation of Amplex Red into resorufin [21][22][23] ) and RP-HPLC.…”

“…[8][9][10][11][12] This assumption is not mere speculation: since molecular oxygen is involved in the main oxidation process (the one that gives rise to oxyluciferin) it is readily available in the enzyme core and it has been observed that the formation of L-AMP by Luc is strongly inhibited when oxygen is scarce. [8,18] However, the nature of the reduced product remained unknown. In previous publications from our group, H 2 O 2 has been pointed to as a possibility.…”

Firefly bioluminescence: A mechanistic approach of luciferase catalyzed reactions

“…The oxygen in the carboxyl group is a nucleophile, and nucleophilic attack of the electrophilic phosphorus at the a-phosphoryl group of ATP displaces inorganic pyrophosphate (PPi), an excellent leaving group, and transfers adenylate (5 0 -AMP) to D-LH 2 . The reaction is, thus, an adenylylation, and produces an enzyme-bound intermediate, luciferyl-adenylate (LH 2 -AMP) (22)(23)(24), a mixed anhydride. The phosphoryl group transfer is also found, for example, in the activation of fatty acids by fatty acyl-coenzyme A synthetases and in the attachment of amino acids to their correspondent tRNA in protein synthesis by aminoacyl-tRNA synthetases (21,25).…”

“…The L-LH 2 isomer can also be adenylylated, giving rise to L-LH 2 -AMP; however it is not used further in light production (24,27). …”