Beetle luciferase, the enzyme responsible for glowing fireflies, and long chain fatty acyl-CoA synthetases (ACSLs), which are expressed in all insects, share a high degree of identity. Both enzymes make adenosine monophosphate (AMP) esters of fatty acids as well as displace the AMP ester with Coenzyme A. However, adding D-luciferin, the substrate for firefly luciferase, to Drosophila melanogaster ACSL does not produce luminescence. Based on Mofford, Reddy and Miller’s earlier work with mutant firefly luciferase and synthetic luciferin substrates, they hypothesized that Drosophila ACSLs could produce light in the presence of the right luciferin substrate. They tested purified Drosophila ACSL CG6178 in the presence of a panel of synthetic luciferin substrates the researchers had previously created to emit red light. The synthetic luciferin substrate CycLuc2 emitted red light when Drosophila ACSL was added, with other closely related substrates producing weaker signals.
Previous experiments with Drosophila ACSL CG6178 showed an inability of ACSL to adenylate D-luciferin. To better understand what might be happening when CG6178 interacts with D-luciferin (lack of binding versus inability to adenylate the substrate), the authors measured luminescence in a reaction with CG6178 + CycLuc2 with increasing amounts of D-luciferin. The firefly luciferase substrate acts as a competitive inhibitor of CycLuc2. So CG6178 can bind D-luciferin with similar affinity as CycLuc2 but is unable to produce the adenylated AMP needed for light production. Due to the nature of ACSL, long chain fatty acids also inhibited luminescence when added to the CG6178 + CycLuc2 reaction.
Examining light emission from CG6178 in the presence of CycLuc2 showed that unlike the characteristic burst phase seen with firefly luciferase and D-luciferin, the light produced is of the glow phase, suggesting light emission is slower than that of firefly luciferase with its substrate. Overall light output for the CG6178 + CycLuc2 reaction was lower than the reaction of firefly luciferase with 100mM CycLuc2; CG6178 + CycLuc2 produced 0.14% of the signal intensity of firefly luciferase and 100mM D-luciferin and 6.7-fold less than the signal for firefly luciferase and 100mM CycLuc2.
To bypass what seems to be an enzymatic roadblock for CG6178, the researchers synthesized adenylated D-luciferin and CycLuc2 (LH2-AMP and CycLuc2-AMP) and incubated these substrates with CG6178. With these substrates, CG6178 emitted light: CycLuc2-AMP at an intensity 4.5-fold higher than with CycLuc2 alone, suggesting adenylation is a rate-limiting step. The comparatively weaker signal with LH2-AMP suggested that the reaction was slower but able to proceed once the adenylation of the substrate occurred. Both adenylated substrates emitted light at 2,100- and 40-fold above known background levels for CycLuc2-AMP and LH2-AMP, respectively, after CG6178 was added.
How does this substrate work in fruit flies? Despite being fed 100μM CycLuc2, fruit flies did not become bioluminescent creatures. However, intact or lysed Drosophila Schneider 2 (S2) cells were luminescent when exposed to CycLuc2 substrate, with a limit of detection of 5,000 cells. D-luciferin produced no light when added to S2 cells. Mofford, Reddy and Miller also transfected CHO cells with CG6178 expressed from a CMV promoter and measured luminescence of live cells in the presence of CycLuc2 and D-luciferin. Like with the S2 cells, luminescence was only measured when CycLuc2 was added to the transfected CHO cells.
This research shows that fruit flies do have latent luminescent activity when the right substrate is used. The homology of firefly luciferase and ASCLs as well as the ability for Drosophila CG6178 to produce light in the presence of CycLuc2 lend more evidence to the possibility that luciferase evolved from ASCLs. However, luminescence was only generated with purified enzymes and in cultured cells so there will be no fruit flies glowing as they cluster around the tomatoes in my kitchen. I doubt there will be much use for glowing fruit flies even if a suitable substrate could be synthesized. Still, I will look at fireflies later this year with greater appreciation and wonder for their unique and enchanting light display.
Mofford, D.M., Reddy, G.R. and Miller, S.C. (2014). Latent luciferase activity in the fruit fly revealed by a synthetic luciferin. Proceedings of the National Academy of Sciences of the United States of America, PMID: 24616520
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