Lessons Learned from Luminous Luciferins and Latent Luciferases.

Compared to the broad palette of fluorescent molecules, there are relatively few structures that are competent to support bioluminescence. Here, we focus on recent advances in the development of luminogenic substrates for firefly luciferase. The scope of this light-emitting chemistry has been found to extend well beyond the natural substrate and to include enzymes incapable of luciferase activity with d-luciferin.

Luciferase Activity of Insect Fatty Acyl-CoA Synthetases with Synthetic Luciferins.

Long-chain fatty acyl-CoA synthetases (ACSLs) are homologues of firefly luciferase but are incapable of emitting light with firefly luciferin. Recently, we found that an ACSL from the fruit fly Drosophila melanogaster is a latent luciferase that will emit light with the synthetic luciferin CycLuc2. Here, we have profiled a panel of three insect ACSLs with a palette of >20 luciferin analogues.

Firefly Luciferase Mutants Allow Substrate-Selective Bioluminescence Imaging in the Mouse Brain.

Bioluminescence imaging is a powerful approach for visualizing specific events occurring inside live mice. Animals can be made to glow in response to the expression of a gene, the activity of an enzyme, or the growth of a tumor. But bioluminescence requires the interaction of a luciferase enzyme with a small-molecule luciferin, and its scope has been limited by the mere handful of natural combinations.

Luciferins behave like drugs.

The light emission chemistry of firefly luciferase can be harnessed to reveal otherwise invisible biological processes occurring in the brains of live animals. Though powerful, the need for the luciferase substrate D-luciferin to traverse the blood-brain barrier poses limitations on the sensitivity and interpretation of these experiments. In this Viewpoint, we discuss bioluminescent imaging probes for the enzyme fatty acid amide hydrolase (FAAH) and the broader implications for optical imaging and drug delivery in the brain.

Luciferin Amides Enable in Vivo Bioluminescence Detection of Endogenous Fatty Acid Amide Hydrolase Activity.

Firefly luciferase is homologous to fatty acyl-CoA synthetases. We hypothesized that the firefly luciferase substrate d-luciferin and its analogs are fatty acid mimics that are ideally suited to probe the chemistry of enzymes that release fatty acid products. Here, we synthesized luciferin amides and found that these molecules are hydrolyzed to substrates for firefly luciferase by the enzyme fatty acid amide hydrolase (FAAH).

Aminoluciferins extend firefly luciferase bioluminescence into the near-infrared and can be preferred substrates over D-luciferin.

Firefly luciferase adenylates and oxidizes d-luciferin to chemically generate visible light and is widely used for biological assays and imaging. Here we show that both luciferase and luciferin can be reengineered to extend the scope of this light-emitting reaction. D-Luciferin can be replaced by synthetic luciferin analogues that increase near-infrared photon flux >10-fold over that of D-luciferin in live luciferase-expressing cells.

Latent luciferase activity in the fruit fly revealed by a synthetic luciferin.

Beetle luciferases are thought to have evolved from fatty acyl-CoA synthetases present in all insects. Both classes of enzymes activate fatty acids with ATP to form acyl-adenylate intermediates, but only luciferases can activate and oxidize d-luciferin to emit light. Here we show that the Drosophila fatty acyl-CoA synthetase CG6178, which cannot use d-luciferin as a substrate, is able to catalyze light emission from the synthetic luciferin analog CycLuc2.

Identification of mutant firefly luciferases that efficiently utilize aminoluciferins.

Firefly luciferase-catalyzed light emission from D-luciferin is widely used as a reporter of gene expression and enzymatic activity both in vitro and in vivo. Despite the power of bioluminescence for imaging and drug discovery, light emission from firefly luciferase is fundamentally limited by the physical properties of the D-luciferin substrate. We and others have synthesized aminoluciferin analogs that exhibit light emission at longer wavelengths than D-luciferin and have increased affinity for luciferase.