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. Firefly luciferase can be mutated to accept and utilize rigid aminoluciferins with high activity in both live and lysed cells yet exhibit 10 000-fold selectivity over the natural luciferase substrate. These new luciferin analogues thus pave the way to an extended family of bioluminescent reporters.

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). In the presence of luciferase, these molecules enable highly sensitive and selective bioluminescent detection of FAAH activity in vitro, in live cells, and in vivo. The potency and tissue distribution of FAAH inhibitors can be imaged in live mice, and luciferin amides serve as exemplary reagents for greatly improved bioluminescence imaging in FAAH-expressing tissues such as the brain.

SUMMARY 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. However, although these substrates can emit an intense initial burst of light that approaches that of D-luciferin, this is followed by much lower levels of sustained light output. We have previously postulated that this behavior is due to product inhibition. Here we describe the creation of mutant luciferases that yield improved sustained light emission with aminoluciferins in both lysed and live mammalian cells, allowing the use of aminoluciferins for cell-based bioluminescence experiments.

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. Bioluminescence can be detected from the purified protein, live Drosophila Schneider 2 cells, and from mammalian cells transfected with CG6178. Thus, the nonluminescent fruit fly possesses an inherent capacity for bioluminescence that is only revealed upon treatment with a xenobiotic molecule. This result expands the scope of bioluminescence and demonstrates that the introduction of a new substrate can unmask latent enzymatic activity that differs significantly from an enzyme's normal function without requiring mutation.evolution | enzymatic promiscuity | imaging | firefly luciferase | chemical biology

Bioluminescence imaging is a powerful approach to visualize 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. Here we show that mutants of firefly luciferase can discriminate between natural and synthetic substrates in the brains of live mice. Using adeno-associated viral (AAV) vectors to express luciferases in the brain, we find that mutant luciferases that are inactive or weakly active with D-luciferin can light up brightly when treated with the aminoluciferins CycLuc1 and CycLuc2 or their respective FAAH-sensitive luciferin amides. Further development of selective luciferases promises to expand the power of bioluminescence and allow multiple events to be imaged in the same live animal.