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bioRxiv · 10.1101/2023.06.30.547126

FruitFire: a luciferase based on a fruit fly metabolic enzyme

Adams, S. T.·Zephyr, J.·Bohn, M. F.·Schiffer, C. A.·Miller, S. C.
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Abstract

Firefly luciferase is homologous to fatty acyl-CoA synthetases from insects that are not bioluminescent. Here, we determined the crystal structure of the fruit fly fatty acyl-CoA synthetase CG6178 to 2.5 [A]. Based on this structure, we mutated a steric protrusion in the active site to create the artificial luciferase FruitFire, which prefers the synthetic luciferin CycLuc2 to D-luciferin by >1000-fold. FruitFire enabled in vivo bioluminescence imaging in the brains of mice using the pro-luciferin CycLuc2-amide. The conversion of a fruit fly enzyme into a luciferase capable of in vivo imaging underscores the potential for bioluminescence with a range of adenylating enzymes from nonluminescent organisms, and the possibilities for application-focused design of enzyme-substrate pairs.

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Adams, S. T., Zephyr, J., Bohn, M. F., Schiffer, C. A., Miller, S. C.. 2023-06-30. FruitFire: a luciferase based on a fruit fly metabolic enzyme. https://doi.org/10.1101/2023.06.30.547126

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