Recombinant Production of Hispidin-3-Hydroxylase: the Key Enzyme in Fungal Luciferin Biosynthesis

Bioluminescence is a phenomenon of light emission resulting from oxidation of a substrate, luciferin, catalyzed by the enzyme luciferase. The fungus Neonothopanus nambi is the first eukaryotic organism with a fully deciphered bioluminescent system: the structure of luciferin was established, the luciferase gene was described, and intermediates and enzymes involved in the luciferin biosynthesis pathway were identified. One of the crucial reactions in this pathway is the formation of luciferin by hydroxylation of hispidin catalyzed by hispidin-3-hydroxylase (nnH3H). To fully understand the mechanism of action and substrate specificity of the enzyme, it is necessary to carry out structural studies of the molecule. To do that, it is necessary to develop a protocol for obtaining a highly purified and functionally active nnH3H in the appropriate quantities. We describe a robust approach to produce a soluble and enzymatically active nnH3H fused with SUMO and coexpressed with GroEL/ES chaperonin at low temperature in Escherichia coli. The yield of recombinant nnH3H achieved was 20 mg per 100 mL of bacterial culture. Additionally, we show for the first time that FAD is a cofactor of fungal hispidin-3-hydroxylase.