Vibrio harveyi flavin reductase--luciferase fusion protein mimics a single-component bifunctional monooxygenase.

Vibrio harveyi luciferase and flavin reductase FRP are, together, a two-component monooxygenase couple. The reduced flavin mononucleotide (FMNH2) generated by FRP must be supplied, through either free diffusion or direct transfer, to luciferase as a substrate. In contrast, single-component bifunctional monooxygenases each contains a bound flavin cofactor and does not require any flavin addition to facilitate catalysis. In this study, we generated and characterized a novel fusion enzyme, FRP-αβ, in which FRP was fused to the luciferase α subunit. Both FRP and luciferase within FRP-αβ were catalytically active. Kinetic properties characteristic of a direct transfer of FMNH2 cofactor from FRP to luciferase in a FRP:luciferase noncovalent complex were retained by FRP-αβ. At submicromolar levels, FRP-αβ was significantly more active than an equal molar mixture of FRP and luciferase in coupled bioluminescence without FMN addition. Importantly, FRP-αβ gave a higher total quantum output without than with exogenou...