Overcoming limitations of Vibrio harveyi flavin reductase P (FRP)-luciferase coupling by protein engineering: Strategies and consequences.

摘要

Vibrio harveyi flavin reductase P (FRP) and luciferase couple is a well characterized donor and acceptor enzyme couple involved in reduced flavin transfer. Detailed studies of their complex have been limited due to inability of FRP to be preserved as a monomer at high concentrations. Two strategies were adopted in this with an initial objective of preserving FRP as a monomer at high concentrations.; In the first part, a novel fusion enzyme FRP-alphabeta was generated and characterized, in which FRP was genetically fused to the luciferase alpha subunit. It was catalytically active, retained kinetic mechanisms and characteristic of a direct transfer of FMNH2 cofactor from FRP to luciferase as in a FRP:luciferase non-covalent complex. At submicromolar levels, FRP-alphabeta was more active than an equal molar mixture of FRP and luciferase in coupled assay without added flavin and gave a higher total quantum output. Moreover, the bioluminescence produced by FRP-alphabeta without flavin was derived from direct transfer of reduced flavin whereas a mixture of FRP and luciferase with or without exogenously added flavin relied on free-diffusing reduced flavin. Therefore, the overall catalytic reaction of FRP-alphabeta without any FMN addition closely mimics that of a single-component bifunctional monooxygenase.; The second part of this work aimed at studying dimer destabilization of FRP using site-directed mutagenesis approach. Based on in silico mutagenesis, residue 99, glutamic acid was chosen for mutation to lysine and the mutant E99K was characterized in vitro for weakening of subunit interaction and kinetic properties. The mutant protein E99K had lower activities compared to wild type FRP in both single enzyme and luciferase couple assay, but the binding affinities for substrates were not significantly altered. E99K retained the kinetic mechanisms and characteristic of much lower Km values for luciferase coupled assay compared to single enzyme assay. Molecular sieve chromatography showed that E99K exhibited monomer-dimer equilibrium similar to FRP but with much weaker affinity for dimerization. Analytical ultracentrifugation showed that the Kd for dimer dissociation in E99K was enhanced by 45 fold, indicating much weaker binding affinity. Thus it was demonstrated that E99 is a critical residue for dimer formation and that its mutation weakens homodimer formation significantly.