title: Vibrio harveyi flavin reductase--luciferase fusion protein mimics a single-component bifunctional monooxygenase.
abstract: 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-alphabeta, in which FRP was fused to the luciferase alpha subunit. Both FRP and luciferase within FRP-alphabeta 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-alphabeta. At submicromolar levels, FRP-alphabeta was significantly more active than an equal molar mixture of FRP and luciferase in coupled bioluminescence without FMN addition. Importantly, FRP-alphabeta gave a higher total quantum output without than with exogenously added FMN. Moreover, effects of increasing concentrations of oxygen on light intensity were investigated using sub-micromolar enzymes, and results indicated that the bioluminescence produced by FRP-alphabeta without added flavin was derived from direct transfer of reduced flavin whereas bioluminescence from 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. This fusion enzyme approach could be useful to other two-component monooxygenases in enhancing the enzyme efficiencies under conditions hindering reduced flavin delivery. Other potential utilities of this approach are discussed.
authors: Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
authors: Navneet
authors: N
authors: Jawanda
authors: Kamran
authors: K
authors: Ahmed
authors: Shiao-Chun
authors: SC
authors: Tu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D038181:FMN Reductase / Q000235:genetics / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D009249:NADP; D005486:Flavin Mononucleotide; D006899:Mixed Function Oxygenases; D008156:Luciferases; D038181:FMN Reductase; D010100:Oxygen