Energy transfer evidence for in vitro and in vivo complexes of Vibrio harveyi flavin reductase P and luciferase.
Conservation of energetically "expensive" metabolites is facilitated by enzymatic intra- and intermolecular channeling mechanisms. Our previous in vitro kinetic studies indicate that Vibrio harveyi reduced nicotinamide adenine dinucleotide phosphate-flavin mononucleotide (NADPH-FMN) oxidoreductase flavin reductase P (FRP) can transfer reduced riboflavin 5'-phosphate (FMNH2) to bacterial luciferase by direct channeling. However, no evidence has ever been reported for such an FMNH2 channeling between these two enzymes in vivo. The formation of a donor-acceptor enzyme complex, stable or transient, is mandatory for direct metabolite channeling between two enzymes regardless of details of the transfer mechanisms. In this study, we have obtained direct evidence of in vitro and in vivo FRP-luciferase complexes that are functionally active. The approach used is a variation of a technique previously described as Bioluminescence Resonance Energy Transfer. Yellow fluorescence protein (YFP) was fused to FRP to generate an active FRP-YFP fusion enzyme, which emits fluorescence peaking at 530 nm. In comparison with the normal 490 nm bioluminescence, an additional 530 nm component was observed in both the in vitro bioluminescence from the coupled reaction of luciferase and FRP-YFP and the in vivo bioluminescence from frp gene-negative V. harveyi cells that expressed FRP-YFP. This 530 nm bioluminescence component was not detected in a control in which a much higher level of YFP was present but not fused to FRP. Such findings indicate an energy transfer from the exited emitter of luciferase to the FRP component of the luciferase-FRP-YFP complex. Hence, the formation of an active complex of luciferase and FRP-YFP was detected both in vitro and in vivo.
Low
John C
JC
Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5001, USA.
Tu
Shiao-Chun
SC
D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D017931:DNA Primers; D004735:Energy Transfer; D008156:Luciferases / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D014733:Vibrio / Q000201:enzymology*
D001426:Bacterial Proteins; D017931:DNA Primers; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D010088:Oxidoreductases; D008156:Luciferases