I. Energy transfer evidence for in vitro and in vivo complexes of Vibrio harveyi flavin reductase P and luciferase. II. Functional roles of conserved residues in the unstructured loop of Vibrio harveyi bacterial luciferase

Direct evidence of in vivo and in vitro interactions between Vibrio harveyi NADPH-FMN oxidoreductase (FRP) and the enzyme Bacterial Luciferase were obtained by variation of a technique previously described as Bioluminescence Resonance Energy Transfer. Yellow Fluorescence Protein (YFP) was fused to FRP to detect complex formation based on the excitation of the reporter chromophore by luciferase emission. Michaelis constants for the fusion protein in a single enzyme assay were 20.9 muM and 48.8 muM for FMN and NADPH, respectively. In the luciferase coupled enzyme assay these values were reduced to 2.8 muM and 0.26 M and the kinetic mechanism was modified to sequential. Difference spectra of light emission in coupled enzyme assays using the FRP-YFP fusion versus FRP or FRP plus YFP reveals a peak at the YFP emission maximum, indicating complex formation. Similar results were obtained replacing native V. harveyi FRP with the tagged enzyme via standard recombination methods.; A highly conserved, protease-labile, disordered loop region comprised of residues 258--291 of the Vibrio harveyi bacterial luciferase alpha subunit is unresolved in the previously determined X-ray structures of the native enzyme. To investigate the role of this region in catalysis, we have performed site-specific mutations of different conserved loop residues. In comparison with Vmax and Vmax/Km,flavin of the native luciferase, the bioluminescence activities of alphaG284P were decreased to 1--2% whereas those of alphaG275P and alphaF261D were reduced by 4 to 6 orders of magnitude. Stopped-flow results indicate that both alphaG275P and alphaF261D were able to form the 4a-hydroperoxyFMN intermediate II but at lower yields. Both mutants also had enhanced rates for the intermediate II non-productive dark decay and significantly compromised abilities to oxidize the decanal substrate. Additional mutations were introduced into the alphaG275 and alphaF261 positions, and the activities of these mutants were characterized. Results indicate that the torsional flexibility of the alphaG275 residue and the bulky and hydrophobic nature of the alphaF261 residue were critical to the luciferase activity. A plausible functional role for the alpha subunit unstructured loop itself is discussed further, in light of these findings.