Functional roles of conserved residues in the unstructured loop of Vibrio harveyi bacterial luciferase.
Residues 257-291 of the Vibrio harveyi bacterial luciferase alpha subunit comprise a highly conserved, protease-labile, disordered loop region, most of which is unresolved in the previously determined X-ray structures of the native enzyme. This loop region has been shown to display a time- dependent proteolysis resistance upon single catalytic turnover and was postulated to undergo conformational changes during catalysis ([AbouKhair, N. K., Ziegler, M. M., and Baldwin, T. O. (1985) Biochemistry 24, 3942-3947]. To investigate the role of this region in catalysis, we have performed site-specific mutations of different conserved loop residues. In comparison with V(max) and V(max)/K(m,flavin) of the native luciferase, the bioluminescence activities of alphaG284P were decreased to 1-2% whereas those of alphaG275P and alphaF261D were reduced by 4-6 orders of magnitude. Stopped-flow results indicate that both alphaG275P and alphaF261D were able to form the 4a-hydroperoxy-FMN intermediate II but at lower yields. Both mutants also had enhanced rates for the intermediate II nonproductive 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 the resulting 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. Our results also support a functional role for the alpha subunit unstructured loop itself, possibly by serving as a mobile gating mechanism in shielding critical intermediates (including the excited flavin emitter) from exposure to medium.
Low
John C
JC
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
Tu
Shiao-Chun
SC
D000595:Amino Acid Sequence; D020134:Catalytic Domain / Q000235:genetics; D017124:Conserved Sequence; D004795:Enzyme Stability; D005486:Flavin Mononucleotide; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D021122:Protein Subunits; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D008156:Luciferases