Functional implications of the unstructured loop in the (beta/alpha)(8) barrel structure of the bacterial luciferase alpha subunit.
Bacterial luciferase catalyzes the conversion of FMNH(2), a long-chain aliphatic aldehyde, and molecular oxygen to FMN, the corresponding carboxylic acid, and H(2)O with the emission of light. The light-emitting species is an enzyme-bound excited state flavin. The enzyme is a heterodimer (alphabeta) of homologous subunits each with an (beta/alpha)(8) barrel structure. A portion of the loop in the alpha subunit that connects beta strand 7 to alpha helix 7 is disordered in the crystal structure. To test the hypothesis that this loop closes over the active site during catalysis and protects the active site from bulk solvent, a mutant was constructed in which the 29 residues that are disordered in the 2.4 A crystal structure were deleted. Deletion of this loop results in a heterodimer with a subunit equilibrium dissociation constant of 1.32 +/- 1.25 microM, whereas the wild-type heterodimer shows no measurable subunit dissociation. This mutant retains its ability to bind substrate flavin and aldehyde with wild-type affinity and can carry out the chemistry of the bioluminescence reaction with nearly wild-type efficiency. However, the bioluminescent quantum yield of the reaction is reduced nearly 2 orders of magnitude from that of the wild-type enzyme.
Sparks
J M
JM
Department of Biochemistry and Molecular Biophysics, The University of Arizona, Tucson, Arizona 85721-0088, USA.
Baldwin
T O
TO
D001665:Binding Sites / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005486:Flavin Mononucleotide / Q000378:metabolism; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion
D021122:Protein Subunits; D011994:Recombinant Proteins; D005486:Flavin Mononucleotide; D008156:Luciferases