Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta subunit.
Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.
Department of Biochemistry and Molecular Biophysics, University of Arizona, 1041 East Lowell Street, Biological Sciences West, Tucson, Arizona 85721-0088, USA.
Zachary T
ZT
Campbell
Andrzej
A
Weichsel
William R
WR
Montfort
Thomas O
TO
Baldwin
D019943:Amino Acid Substitution; D055162:Biocatalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005486:Flavin Mononucleotide / Q000737:chemistry* / Q000378:metabolism; D006860:Hydrogen Bonding; D006873:Hydroquinones / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D049453:Luciferases, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics; D014733:Vibrio / Q000201:enzymology*
D006873:Hydroquinones; D021122:Protein Subunits; D011994:Recombinant Proteins; C540087:flavin mononucleotide hydroquinone; D014443:Tyrosine; D005486:Flavin Mononucleotide; D049453:Luciferases, Bacterial