Abstract
Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic R subunit and a homologous but noncatalytic β 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 Å. 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 [Fisher, A. J., Raushel, F. M., Baldwin, T. O., and Rayment, I. (1995) Biochemistry 34, 6581-6586; Fisher, A. J., Thompson, T. B., Thoden, J. B., Baldwin, T. O., and Rayment, I. (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 R subunit, not seen in the previous structures, and Tyr151 of the β subunit. Substitutions at position 151 on the β subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH2). These findings partially address the long-standing question of how the β subunit stabilizes the active conformation of the R subunit, thereby participating in the catalytic mechanism.
Original language | English (US) |
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Pages (from-to) | 6085-6094 |
Number of pages | 10 |
Journal | Biochemistry |
Volume | 48 |
Issue number | 26 |
DOIs | |
State | Published - Jul 7 2009 |
ASJC Scopus subject areas
- Biochemistry
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Crystal Structure of the Bacterial Luciferase:Flavin Complex Reveals the Basis of Intersubunit Communication
Campbell, Z. T. (Contributor), Weichsel, A. (Contributor), Montfort, W. R. (Contributor) & Baldwin, T. O. (Contributor), Protein Data Bank (PDB), May 26 2009
DOI: 10.2210/pdb3FGC/pdb, https://www.wwpdb.org/pdb?id=pdb_00003fgc
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