Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the β subunit

Zachary T. Campbell, Andrzej Weichsel, William "Bill" Montfort, Thomas O. Baldwin

Research output: Contribution to journalArticle

46 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)6085-6094
Number of pages10
JournalBiochemistry
Volume48
Issue number26
DOIs
StatePublished - Jul 7 2009

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Bacterial Luciferases
Flavin Mononucleotide
Conformations
Crystal structure
Biochemistry
Vibrio
Oxygenation
Quantum yield
Luciferases
Catalytic Domain
Substitution reactions
Peptides
Proteins
4,6-dinitro-o-cresol

ASJC Scopus subject areas

  • Biochemistry

Cite this

Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the β subunit. / Campbell, Zachary T.; Weichsel, Andrzej; Montfort, William "Bill"; Baldwin, Thomas O.

In: Biochemistry, Vol. 48, No. 26, 07.07.2009, p. 6085-6094.

Research output: Contribution to journalArticle

Campbell, Zachary T. ; Weichsel, Andrzej ; Montfort, William "Bill" ; Baldwin, Thomas O. / Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the β subunit. In: Biochemistry. 2009 ; Vol. 48, No. 26. pp. 6085-6094.
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