title: Bioluminescence is produced from a trapped firefly luciferase conformation predicted by the domain alternation mechanism.
abstract: According to the domain alternation mechanism and crystal structure evidence, the acyl-CoA synthetases, one of three subgroups of a superfamily of adenylating enzymes, catalyze adenylate- and thioester-forming half-reactions in two different conformations. The enzymes accomplish this by presenting two active sites through an ~140° rotation of the C-domain. The second half-reaction catalyzed by another subgroup, the beetle luciferases, is a mechanistically dissimilar oxidative process that produces bioluminescence. We have demonstrated that a firefly luciferase variant containing cysteine residues at positions 108 and 447 can be intramolecularly cross-linked by 1,2-bis(maleimido)ethane, trapping the enzyme in a C-domain-rotated conformation previously undocumented in the available luciferase crystal structures. The cross-linked luciferase cannot adenylate luciferin but is nearly fully capable of bioluminescence with synthetic luciferyl adenylate because it retains the ability to carry out the oxidative half-reaction. The cross-linked luciferase is apparently trapped in a conformation similar to those adopted by acyl-CoA synthetases as they convert acyl adenylates into the corresponding CoA thioesters.
authors.affiliation: Department of Chemistry, Connecticut College, New London, Connecticut 06320, USA. brbra@conncoll.edu
authors.forename: Bruce R
authors.initials: BR
authors.lastname: Branchini
authors.forename: Justin C
authors.initials: JC
authors.lastname: Rosenberg
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Fontaine
authors.forename: Tara L
authors.initials: TL
authors.lastname: Southworth
authors.forename: Curran E
authors.initials: CE
authors.lastname: Behney
authors.forename: Lerna
authors.initials: L
authors.lastname: Uzasci
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D048888:Fireflies / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D049454:Firefly Luciferin / Q000378:metabolism; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049449:Luminescence; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D049454:Firefly Luciferin; D049409:Luciferases, Firefly