Bioluminescence Is Produced from a Trapped Firefly Luciferase Conformation Predicted by the Domain Alternation Mechanism

摘要

According to the domain alternation mechanism and crystal structure evidence, the acyl-CoA synthetases, one of three subgroups of a superfamiry of adenylating enzymes, catalyze adenykte- 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 mat a firefly luciferase variant containing cysteine residues at positions 108 and 447 can be intramolecularly crosslinked by l,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 adenykte luciferin but is nearly fully capable of bioluminescence with synthetic ludferyi adenylate because it retains die 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.