title: Color-shifting mutations in the C-domain of L. mingrelica firefly luciferase provide new information about the domain alternation mechanism.
abstract: We identified three color-shifting mutations-Phe467Ser, Glu490Val, and Glu490Lys-in the C-domain of the wild-type recombinant L. mingrelica luciferase. These mutations had moderate effect on the specific activity and thermal stability of the enzyme but changed the pH-dependence of its bioluminescence spectra. We constructed the model structures of the enzyme in three known conformations (open, adenylation, and oxidation conformation). The structural analysis and experimental data provided no evidences that these residues participate in structure-forming interactions in the open or oxidation conformation or that their mutations alter the overall structure of the enzyme. Given that the bioluminescence spectra reflect the microenvironment of the emitter (oxyluciferin in an electronically excited state), we concluded that the mutated residues affect the active site during the emission of light via short-range interactions. We found that it is only in the adenylation conformation that the residues Phe467 and Glu490 approach the N-domain, whereas the domain rotation associated with the oxidation conformation completely removes them from the active site. Therefore, the emission most likely occurs from the adenylation conformation.
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, Moscow State University, Moscow 119991, Russia; Lumtek LLC, ul. Vorob'ievy Gory 1/77, Moscow 119992, Russia. Electronic address: jmodestova@yahoo.com.
authors.forename: Yulia
authors.initials: Y
authors.lastname: Modestova
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, Moscow State University, Moscow 119991, Russia; Lumtek LLC, ul. Vorob'ievy Gory 1/77, Moscow 119992, Russia.
authors.forename: Natalia N
authors.initials: NN
authors.lastname: Ugarova
mesh_terms: D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D003116:Color; D004795:Enzyme Stability; D048888:Fireflies / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008163:Luminescent Measurements; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D055668:Photochemical Processes; D011487:Protein Conformation; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D049409:Luciferases, Firefly
keywords: Bioluminescence; C-domain; Domain alternation mechanism; Firefly luciferase; pH-sensitivity