title: Point mutations in firefly luciferase C-domain demonstrate its significance in green color of bioluminescence.
abstract: Firefly luciferase is a two-domain enzyme that catalyzes the bioluminescent reaction of firefly luciferin oxidation. Color of the emitted light depends on the structure of the enzyme, yet the exact color-tuning mechanism remains unknown by now, and the role of the C-domain in it is rarely discussed, because a very few color-shifting mutations in the C-domain were described. Recently we reported a strong red-shifting mutation E457K in the C-domain; the bioluminescence spectra of this enzyme were independent of temperature or pH. In the present study we investigated the role of the residue E457 in the enzyme using the Luciola mingrelica luciferase with a thermostabilized N-domain as a parent enzyme for site-directed mutagenesis. We obtained a set of mutants and studied their catalytic properties, thermal stability and bioluminescence spectra. Experimental spectra were represented as a sum of two components (bioluminescence spectra of putative "red" and "green" emitters); λmax of these components were constant for all the mutants, but the ratio of these emitters was defined by temperature and mutations in the C-domain. We suggest that each emitter is stabilized by a specific conformation of the active site; thus, enzymes with two forms of the active site coexist in the reactive media. The rigid structure of the C-domain is crucial for maintaining the conformation corresponding to the "green" emitter. We presume that the emitters are the keto- and enol forms of oxyluciferin.
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, Moscow State University, Moscow, 119991 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.
authors.forename: Mikhail I
authors.initials: MI
authors.lastname: Koksharov
authors.affiliation: Department of Chemical Enzymology, Faculty of Chemistry, Moscow State University, Moscow, 119991 Russia.
authors.forename: Natalia N
authors.initials: NN
authors.lastname: Ugarova
mesh_terms: D000818:Animals; D020134:Catalytic Domain; D003116:Color; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D048888:Fireflies / Q000737:chemistry* / Q000201:enzymology; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007211:Indoles / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011719:Pyrazines / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013696:Temperature; D013816:Thermodynamics
chemical_list: D007211:Indoles; D011719:Pyrazines; D011994:Recombinant Proteins; C014730:oxyluciferin; D049409:Luciferases, Firefly
keywords: Bioluminescence; Color-tuning mechanism; Firefly luciferase; Oxyluciferin; Site-directed mutagenesis