title: Implication of an unfavorable residue (Thr346) in intrinsic flexibility of firefly luciferase.
abstract: In order to better understand the functional role of an unusual residue (Thr346) of firefly luciferase mutagenesis at this residue was performed. Firefly luciferase, catalyzes the bioluminescence reaction and is an excellent tool as a reporter in nano-system biology studies. Nonetheless, the enzyme rapidly loses its activity at temperatures above 30 °C and this leads to reduced sensitivity and precision in analytical applications. Residue Thr346 in a connecting loop (341-348) of firefly luciferase is located in a disallowed region of Ramachandran plot. In this study, we have substituted this residue (T346) with anomalous dihedral angles with Val, Gly and Pro to clarify the role of this residue in structure and function of the enzyme using site-directed mutagenesis. Substitution of this unfavorable residue (T346) with atypical dihedral angles (ψ, φ) with other residues brought about an increase of thermostability and decrease of specific activity. Structural and functional properties of the mutants were analyzed using different spectroscopic methods. It seems that this residue is a critically conserved residue to support the functional flexibility for a fast kinetic bioluminescence reaction at the expense of lower stability.
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Maryam
authors.initials: M
authors.lastname: Moradi
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
authors.forename: Rahman
authors.initials: R
authors.lastname: Emamzadeh
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002942:Circular Dichroism; D017124:Conserved Sequence; D004795:Enzyme Stability; D048888:Fireflies / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D049409:Luciferases, Firefly / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship; D013696:Temperature; D013912:Threonine / Q000737:chemistry*
chemical_list: D013912:Threonine; D049409:Luciferases, Firefly