title: Proposed ionic bond between Arg300 and Glu270 and Glu271 are not involved in inactivation of a mutant firefly luciferase (LRR).
abstract: The weakness of firefly luciferase is its rapid inactivation. Many studies have been done to develop thermostable luciferases. One of these modifications was LRR mutant in which the Leu300 was substituted with Arg in the E(354)RR(356)Lampyris turkestanicus luciferase as template. LRR was more thermostable than the wild type but with only 0.02% activity. In this study, site-directed mutagenesis was used to change the proposed ionic bond between the Arg and two neighboring residues (Glu270 and Glu271), to understand if the induced interactions were responsible for inactivation in LRR. Our results showed that substitution of Glu270 and 271 with Ala removed the interactions but the activity of enzyme did not return. The E270A mutant was more active than LRR but the E271A and E270A/E271A mutants were inactive. Fluorescence and CD measurements showed that these mutations were accompanied by conformational changes. Extrinsic fluorescence measurement and obtained quenching data by KI and acrylamide also confirmed that the mutants were less compact than the LRR enzyme. In conclusion, in LRR, the interactions between Arg300 and Glu270 and Glu271 were not responsible for the enzyme inactivation and it is proposed that the enzyme inactivation is due to conformational changes of LRR mutant of firefly luciferase.
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: Sobhanizahra10@gmail.com.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Sobhani-Damavandifar
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: saman_h@modares.ac.ir.
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran. Electronic address: sajedi_r@modares.ac.ir.
authors.forename: Reza H
authors.initials: RH
authors.lastname: Sajedi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D004795:Enzyme Stability; D048888:Fireflies / Q000201:enzymology / Q000235:genetics; D057927:Hydrophobic and Hydrophilic Interactions; D019476:Insect Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D007700:Kinetics; D049409:Luciferases, Firefly / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D019476:Insect Proteins; D011994:Recombinant Proteins; D049409:Luciferases, Firefly
keywords: Firefly luciferase; LRR mutant; Mutagenesis; Reactivation; Thermostable