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Proposed ionic bond between Arg300 and Glu270 and Glu271 are not involved in inactivation of a mutant firefly luciferase (LRR)

Paper ID Volume ID Publish Year Pages File Format Full-Text
16793 42613 2016 8 PDF Available
Title
Proposed ionic bond between Arg300 and Glu270 and Glu271 are not involved in inactivation of a mutant firefly luciferase (LRR)
Abstract

•LRR is more thermostable than the wild-type luciferase.•LRR has only 0.02% of native activity.•Disruption of LRR ionic bond did not return activity.•Disruption of LRR ionic bond reduced rigidity of luciferase.

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 E354RR356Lampyris 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.

Keywords
Firefly luciferase; LRR mutant; Reactivation; Mutagenesis; Thermostable
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Proposed ionic bond between Arg300 and Glu270 and Glu271 are not involved in inactivation of a mutant firefly luciferase (LRR)
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Publisher
Database: Elsevier - ScienceDirect
Journal: Enzyme and Microbial Technology - Volume 86, May 2016, Pages 17–24
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us