Mutagenesis of Solvent-exposed Amino Acids in Photinus Pyralis Luciferase Improves Thermostability and PH-tolerance

Overview

Journal Biochem J

Specialty Biochemistry

Date 2006 Mar 23

PMID 16551268

Citations 26

Authors

G H Erica Law

Olga A Gandelman

Laurence C Tisi

Christopher R Lowe

James A H Murray

Affiliations

Soon will be listed here.

Abstract

Firefly luciferase catalyses a two-step reaction, using ATP-Mg2+, firefly luciferin and molecular oxygen as substrates, leading to the efficient emission of yellow-green light. We report the identification of novel luciferase mutants which combine improved pH-tolerance and thermostability and that retain the specific activity of the wild-type enzyme. These were identified by the mutagenesis of solvent-exposed non-conserved hydrophobic amino acids to hydrophilic residues in Photinus pyralis firefly luciferase followed by in vivo activity screening. Mutants F14R, L35Q, V182K, I232K and F465R were found to be the preferred substitutions at the respective positions. The effects of these amino acid replacements are additive, since combination of the five substitutions produced an enzyme with greatly improved pH-tolerance and stability up to 45 degrees C. All mutants, including the mutant with all five substitutions, showed neither a decrease in specific activity relative to the recombinant wild-type enzyme, nor any substantial differences in kinetic constants. It is envisaged that the combined mutant will be superior to wild-type luciferase for many in vitro and in vivo applications.

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