Mutagenesis of solvent-exposed amino acids in Photinus pyralis luciferase improves thermostability and pH-tolerance

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.

Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

“…40 In addition, I212L/S463R was much more stable than I212L, possibly because of an additive effect of S463R because the stability of S463R itself is slightly higher than that of WT. Finally, we noted that N351K, unlike its counterparts in Ppy (E354K) 32 and in Hpa (E356R), 35 had lower stability than WT.…”

“…51 Unlike N351K in this study, mutations of the corresponding positions in other green-yellow-emitting firefly luciferases caused a small red shift. 32,38 Materials and Methods…”

“…10,11 Nevertheless, the poor thermostability of wild-type (WT) PhRED, which is also a common problem for all other beetle luciferases 23 and can affect the detection efficiency of in vivo BLI, 29 limits its application in demanding circumstances. Some attempts have been made to improve firefly luciferase thermostability by mutagenesis (e.g., Ppy, [30][31][32] Lcr, 33 Luciola lateralis (Lla), 34 Hotaria parvula (Hpa) 35 ). Among these, mutations of T217 (Lcr) and A217 (Lla) (i.e., I212 of PhRED) to the three most hydrophobic residues (I, V, and L) produced mutants with greater thermostability.…”

“…35 In addition, mutations of solvent-exposed nonconserved hydrophobic residues to hydrophilic residues in Ppy (e.g., F465R: i.e., S463 of PhRED) improved the thermostability. 32 In this study, we aimed at optimizing PhRED mutants for applications. We constructed five mutants (I212L, N351K, S463R, I212L/N351K, and I212L/S463R) and investigated their spectra, extract-based activity, thermostability at 37 C, and their practicability in cell-based real-time monitoring.…”

A Dual‐Color Far‐Red to Near‐Infrared Firefly Luciferin Analogue Designed for Multiparametric Bioluminescence Imaging

“…This highlights the importance of retaining the 6'-hydroxy group for color modulation. [22][23][24][25][26][27][28] In vitro bioluminescence spectra of iLH 2 6 ethyl ester (saponified with PLE (pig liver esterase) in situ immediately prior to use) with purified wild-type (WT) Fluc, the x5 Fluc mutant (a thermostable Fluc with similar properties to WT but with higher quantum yields), [35,36] and the x5 S284T Fluc mutant (a bright red-shifted point mutant of x5) [37,38] showed marked red-shifted peak maxima of 100 nm magnitude compared to the l max of each enzyme with 1 ( Figure 2, Table 1). This effect is remarkable considering that these mutants were originally engineered for different emission …”