The Role of Active Site Residue Arginine 218 in Firefly Luciferase Bioluminescence

Abstract

Firefly luciferase catalyzes the highly efficient emission of yellow-green light from substrate firefly luciferin by a sequence of reactions that require Mg-ATP and molecular oxygen. We had previously developed a working model of the luciferase active site based on the X-ray structure of the enzyme without bound substrates. In our model, the side chain guanidinium group of Arg218 appears to be located in close proximity to the substrate's hydroxyl group at the bottom of the luciferin binding pocket. A similar role for Arg337 also has been proposed. We report here the construction, purification, and characterization of mutant luciferases R218A, R218Q, R218K, R337Q, and R337K. Alteration of the Arg218 side chain produced enzymes with 15-20-fold increases in the Km values for luciferin. The contrasting near-normal Km values for luciferin determined with the Arg337 enzymes support our proposal that Arg218 (and not Arg337) is an essential luciferin binding site residue. Bioluminescence emission studies indicated that in the absence of a positively charged group at position 218, red bioluminescence was produced. Based on this result and those of additional fluorescence experiments, we speculate that Arg218 maintains the polarity and rigidity of the emitter binding site necessary for the normal yellow-green emission of P. pyralis luciferase. The findings reported here are interpreted in the context of the firefly luciferase X-ray structures and computational-based models of the active site.

The Ribosome-Bound Chaperones RAC and Ssb1/2p Are Required for Accurate Translation in Saccharomyces cerevisiae

“…In order to test whether the mutant strains were specifically affected in translation termination or displayed a more general translational defect, we devised an in vivo misincorporation assay. Replacing arginine in position 218 of luciferase with serine inactivates the enzyme (6). To test whether luciferase-R218S was suited as a misincorporation reporter, two different versions were generated.…”

Active-Site Properties of Phrixotrix Railroad Worm Green and Red Bioluminescence-Eliciting Luciferases

“…Click beetle luciferases, which produce different colours, have also been cloned (17,18). Mutagenesis studies identified the residues important for the bioluminescence activity and colours of these luciferases (19)(20)(21)(22)(23)(24). Three mechanisms have been proposed to explain bioluminescence colour modulation by beetle luciferases: (I) non-specific solvent and orientation polarizability effects (15,16); (II) specific active-site interactions with the emitter (25); and (III) the effect of the conformation of the active site on the rotational properties of the oxyluciferin thiazine rings (26).…”

Thermostabilization of Firefly Luciferases Using Genetic Engineering

“…These models were further confirmed by the independent studies: the residual activity of the luciferase mutant, in which Lys529 was changed to Ala, was less than 0.1% (Branchini et al, 2000). The role of others residues of the luciferase active site was supported by mutagenesis methods (Branchini et al, 1999(Branchini et al, , 2000(Branchini et al, , 2001(Branchini et al, , 2003. Fig.…”

“…The residue R220 (the residue R218 in P.pyralis luciferase) is highly conservative and necessary for the green emission of firefly luciferases. Its substitutions led to the red bioluminescence, 3-15-fold decrease in activity, extended luminescence decay times and dramatic increase in K m values (Branchini et al, 2001). The G216N/A217L double substitution in L. mingrelica luciferase caused the similar type of effects but of less extent.…”