IMPROVED THERMOSTABILITY OF THE NORTH-AMERICAN FIREFLY LUCIFERASE - SATURATION MUTAGENESIS AT POSITION-354

We have used random chemical mutagenesis and a simple genetic screen t o generate and isolate a thermostable mutant of luciferase from the No rth American firefly (Photinus pyralis). A single G-to-A transition mu tation, resulting in the substitution of a glutamate for a lysine resi due at position 354 in the protein sequence, was shown to be responsib le for this enhanced thermostability. Replacement of Glu-354 with all possible amino acid residues was achieved using directed mutagenesis, and produced mutant enzymes with a range of thermostabilities. The mut ations E354K and E354R conferred the largest increases in thermostabil ity, suggesting that side-chain size and hydrophobicity, as well as ch arge, may also be important contributors to the overall thermostabilit y of the polypeptide chain at this position, Unusually for such mutati ons, biochemical studies suggest that this position is on the surface of the protein and exposed to solvent.