We have used random chemical mutagenesis and a simple genetic screen to generate and isolate a thermostable mutant of luciferase from the North American firefly (Photinus pyralis). A single G-to-A transition mutation, resulting in the substitution of a glutamate for a lysine residue at position 354 in the protein sequence, was shown to be responsible 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 mutations E354K and E354R conferred the largest increases in thermostability, suggesting that side-chain size and hydrophobicity, as well as charge, may also be important contributors to the overall thermostability of the polypeptide chain at this position. Unusually for such mutations, biochemical studies suggest that this position is on the surface of the protein and exposed to solvent.

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