STRUCTURALLY MODIFIED FIREFLY LUCIFERASE. EFFECTS OF AMINO ACID SUBSTITUTION AT POSITION 286

Ser was replaced at position 286 of firefly luciferase (Luciola mingrelica) by a series of naturally occurring and unnatural amino acids. The effect of these substitutions on the properties of luciferase, such as thermostability, pH dependence, and color of light emitted, was investigated. For these purposes, the Ser286 codon (AGT) was replaced by an amber stop codon (TAG) within the luciferase gene and transformed into Escherichia coli strains producing specific amber suppressor tRNA's to express luciferase with different substitutions at this position. The incorporation of Leu, Lys, Tyr, or Gln at this position reduced the thermostability of mutated luciferases. The color of emitted light changed upon substitution from yellow-green (λmax 582 nm) for the wild-type enzyme having Ser286 to, for example, red (λmax 622 nm) for luciferase having Leu286. For further evaluation of the structural relationship between the amino acid position at 286 and the wavelength of emitted light, we used the method of in vit...