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Luciferase knockout mutants by site-directed mutagenesis of ATP binding site Gabriela Ispas*2; Ivan Famelaer1; Fatima1; Michael Jacobs1 (1) Free University Brussels, Institute of Molecular B, Laboratory of Plant Genetics, B 1640 Sint Genesius Rode, Belgium; (2) Cluj-Napoca University, Faculty of Biology, Laboratory of Plant Genetic Engineering, 3400 Cluj-Napoca, Romania      *(gsgispas@vub.ac.be) The firefly luciferase has been intensively studied in the past and the ATP binding domain, peroxisome tagging sequence, as well as some of the amino acids involved in the mechanism of light emission, have been identified. In our research, two amino acids from luciferase ATP binding domain have been mutated: S199 to L and G200 to E. Consequently a knocked-out luciferase has been obtained. In an attempt to asses which of the two mutations are essential for the switching of luciferase activity, the S199 has been restored and a second luciferase mutant conserving the mutation G200 to E has been produced. In both mutants the luciferase is inactive. These experiments confirm that glycine presents in the ATP binding domain has an essential role for luciferase activity either via a direct mechanism conditioning the ATP binding, or indirectly by determining the corresponding protein structure required for light emission. [Poster: famelaer.ivan.26632]

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