The mutation of Leu300 to Arg in Lampyris turkestanicus leads to inactivation of the enzyme. Modeling studies had shown that Arg300 side chain have ion interactions with side chains of glutamate 270 and 271 that lead to very low activity of the enzyme. It seems that low activity is a result of high rigidity of the enzyme structure. According to these reasons, the roles of these residues are investigated on function of luciferase. Mutations were created using Quick Change Site-Directed Mutagenesis on pET28a plasmid containing cloned luciferase. Mutations were confirmed by standard sequencing. Mutant plasmids were transformed to competent E.coli BL21 cells for protein expression and purification using affinity chromatography. Luciferase activity was measured by adding enzyme solution to a complex containing 50 mM Tris, 5.0 mM luciferin, 40 mM ATP and 50 mM MgSO4. The mutant forms of luciferase, surprisingly, were almost fully inactivated before purification. Low activity was observed after purification of mutants. SDS-PAGE analysis of purified mutant proteins showed decreased expression of mutant luciferases relative to native luciferase. The Km values of mutants for luciferin was almost the same as that of wild-type luciferase. It may be suggested part of low activity is due to decreased expression. It seems, these mutations have decreased mRNA stability or translation of the luciferase. Further experiments are underway to identify the effects of these mutations on level of expression, structure and function of the enzyme.
