title: Directed improvement of luciferin regenerating enzyme binding properties: implication of some conserved residues in luciferin-binding domain.
abstract: Luciferin regenerating enzyme (LRE) contributes to in vitro recycling of d-luciferin to produce persistent and longer light emission by luciferase. Luciferin binding domains I and II among LREs regarded as potential candidates for luciferin-binding sites. In this study, for the first time, amino acids T69, G75 and K77 located at luciferin binding domain I of LRE from L. turkestanicus (T-LRE) substituted by using site-directed mutagenesis. Single mutant T(69)R increased luciferase light output more than two-fold over a longer time in comparison with a wild-type and other mutants of T-LRE. Nevertheless, double mutant (K(77)E/T(69)R) increased the amount of bioluminescent signal more than two-fold over a short time. In addition, G(75)E, K(77)E and G(75)E/T(69) R mutants did not improve luciferin-luciferase in vitro bioluminescence. Based on our results, addition of K(77)E/G(75)E and K(77)E/G(75)E/T(69)R mutants caused intermediate changes in bioluminescence from in vitro luciferin-luciferase reaction. These findings indicated that the amino acids in question are possible to be located within T-LRE active site. It may also be suggested that substituted Arg69 (Arg218) plays an important role in luciferin binding and the existence of Gly75 as well as Lys77 is essential for T-LRE which has already evolved to have different functions in nature.
authors.affiliation: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors.forename: Roohullah
authors.initials: R
authors.lastname: Hemmati
authors.forename: Reza H
authors.initials: RH
authors.lastname: Sajedi
authors.forename: Nuredin
authors.initials: N
authors.lastname: Bakhtiari
authors.forename: Saman
authors.initials: S
authors.lastname: Hosseinkhani
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D049454:Firefly Luciferin / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D008025:Ligases / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D019476:Insect Proteins; D049454:Firefly Luciferin; D008025:Ligases; C471161:luciferin-regenerating enzyme, insect