title: Luciferin-Regenerating Enzyme Mediates Firefly Luciferase Activation Through Direct Effects of D-Cysteine on Luciferase Structure and Activity.
abstract: Luciferin-regenerating enzyme (LRE) contributes to in vitro recycling of D-luciferin. In this study, reinvestigation of the luciferase-based LRE assay is reported. Here, using quick change site-directed mutagenesis seven T-LRE (Lampyris turkestanicusLRE) mutants were constructed and the most functional mutant of T-LRE (T(69)R) was selected for this research and the effects of D- and L-cysteine on T(69)R T-LRE-luciferase-coupled assay are examined. Our results demonstrate that bioluminescent signal of T(69)R T-LRE-luciferase-coupled assay increases and then reach equilibrium state in the presence of 5 mm D-cysteine. In addition, results reveal that 5 mm D- and L-cysteine in the absence of T(69)R T-LRE cause a significant increase in bioluminescence intensity of luciferase over a long time as well as decrease in decay rate. Based on activity measurements, far-UV CD analysis, ANS fluorescence and DLS (Dynamic light scattering) results, D-cysteine increases the activity of luciferase due to weak redox potential, antiaggregatory effects, induction of changes in conformational structure and kinetics properties. In conclusion, in spite of previous reports on the effect of LRE on luciferase bioluminescent intensity, the majority of increase in luciferase light output and time-course originate from the direct effects of D-cysteine on structure and activity of firefly luciferase.
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Roohullah
authors: R
authors: Hemmati
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Saman
authors: S
authors: Hosseinkhani
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Reza H
authors: RH
authors: Sajedi
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Taha
authors: T
authors: Azad
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Amin
authors: A
authors: Tashakor
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Nuredin
authors: N
authors: Bakhtiari
authors: Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
authors: Farangis
authors: F
authors: Ataei
mesh_terms: D003545:Cysteine / Q000378:metabolism*; D004789:Enzyme Activation; D019476:Insect Proteins / Q000378:metabolism*; D008025:Ligases / Q000378:metabolism*; D008156:Luciferases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D019476:Insect Proteins; D008156:Luciferases; D008025:Ligases; C471161:luciferin-regenerating enzyme, insect; D003545:Cysteine