Isolation and characterization of the transient,luciferase-bound flavin-4a-hydroxide in the bacterial luciferase reaction |
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| Institution: | 1. Department of Cellular and Developmental Biology, Harvard University, Cambridge, MA U.S.A.;2. Department of Biology, University of Konstanz, Konstanz F.R.G.;1. Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia;2. Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia;3. BioChemMack JSC, Lomonosovsky Pr., 29/1, Moscow 119192, Russia;1. Bio-energy Corporation, Research & Development Laboratory, 2-9-7 Minaminanamatsu, Amagasaki 660-0053, Japan;2. Kansai Chemical Engineering Co. Ltd., 2-9-7 Minaminanamatsu, Amagasaki 660-0053, Japan;3. Graduate School of Science, Technology and Innovation, Kobe University, 1-1 Rokkodai, Nada, Kobe 657-8501, Japan;1. Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Stockholm, 14186, Sweden;2. Division of Gastroenterology and Hepatology, Joan & Sanford I. Weill Department of Medicine, Weill Cornell Medical College, New York, NY 10021, USA;1. Institute of Applied Chemistry, Department of Chemical Engineering, Tsinghua University, Beijing 100084, China;2. College of Life Sciences/Engineering Research Center of Industrial Microbiology, Fujian Normal University, Fuzhou 350108, China;3. School of Chemical Engineering, Purdue University, 480 Stadium Mall Drive, West Lafayette, IN 47907, USA |
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| Abstract: | Procedures and conditions have been established such that the unstable enzyme-bound flavin intermediate produced in the bacterial luciferase reaction can be isolated as approximately 70% of the flavin product, the remaining being the final product, FMN. The structure of the intermediate is proposed to be that of a luciferase-bound 4a,5-dihydroflavin-4a-hydroxide. The intermediate has a half-life of 33 min at 2°C and decays spontaneously to give H2O and luciferase-bound FMN with an activation enthalpy of about 120 kJ/mol. It has an absorption spectrum (λmax = 360 nm) that is consistent with the proposed structure, and a fluorescence emission (λmax = 485 nm) that matches the bioluminescence emission closely. |
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