Identification and characterization of a second chromophore of DNA photolyase from Thermus thermophilus HB27.
Cyclobutane pyrimidine dimer (CPD) photolyases use light to repair CPDs. For efficient light absorption, CPD photolyases use a second chromophore. We purified Thermus thermophilus CPD photolyase with its second chromophore. UV-visible absorption spectra, reverse-phase HPLC, and NMR analyses of the chromophores revealed that the second chromophore of the enzyme is flavin mononucleotide (FMN). To clarify the role of FMN in the CPD repair reaction, the enzyme without FMN (Enz-FMN(-) and that with a stoichiometric amount of FMN (Enz-FMN(+)) were both successfully obtained. The CPD repair activity of Enz-FMN(+) was higher than that of Enz-FMN(-), and the CPD repair activity ratio of Enz-FMN(+) and Enz-FMN(-) was dependent on the wavelength of light. These results suggest that FMN increases the light absorption efficiency of the enzyme. NMR analyses of Enz-FMN(+) and Enz-FMN(-) revealed that the binding mode of FMN is similar to that of 7,8-didemethyl-8-hydroxy-5-deazariboflavin in Anacystis nidulans CPD photolyase, and thus a direct electron transfer between FMN and CPD is not likely to occur. Based on these results, we concluded that FMN acts as a highly efficient light harvester that gathers light and transfers the energy to FAD.
Ueda
Takumi
T
Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Kato
Akira
A
Kuramitsu
Seiki
S
Terasawa
Hiroaki
H
Shimada
Ichio
I
D002482:Cellulose / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000502:physiology*; D019281:Dimerization; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D005486:Flavin Mononucleotide / Q000737:chemistry; D008027:Light; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010100:Oxygen / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D016963:Thermus thermophilus / Q000378:metabolism*; D014466:Ultraviolet Rays
D005486:Flavin Mononucleotide; D002482:Cellulose; D004247:DNA; D004255:Deoxyribodipyrimidine Photo-Lyase; D010100:Oxygen