Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro.
DNA photolyases repair cyclobutadipyrimidines (Pyr()Pyr) in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains methenyltetrahydrofolate (MTHF), which functions as photoantenna, and FADH2, which is the redox-active cofactor. During purification, FADH2 is oxidized to the blue neutral radical form, FADH., which has greatly diminished activity. Previous nanosecond flash photolysis studies [Heelis, P.F., Okamura, T., & Sancar, A. (1990) Biochemistry 29, 5694-5698] indicated that excitation of FADH. either directly by absorbing a photon or indirectly by electronic energy transfer from MTHF excited singlet state yielded an FADH. quartet which abstracted a hydrogen atom from a nearby tryptophan to generate the catalytically competent FADH2 from of the enzyme. Using site-directed mutagenesis, we replaced all 15 photolyase tryptophan residues by phenylalanine, individually, in order to identify the internal hydrogen atom donor responsible for photoreduction. We found that W306F mutation abolished photoreduction of FADH. without affecting the excited-state properties of FADH. or the substrate binding (KA approximately 10(9) M-1) of the enzyme. The specificity constant (kcat/km) was approximately 0 for the mutant enzyme in the absence of reducing agents in the reaction mixture, indicating that photoreduction of FADH. is an essential step for photorepair by photolyase in vitro. Chemical reduction of FADH. of the mutant enzyme restored the specificity constant to the wild-type level.
Li
Y F
YF
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Heelis
P F
PF
Sancar
A
A
D001665:Binding Sites; D002384:Catalysis; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004735:Energy Transfer; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005609:Free Radicals; D006859:Hydrogen / Q000378:metabolism; D007834:Lasers; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010782:Photolysis; D012689:Sequence Homology, Nucleic Acid; D014364:Tryptophan / Q000378:metabolism*
D005609:Free Radicals; D005182:Flavin-Adenine Dinucleotide; D006859:Hydrogen; D014364:Tryptophan; D004255:Deoxyribodipyrimidine Photo-Lyase