Active site of Escherichia coli DNA photolyase: mutations at Trp277 alter the selectivity of the enzyme without affecting the quantum yield of photorepair.
Escherichia coli DNA photolyase repairs pyrimidine dimers by a photoinduced electron-transfer reaction. The enzyme binds to UV-damaged DNA independent of light (the dark reaction) and upon absorbing a 300-500-nm photon breaks the cyclobutane ring of the dimer (the light reaction) and thus restores the DNA. No structural information on the enzyme is available at present. However, comparison of the sequences of photolyases from five different organisms has identified highly conserved regions of homology. These regions are presumably involved in chromophore (flavin and folate) and substrate binding or catalysis. Trp277 (W277) in E. coli photolyase is conserved in all photolyases sequenced to date. We replaced this residue with Arg, Glu, Gln, His, and Phe by site-specific mutagenesis. Properties of the mutant proteins indicate that W277 is involved in binding to DNA but not in chromophore binding or catalysis. Of particular significance is the finding that compared to wild type W277R and W277E mutants have about 300- and 1000-fold lower affinity, respectively, for substrate but were indistinguishable from wild-type enzyme in their photochemical and photocatalytic properties.
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
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D000595:Amino Acid Sequence; D001665:Binding Sites; D004260:DNA Repair; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D008190:Lyases / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D010782:Photolysis; D011740:Pyrimidine Dimers / Q000096:biosynthesis; D011789:Quantum Theory; D013379:Substrate Specificity; D014364:Tryptophan / Q000235:genetics
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; D014364:Tryptophan; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase