title: Active site of Escherichia coli DNA photolyase: Asn378 is crucial both for stabilizing the neutral flavin radical cofactor and for DNA repair.
abstract: Escherichia coli DNA photolyase repairs cyclobutane pyrimidine dimer (CPD) in UV-damaged DNA through a photoinduced electron transfer mechanism. The catalytic activity of the enzyme requires fully reduced FAD (FADH (-)). After purification in vitro, the cofactor FADH (-) in photolyase is oxidized into the neutral radical form FADH (*) under aerobic conditions and the enzyme loses its repair function. We have constructed a mutant photolyase in which asparagine 378 (N378) is replaced with serine (S). In comparison with wild-type photolyase, we found N378S mutant photolyase containing oxidized FAD (FAD ox) but not FADH (*) after routine purification procedures, but evidence shows that the mutant protein contains FADH (-) in vivo as the wild type. Although N378S mutant photolyase is photoreducable and capable of binding CPD in DNA, the activity assays indicate the mutant protein is catalytically inert. We conclude that the Asn378 residue of E. coli photolyase is crucial both for stabilizing the neutral flavin radical cofactor and for catalysis.
authors.affiliation: School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230027, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Xu
authors.forename: Wanmeng
authors.initials: W
authors.lastname: Mu
authors.forename: Yanwei
authors.initials: Y
authors.lastname: Ding
authors.forename: Zhaofeng
authors.initials: Z
authors.lastname: Luo
authors.forename: Qingkai
authors.initials: Q
authors.lastname: Han
authors.forename: Fuyong
authors.initials: F
authors.lastname: Bi
authors.forename: Yuzhen
authors.initials: Y
authors.lastname: Wang
authors.forename: Qinhua
authors.initials: Q
authors.lastname: Song
mesh_terms: D001216:Asparagine / Q000737:chemistry*; D001665:Binding Sites; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology*; D004269:DNA, Bacterial; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005415:Flavins / Q000737:chemistry*; D009154:Mutation; D011487:Protein Conformation
chemical_list: D004269:DNA, Bacterial; D005415:Flavins; D001216:Asparagine; D004255:Deoxyribodipyrimidine Photo-Lyase