Affinity probing of flavin binding sites. 2. Identification of a reactive cysteine in the flavin domain of Escherichia coli DNA photolyase.
8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys293) in the flavin domain of Escherichia coli DNA photolyase to form an 8-(cysteinyl)FAD derivative covalently bound to the protein. About 80% protection against covalent attachment with 8-(methylsulfonyl)FAD was observed in the presence of an equimolar amount of FAD. Flavinylated photolyase retains the ability to repair pyrimidine dimers (15% of native activity) and to bind its antenna chromophore, 5,10-methenyltetrahydrofolate. Comparison of the properties of flavinylated enzyme with photolyase containing noncovalently bound 8-(methylthio)-FAD indicate that a perturbation is necessary to accommodate covalent bond formation. 8-(Methylthio)-FAD-reconstituted enzyme exhibits 95% of native activity. The aerobic stability of fully reduced and radical forms of 8-(methylthio)FAD enzyme is similar to that of native enzyme, whereas a radical form is not detected with flavinylated enzyme and the fully reduced enzyme is more easily oxidized by oxygen. The flavin in 8-(methylthio)FAD enzyme or flavinylated photolyase is shielded from solvent. However, the flavin environment in flavinylated enzyme is less hydrophobic as judged by spectral comparison with model 8-(alkylthio)flavins in various solvents. Enzyme containing noncovalently bound 8-(methylsulfonyl)-FAD was prepared by reconstitution with the fully reduced flavin which does not undergo covalent attachment. Covalent attachment was observed after reoxidation but probably involved dissociation and rebinding of oxidized 8-(methylsulfonyl)FAD. The results show that 8-(cysteinyl)FAD in flavinylated photolyase is at or near the normal flavin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
Raibekas
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Department of Biological Chemistry, Hahnemann University School of Medicine, Philadelphia, Pennsylvania 19102.
Jorns
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000378:metabolism; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011740:Pyrimidine Dimers / Q000378:metabolism; D013763:Tetrahydrofolates / Q000378:metabolism
C090004:8-(cysteinyl)FAD; C090005:8-(methylthio)FAD; D011740:Pyrimidine Dimers; D013763:Tetrahydrofolates; D005182:Flavin-Adenine Dinucleotide; C090003:8-(methylsulfonyl)FAD; C018092:5,10-methenyltetrahydrofolate; D004255:Deoxyribodipyrimidine Photo-Lyase; D003545:Cysteine