Reconstitution of Escherichia coli photolyase with flavins and flavin analogues.
Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). A procedure was developed for reversible resolution of apophotolyase and its chromophores. To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted. Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase. The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD. Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.
Payne
G
G
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599.
Wills
M
M
Walsh
C
C
Sancar
A
A
D001051:Apoenzymes / Q000378:metabolism*; D001059:Apoproteins / Q000378:metabolism*; D001665:Binding Sites; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology*; D005486:Flavin Mononucleotide / Q000493:pharmacokinetics; D005182:Flavin-Adenine Dinucleotide / Q000493:pharmacokinetics; D005415:Flavins / Q000493:pharmacokinetics*; D008190:Lyases / Q000378:metabolism*; D010782:Photolysis; D011740:Pyrimidine Dimers / Q000378:metabolism; D013379:Substrate Specificity
D001051:Apoenzymes; D001059:Apoproteins; D005415:Flavins; D011740:Pyrimidine Dimers; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase