DNA photoreactivating enzyme from the cyanobacterium Anacystis nidulans.
Photoreactivating enzyme, which specifically monomerizes pyrimidine dimers in UV-irradiated DNA, was purified 21,000-fold from the cyanobacterium Anacystis nidulans to apparent homogeneity with 41% overall yield. The enzyme consists of a single protein chain with 53,000 molecular weight. Maximal activity was found at pH 6.2 and 0.1 M NaCl. Purified photoreactivating enzyme exhibits a marked absorption spectrum with a main band in the blue region (maximum 437 nm), a protein band (maximum 266 nm), and a low intensity band above 500 nm. The molar extinction coefficient of native enzyme was estimated 53,000 at 437 nm. The action spectrum for photoreactivation shows maximal activity at 440 nm and correlates closely with the 437-nm absorption band. The enzyme contains two different intrinsic chromophores in equimolar amounts, which were identified as 7,8-didemethyl-8-hydroxy-5-deazariboflavin (FO) and (reduced) FAD. The low intensity absorption band of native photoreactivating enzyme exhibits a shoulder at 498 and maxima at 588 and 634 nm. This band is attributed to a neutral FAD semiquinone radical which accounts for the major part of the FAD present in dark equilibrated enzyme. Preillumination at 585 nm bleaches the semiquinone spectrum due to formation of fully reduced FAD, but exposure to air in the dark restores the spectrum completely. On preillumination at 437 nm the disappearance of FAD semiquinone is more rapid, indicating that the photoreduction is sensitized by the 8-hydroxy-5-deazaflavin chromophore. The 8-hydroxy-5-deazaflavin and possibly also the reduced FAD chromophore appear to act as a primary photon acceptor in the photoreactivation process.
Eker
A P
AP
Biochemical and Biophysical Laboratory, Delft University of Technology, The Netherlands.
Kooiman
P
P
Hessels
J K
JK
Yasui
A
A
D000595:Amino Acid Sequence; D002845:Chromatography; D000458:Cyanobacteria / Q000201:enzymology*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000302:isolation & purification*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D008027:Light; D008190:Lyases / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010777:Photochemistry; D011489:Protein Denaturation; D012256:Riboflavin / Q000031:analogs & derivatives / Q000032:analysis; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D005182:Flavin-Adenine Dinucleotide; C007701:factor 420; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase; D012256:Riboflavin