Identification of the second chromophore of Escherichia coli and yeast DNA photolyases as 5,10-methenyltetrahydrofolate.
Denaturation of DNA photolyase (deoxyribodipyrimidine photolyase, EC 4.1.99.3) from Escherichia coli with guanidine hydrochloride or acidification to pH 2 released, in addition to FAD, a chromophore with the spectral and chromatographic properties of a reduced pterin. Treatment of the enzyme with iodine prior to acidification converted the chromophore to a stable, oxidized derivative, which was resolved by HPLC into four species with identical spectral properties. The same species, in the same distribution, were obtained from the yeast enzyme. The material isolated from the iodine-oxidized enzyme was shown to be a pterin by conversion to pterin-6-carboxylic acid with alkaline permanganate and was found to release glutamate upon acid hydrolysis. The presence of 10-formylfolate in the isolated, oxidized chromophore was demonstrated by absorption and fluorescence spectroscopy and by deformylation and conversion to folic acid. Analysis of the distribution of polyglutamates revealed that the four species identified by HPLC corresponded to the tri-, tetra-, penta-, and hexaglutamate derivatives of 10-formylfolate. The results were consistent with gamma linkages in the triglutamate derivative with additional glutamates linked via the alpha-carboxyl group of the preceding residue. Treatment with rat plasma hydrolase produced the monoglutamate derivative of 10-formylfolate. The native, enzyme-bound form of the folate cofactor was identified as 5,10-methenyltetrahydrofolylpolyglutamate by effecting release and isolation at low pH to protect the 5,10-methenyl bridge and preserve the reduced pyrazine ring structure.
D001426:Bacterial Proteins / Q000032:analysis*; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology*; D005656:Fungal Proteins / Q000032:analysis*; D005971:Glutamates / Q000032:analysis; D018698:Glutamic Acid; D006868:Hydrolysis; D008190:Lyases / Q000032:analysis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013763:Tetrahydrofolates / Q000302:isolation & purification*
D001426:Bacterial Proteins; D005656:Fungal Proteins; D005971:Glutamates; D013763:Tetrahydrofolates; D018698:Glutamic Acid; C018092:5,10-methenyltetrahydrofolate; D008190:Lyases; D004255:Deoxyribodipyrimidine Photo-Lyase