title: An unnatural folate stereoisomer is catalytically competent in DNA photolyase.
abstract: The folate chromophore in native Escherichia coli DNA photolyase ([6R]-5,10-CH+-H4Pte(Glu)n=3-6) serves as an antenna, transferring light energy to the fully reduced flavin (FADH2) reaction center at high efficiency (EET = 0.92). Apophotolyase reconstituted after an overnight incubation with [6R,S]-5,10-CH+-H4folate (a monoglutamate analogue of the native cofactor) contains equimolar amounts of the [6R]- and [6S]-isomers, suggesting similar binding affinities. A rapid, biphasic increase in fluorescence (approximately 100-fold) is observed upon binding of 5,10-CH+-H4folate to apophotolyase at 5 degrees C; the [6S]-isomer binds about 25-fold faster than the [6R]-isomer. Although identical absorption and fluorescence emission maxima are observed for enzyme reconstituted with [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate, folate fluorescence quantum yield values vary depending on the stereochemical configuration at the 6 position (theta = 0.18, 0.82, or 0.46, respectively, at 5 degrees C), a feature not seen with free folate. The fluorescence of enzyme-bound folate is quenched upon flavin binding; the efficiency of quenching by flavin radical (EQ = 0.96) or FADH2 (EQ = 0.89) is the same for both folate isomers. In contrast, energy transfer from folate to FADH2 is sensitive to the stereochemical configuration at the 6 position. The efficiency of energy transfer observed for enzyme containing FADH2 and [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate (theta = 0.26, 0.66, or 0.44, respectively) is directly proportional to the fluorescence quantum yield observed for folate in the absence of FADH2, as expected for Förster-type energy transfer. Although less efficient, the unnatural [6S]-isomer is catalytically functional, a feature not previously observed with other folate-dependent enzymes. Fluorescence quantum yield studies at 77 K with free (theta = 0.67) and enzyme-bound (theta = 1.0) folate suggest that differences in solvent exposure may contribute to the fluorescence efficiency differences observed with the enzyme-bound folate isomers at 5 degrees C.
authors.affiliation: Department of Biochemistry, Medical College of Pennsylvania, Philadelphia, USA.
authors.forename: R S
authors.initials: RS
authors.lastname: Lipman
authors.forename: M S
authors.initials: MS
authors.lastname: Jorns
mesh_terms: D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005492:Folic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D007700:Kinetics; D010084:Oxidation-Reduction; D011789:Quantum Theory; D013050:Spectrometry, Fluorescence; D013237:Stereoisomerism
chemical_list: D001051:Apoenzymes; D005182:Flavin-Adenine Dinucleotide; D005492:Folic Acid; D004255:Deoxyribodipyrimidine Photo-Lyase