Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.99.3 (PRE)
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Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyl-tetrahydrofolate (5,10-CH+-H4folate). Both chromophores are fluorescent, and either can function as a sensitizer in catalysis. At 77 K separate fluorescence emission bands are observed for FADH2 (lambda max = 505 nm, shoulder at 540 nm) and 5,10-CH+-H4folate (lambda max = 465, 440 nm) whereas at 5 degrees C only a shoulder at 505 nm is attributable to FADH2. Formation of an enzyme-substrate complex with various dimer-containing oligothymidylates [UV-oligo(dT)n] quenches the fluorescence due to FADH2 at 5 degrees C or 77 K and also stabilizes FADH2 against air oxidation. The fluorescence of 5,10-CH+-H4folate is unaffected by substrate. Reduction of the pterin chromophore eliminates the chromophore's fluorescence but does not affect catalytic activity or the ability of substrate to quench FADH2 fluorescence. Quenching of FADH2 fluorescence is fully reversible upon dimer repair. The results are consistent with the proposal that the singlet state of FADH2 functions as an intermediate in catalysis. Fluorometric titrations indicate that the enzyme has a similar affinity for dimers in UV-oligo(dT)4 (KD = 2.5 X 10(-7) M, delta G = 8.4 kcal/mol at 5 degrees C) or UV-oligo(dT)6, except for dimers located at the unphosphorylated 3' end of the oligomers where binding is considerably weaker.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Evidence for a singlet intermediate in catalysis by Escherichia coli DNA photolyase and evaluation of substrate binding determinants. 306 30

We have placed the PHR1 gene of Saccharomyces cerevisiae under the transcriptional and translational control of the tac expression cartridge. Under inducing conditions Escherichia coli cells harboring plasmids carrying this construct accumulate approximately 8% of total cellular protein as the Phr1 photolyase. Using a strain devoid of E. coli photolyase activity, we have obtained milligram quantities of the yeast enzyme at greater than 95% purity and have characterized the enzyme. Phr1 photolyase is a monomer in solution with an Mr of 60,000, has a turnover number of 0.7 dimers min-1 molecule-1 in vitro, exhibits absorbance maxima at lambda = 277 and 377 nm, and has a fluorescence excitation maximum at 390 nm and an emission maximum at 475 nm. The near UV absorbance peak is shown to reflect the contributions of two intrinsic chromophores which are noncovalently bound to the enzyme. Spectroscopic, fluorescence, and thin layer chromatographic studies indicate that one of these chromophores is 1,5-reduced FAD rather than 4a,5-reduced FAD as previously proposed (Iwatsuki, N., Joe, C. O., and Werbin, H. (1980) Biochemistry 19, 1172-1176), while the other chromophore has properties similar to the second chromophore of E. coli photolyase. The fact that yeast and E. coli photolyases are similar both with respect to amino acid sequence and chromophore composition provides strong evidence that the enzymes share a common action mechanism which may also be utilized by photolyases from other organisms throughout the phylogenetic tree.
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PMID:Purification of the yeast PHR1 photolyase from an Escherichia coli overproducing strain and characterization of the intrinsic chromophores of the enzyme. 331 99

DNA photolyase from Escherichia coli contains FAD plus a partially characterized, second chromophore. In vivo, the flavin is fully reduced (FADH2), but oxidation to a stable, blue radical (FADH.) occurs during enzyme isolation. The second chromophore is irreversibly reduced by reaction of the enzyme with sodium borohydride or by photoreduction in the presence of dithiothreitol. A similar reaction occurs with the protein-free chromophore and sodium cyanoborohydride. Reduction of the second chromophore is accompanied by a complete loss of the chromophore's visible absorption and fluorescence but does not significantly affect catalytic activity. The results show that the enzyme can repair dimers by a pathway involving only FADH2. Enzyme-bound FADH2 is fluorescent and exhibits emission (505 nm) and absorption (360 nm) maxima similar to that expected for a 1,5-dihydroflavin derivative. It is proposed that dimer cleavage via the second chromophore independent pathway involves electron donation from excited FADH2 to pyrimidine dimer. Pyrimidine dimer radicals are unstable and spontaneously monomerize. Unmodified second chromophore can also act as a sensitizer in a pathway that requires FADH2. This pathway may be similar to that proposed for the second chromophore independent reaction except that excited FADH2 would be produced via energy transfer from the excited second chromophore. The fluorescence observed for enzyme-bound, unmodified second chromophore is quenched by FADH. and increases 6-fold when the latter is reduced, but the absorption spectrum (lambda max = 390 nm epsilon 390 = 12.7 x 10(3) M-1 cm-1) is independent of the redox state of the flavin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:DNA repair catalyzed by Escherichia coli DNA photolyase containing only reduced flavin: elimination of the enzyme's second chromophore by reduction with sodium borohydride. 332 90

DNA photolyase repairs pyrimidine dimers in DNA in a reaction that requires visible light. Photolyase from Escherichia coli is normally isolated as a blue protein and contains 2 chromophores: a blue FAD radical plus a second chromophore that exhibits an absorption maximum at 360 nm when free in solution. Oxidation of the FAD radical is accompanied by a reversible loss of activity which is proportional to the fraction of the enzyme flavin converted to FADox. Quantitative reduction of the radical to fully reduced FAD causes a 3-fold increase in activity. The results show that a reduced flavin is required for activity and suggest that flavin may act as an electron donor in catalysis. Comparison of the absorption spectrum calculated for the protein-bound second chromophore (lambda max = 390 nm) with fluorescence data and with the relative action spectrum for dimer repair indicates that the second chromophore is the fluorophore in photolyase and that it does act as a sensitizer in catalysis. On the other hand, enzyme preparations containing diminished amounts of the second chromophore do not exhibit correspondingly lower activity. This suggests that reduced flavin may also act as a sensitizer in catalysis. The blue color of the enzyme is lost upon reduction of the FAD radical. The fully reduced E. coli enzyme exhibits absorption and fluorescence properties very similar to yeast photolyase. This indicates that the two enzymes probably contain similar chromophores but are isolated in different forms with respect to the redox state of the flavin.
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PMID:Action mechanism of Escherichia coli DNA photolyase. II. Role of the chromophores in catalysis. 353 40

The absolute action spectrum of Escherichia coli DNA photolyase was determined in vitro. In vivo the photoreactivation cross-section (epsilon phi) is 2.4 X 10(4) M-1 cm-1 suggesting that the quantum yield (phi) is about 1.0 if one assumes that the enzyme has the same spectral properties (e.g. epsilon 384 = 1.8 X 10(4) M-1 cm-1) in vivo as those of the enzyme purified to homogeneity. The relative action spectrum of the pure enzyme (blue enzyme that contains FAD neutral semiquinone radical) agrees with the relative action spectrum for photoreactivation of E. coli, having lambda max = 384 nm. However, the absolute action spectrum of the blue enzyme yields a photoreactivation cross-section (epsilon phi = 1.2 X 10(3) at 384 nm) that is 20-fold lower than the in vivo values indicative of an apparent lower quantum yield (phi approximately equal to 0.07) in vitro. Reducing the enzyme with dithionite results in reduction of the flavin semiquinone and a concomitant 12-15-fold increase in the quantum yield. These results suggest that the flavin cofactor of the enzyme is fully reduced in vivo and that, upon absorption of a single photon in the 300-500 nm range, the photolyase chromophore (which consists of reduced FAD plus the second chromophore) donates an electron to the pyrimidine dimer causing its reversal to two pyrimidines. The reduced chromophore is regenerated at the end of the photochemical step thus enabling the enzyme to act catalytically.+
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PMID:Action mechanism of Escherichia coli DNA photolyase. III. Photolysis of the enzyme-substrate complex and the absolute action spectrum. 353 41

DNA photolyase from Escherichia coli contains folate ([6S]-5,10-CH(+)-H4Pte(Glu)n = 3-6) and reduced FAD. The folate chromophore acts as an antenna, harvesting light energy which is transferred to the reduced flavin where DNA repair occurs. The folate binding stereospecificity of the enzyme was investigated by reconstituting the apoenzyme with [6R,S]-5,10-CH(+)-H4folate and reduced FAD. The isomer composition of [methyl-3H]-5-CH3-H4folate, released into solution upon reduction of the reconstituted enzyme with [3H]NaBH4, was analyzed by enzymatic and chiral chromatographic methods. Both methods showed that the reconstituted enzyme contained nearly equimolar amounts of [6R]- and [6S]-5,10-CH(+)-H4folate.
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PMID:Stereospecificity of folate binding to DNA photolyase from Escherichia coli. 766 79

DNA photolyase specifically repairs UV light-induced cyclobutane-type pyrimidine dimers in DNA through a light-dependent reaction mechanism. We have obtained photolyase genes from Drosophila melanogaster (fruit fly), Oryzias latipes (killifish) and the marsupial Potorous tridactylis (rat kangaroo), the first photolyase gene cloned from a mammalian species. The deduced amino acid sequences of these higher eukaryote genes show only limited homology with microbial photolyase genes. Together with the previously cloned Carassius auratus (goldfish) gene they form a separate group of photolyase genes. A new classification for photolyases comprising two distantly related groups is proposed. For functional analysis P.tridactylis photolyase was expressed and purified as glutathione S-transferase fusion protein from Escherichia coli cells. The biologically active protein contained FAD as light-absorbing cofactor, a property in common with the microbial class photolyases. Furthermore, we found in the archaebacterium Methanobacterium thermoautotrophicum a gene similar to the higher eukaryote photolyase genes, but we could not obtain evidence for the presence of a homologous gene in the human genome. Our results suggest a divergence of photolyase genes in early evolution.
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PMID:A new class of DNA photolyases present in various organisms including aplacental mammals. 781 51

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)
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PMID:Affinity probing of flavin binding sites. 2. Identification of a reactive cysteine in the flavin domain of Escherichia coli DNA photolyase. 791 92

A phr-gene from the filamentous fungus Neurospora crassa was overexpressed in Escherichia coli cells, yielding a biologically active photolyase. After purification till apparent homogeneity, the 66 kDa protein was found to contain equimolar amounts of 5,10-methenyltetrahydrofolic acid (MTHF) and FAD, classifying it as an MTHF-type photolyase. Compared to other MTHF photolyases the absorption maximum of Neurospora photolyase is shifted from ca 380 nm to 391 nm (epsilon = 34,800), while an additional shoulder is present at 465 nm. In dark-adapted enzyme the FAD chromophore is predominantly present in the oxidized form, in contrast with E. coli and Saccharomyces cerevisiae photolyase, which contain mainly semiquinone or fully reduced FAD, respectively. Preillumination or dithionite treatment converted oxidized FAD in Neurospora photolyase into the fully reduced form, with a concomitant shift of the absorption maximum from 391 to 396 nm and disappearance of the 465 nm shoulder. The action spectrum of photoreactivation coincides with the absorption spectrum of preilluminated (reduced) photolyase, extending the spectral region of MTHF-type photolyases from 380 till 396 nm. A quantum yield of 0.57 was obtained for the overall repair reaction. Comparison of spectral properties of FAD in Neurospora photolyase and the model compound lumiflavin points to an apolar microenvironment of photolyase-bound FAD. Neurospora photolyase has distinct advantages over E. coli photolyase as it is more stable and contains a full complement of chromophores.
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PMID:DNA photolyase from the fungus Neurospora crassa. Purification, characterization and comparison with other photolyases. 793 8

The gene for the apoenzyme of Bacillus firmus photolyase was cloned and sequenced. The enzyme was overproduced in Escherichia coli, purified, and characterized. It has the unique property of having the maximum activity over a wavelength range where all other known photolyases exhibit modest activity. The enzyme contains reduced FAD and methenyltetrahydrofolate and has an absorption and action spectrum peak at 410 nm, and it repairs DNA with a quantum yield of phi approximately 0.75.
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PMID:Characterization of a medium wavelength type DNA photolyase: purification and properties of photolyase from Bacillus firmus. 803 61


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