flavin-adenine-dinucleotide and 5-10-methenyltetrahydrofolate

Photoreceptors in the eyes of mammals synchronize the innate circadian rhythm to the solar light-dark cycle. They differ from the photoreceptors serving vision in rods and cones of the retina and are located in the ganglion cells of the retina. They consist of melanopsin, a protein homologous to rhodopsin, with retinal as the light-sensitive prosthetic group; and cryptochromes 1 and 2, proteins combined with methenyltetrahydrofolate and flavin adenine dinucleotide, which function as blue light-sensitive photoreceptors.

Topics: Animals; Circadian Rhythm; Cryptochromes; Drosophila Proteins; Eye Proteins; Flavin-Adenine Dinucleotide; Flavoproteins; Homeostasis; Humans; Light; Photoreceptor Cells; Photoreceptor Cells, Invertebrate; Receptors, G-Protein-Coupled; Rod Opsins; Tetrahydrofolates; Vitamins

Members of the cryptochrome/photolyase family (CPF) of proteins utilize noncovalently bound light-absorbing cofactors for their biological function. Usually, the identity of these cofactors is determined after expression in heterologous systems leaving the question unanswered whether these cofactors are identical to the indigenous ones. Here, cryptochrome 3 from Arabidopsis thaliana was expressed as a fusion with the green fluorescent protein in Arabidopsis plants. Besides the confirmation of the earlier report of its localization in chloroplasts, our data indicate that fractions of the fusion protein are present in the stroma and associated with thylakoids, respectively. Furthermore, it is shown that the fusion protein expressed in planta contains the same cofactors as the His

Topics: Arabidopsis; Chromatography, Reverse-Phase; Cryptochromes; Escherichia coli; Flavin-Adenine Dinucleotide; Green Fluorescent Proteins; Immunoprecipitation; Plants, Genetically Modified; Recombinant Fusion Proteins; Spectrometry, Fluorescence; Tetrahydrofolates

Photolyases are proteins with an FAD chromophore that repair UV-induced pyrimidine dimers on the DNA in a light-dependent manner. The cyclobutane pyrimidine dimer class III photolyases are structurally unknown but closely related to plant cryptochromes, which serve as blue-light photoreceptors. Here we present the crystal structure of a class III photolyase termed photolyase-related protein A (PhrA) of Agrobacterium tumefaciens at 1.67-Å resolution. PhrA contains 5,10-methenyltetrahydrofolate (MTHF) as an antenna chromophore with a unique binding site and mode. Two Trp residues play pivotal roles for stabilizing MTHF by a double π-stacking sandwich. Plant cryptochrome I forms a pocket at the same site that could accommodate MTHF or a similar molecule. The PhrA structure and mutant studies showed that electrons flow during FAD photoreduction proceeds via two Trp triads. The structural studies on PhrA give a clearer picture on the evolutionary transition from photolyase to photoreceptor.

Topics: Agrobacterium tumefaciens; Binding Sites; Crystallography, X-Ray; Cytochromes; Deoxyribodipyrimidine Photo-Lyase; DNA Damage; Enzyme Stability; Evolution, Molecular; Flavin-Adenine Dinucleotide; Models, Molecular; Nucleic Acid Conformation; Oxidation-Reduction; Protein Structure, Tertiary; Pyrimidine Dimers; Tetrahydrofolates; Ultraviolet Rays

The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FAD(ox)), semiquinone form (FADH(.)), and anionic hydroquinone form (FAD(red)H(-)). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FAD(ox) to the anionic semiquinone form (FAD()(-)) with a quantum efficiency of about 2% and a back recovery time of about 10s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FAD(ox) to FADH(.), FADH(.) to FAD(red)H(-), and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.

Topics: Amino Acid Substitution; Arabidopsis; Arabidopsis Proteins; Cryptochromes; Flavin-Adenine Dinucleotide; Mutagenesis, Site-Directed; Recombinant Proteins; Spectrometry, Fluorescence; Tetrahydrofolates

Escherichia coli photolyase catalyzes the repair of cyclobutane pyrimidine dimers (CPD) in DNA under near UV/blue-light irradiation. The enzyme contains flavin adenine dinucleotide (FAD) and methenyltetrahydrofolate (MTHF) as noncovalently bound light sensing cofactors. To study the apoprotein-chromophore interactions we developed a new procedure to prepare apo-photolyase. MTHF-free photolyase was obtained by binding the C-terminal His-tagged holoenzyme to a metal-affinity column at neutral pH and washing the column with deionized water. Under these conditions the flavin remains bound and the defolated enzyme can be released from the column with 0.5 M imidazole pH 7.2. The MTHF-free protein was still capable of DNA repair, showing 70% activity of native enzyme. Fluorescence polarization experiments confirmed that MTHF binding is weakened at low ionic strength. Apo-photolyase was obtained by treating the His-tagged holoenzyme with 0.5 M imidazole pH 10.0. The apo-photolyase thus obtained was highly reconstitutable and bound nearly stoichiometric amounts of FAD(ox). Photolyase reconstituted with FAD(ox) had about 34% activity of native enzyme, which increased to 83% when FAD(ox) was reduced to FADH(-). Reconstitution kinetics performed at 20 degrees C showed that apo-photolyase associates with FADH(-) much faster (k(obs) approximately 3,000 M(-1) s(-1)) than with FAD(ox) (k(obs)=16 [corrected] M(-1) s(-1)). The dissociation constant of the photolyase-FAD(ox) complex is about 2.3 microM and that of E-FADH(-) is not higher than 20 nM (pH 7.2).

Topics: Apoenzymes; Deoxyribodipyrimidine Photo-Lyase; Escherichia coli; Flavin-Adenine Dinucleotide; Fluorescence Polarization; Histidine; Kinetics; Spectrophotometry; Tetrahydrofolates

Topics: Amino Acid Sequence; Apoenzymes; Aspergillus nidulans; Binding Sites; Catalysis; Chromatography; Chromatography, Affinity; Chromatography, Gel; Coenzymes; Deoxyribodipyrimidine Photo-Lyase; Deuterium; DNA Repair; Durapatite; Electron Spin Resonance Spectroscopy; Escherichia coli; Flavin-Adenine Dinucleotide; Pyrimidine Dimers; Radioisotope Dilution Technique; Riboflavin; Spectrophotometry; Spectrophotometry, Ultraviolet; Tetrahydrofolates; Tryptophan

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)

Topics: Amino Acid Sequence; Binding Sites; Catalysis; Cysteine; Deoxyribodipyrimidine Photo-Lyase; Escherichia coli; Flavin-Adenine Dinucleotide; Molecular Sequence Data; Pyrimidine Dimers; Tetrahydrofolates

Photolyase repairs UV-induced cyclobutane-pyrimidine dimers in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains 5,10-methenyltetrahydrofolate, which functions as the light-harvesting chromophore, and fully reduced flavin adenine dinucleotide (FAD), which functions as the redox catalyst. During enzyme preparation, the flavin is oxidized to FADH0, which is catalytically inert. Illumination of the enzyme with 300- to 600-nm light converts the flavin to the fully reduced form in a reaction that involves photooxidation of an amino acid in the apoenzyme. The results of earlier optical studies had indicated that the redox-active amino acid in this photoactivation process was tryptophan. We have now used time-resolved electron paramagnetic resonance (EPR) spectroscopy to investigate the photoactivation reaction. Excitation of the flavin-radical-containing inactive enzyme produces a spin-polarized radical that we identify by 2H and 15N labeling as originating from a tryptophan residue, confirming the inferences from the optical work. These results and Trp-->Phe replacement by site-directed mutagenesis reveal that flavin radical photoreduction is achieved by electron abstraction from Trp-306 by the excited-state FADH0. Analysis of the hyperfine couplings and spin density distribution deduced from the isotopic-labeling results shows that the product of the light-driven redox chemistry is the Trp-306 cation radical. The results strongly suggest that the active form of photolyase contains FADH- and not FADH2.

Topics: Amino Acid Sequence; Deoxyribodipyrimidine Photo-Lyase; Electron Spin Resonance Spectroscopy; Escherichia coli; Flavin-Adenine Dinucleotide; Kinetics; Magnetic Resonance Spectroscopy; Oxidation-Reduction; Tetrahydrofolates; Time Factors; Tryptophan

DNA photolyase from Escherichia coli (M(r) 54,000) consists of a polypeptide chain of 471 amino acids and the non-covalently bound cofactors methenyltetrahydrofolate (MTHF) and flavin adenine dinucleotide (FADH2). Two crystal forms of the enzyme were obtained; both have symmetry of space group P1. Form I has the unit cell dimensions a = 89.4 A, b = 97.3 A, c = 62.1 A, alpha = 108.3 degrees, beta = 97.4 degrees and gamma = 90.0 degrees. Diffraction from this form extends beyond 3 A resolution, but the crystals are radiation-sensitive and difficult to reproduce. Form II has the unit cell dimensions a = 62.6 A, b = 72.2 A, c = 58.5 A, alpha = 99.1 degrees, beta = 101.5 degrees and gamma = 72.0 degrees; most likely, the unit cell contains two molecules. High diffraction quality and reproducibility make form II suitable for structure analysis.

Topics: Crystallization; Deoxyribodipyrimidine Photo-Lyase; Escherichia coli; Flavin-Adenine Dinucleotide; Tetrahydrofolates; X-Ray Diffraction

The active form of native Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate [5,10-CH(+)-H4Pte(Glu)n]. Enzyme containing FADH2 and/or 5,10-methyltetrahydrofolate (5,10-CH(+)-H4folate) can be prepared in reconstitution experiments. Fluorescence quantum yield measurements at various wavelengths with native or reconstituted enzyme provide a simple method for detecting singlet-singlet energy transfer from pterin to FADH2, a key step in the proposed catalytic mechanism. The data satisfy the following criteria: (1) Wavelength-independent quantum yield values are observed for 5,10-CH(+)-H4folate in the absence (0.434) or presence (3.57 X 10(-2)) of FADH2, for 5,10-CH(+)-H4Pte(Glu)n in the presence of FADH2 (5.58 X 10(-2)) and for FADH2 in the absence of pterin (5.34 X 10(-3)); (2) The observed decrease in pterin fluorescence quantum yield in the presence of FADH2 can be used to estimate the efficiency of pterin fluorescence quenching (EQ = 0.918 or 0.871 with 5,10-CH(+)-H4folate or 5,10-CH(+)-H4Pte(Glu)n, respectively); (3) The fluorescence quantum yield of FADH2 is increased in the presence of pterin and varies depending on the excitation wavelength, in agreement with the predicted effect of energy transfer on acceptor fluorescence quantum yield [phi acceptor (+ donor)/phi acceptor (alone) = 1 + EET(epsilon donor/epsilon acceptor), where EET is the efficiency of the energy transfer process]. With 5,10-CH(+)-H4Pte(Glu)n in native enzyme the value obtained for EET (0.92) is similar to EQ, whereas with 5,10-CH(+)-H4folate in reconstituted enzyme the value obtained for EET (0.46) is 2-fold smaller than EQ.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Anaerobiosis; Deoxyribodipyrimidine Photo-Lyase; Energy Transfer; Escherichia coli; Flavin-Adenine Dinucleotide; Kinetics; Oxidation-Reduction; Protein Binding; Spectrometry, Fluorescence; Substrate Specificity; Tetrahydrofolates

Escherichia coli DNA photolyase mediates photorepair of pyrimidine dimers occurring in UV-damaged DNA. The enzyme contains two chromophores, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolylpolyglutamate (MTHF). To define the roles of the two chromophores in the photochemical reaction(s) resulting in DNA repair and the effect of DNA structure on the photocatalytic step, we determined the absolute action spectra of the enzyme containing only FADH2 (E-FADH2) or both chromophores (E-FADH2-MTHF), with double- and single-stranded substrates and with substrates of different sequences in the immediate vicinity of the thymine dimer. We found that the shape of the action spectrum of E-FADH2 matches that of the absorption spectrum with a quantum yield phi (FADH2) = 0.69. The action spectrum of E-FADH2-MTHF is also in a fairly good agreement with the absorption spectrum with phi (FADH2-MTHF) = 0.59. From these values and from the previously established properties of the two chromophores, we propose that MTHF transfers energy to FADH2 with a quantum yield of phi epsilon T = 0.8 and that 1FADH2 singlet transfers an electron to or from the dimer with a quantum yield phi ET = 0.69. The chemical nature of the chromophores did not change after several catalytic cycles. The enzyme repaired a thymine dimer in five different sequence contexts with the same efficiency. Similarly, single- and double-stranded DNAs were repaired with the same overall quantum yield.

Topics: Base Sequence; Deoxyribodipyrimidine Photo-Lyase; DNA Repair; Escherichia coli; Flavin-Adenine Dinucleotide; Folic Acid; Molecular Sequence Data; Protein Denaturation; Pyrimidine Dimers; Spectrophotometry, Ultraviolet; Substrate Specificity; Tetrahydrofolates

Native DNA photolyase, as isolated from Escherichia coli, contains a neutral flavin radical (FADH.) plus a pterin chromophore (5,10-methenyltetrahydropteroylpolyglutamate) and can be converted to its physiologically significant form by reduction of FADH. to fully reduced flavin (FADH2) with dithionite or by photoreduction. Either FADH2 or the pterin chromophore in dithionite-reduced native enzyme can function as a sensitizer in catalysis. Various enzyme forms (EFADox, EFADH., EFADH2, EPteFADox, EPteFADH., EPteFADH2, EPte) containing stoichiometric amounts of FAD in either of its three oxidation states and/or 5,10-methenyltetrahydrofolate (Pte) have been prepared in reconstitution experiments. Studies with EFADox and EPte showed that these preparations retained the ability to bind the missing chromophore. The results suggest that there could be considerable flexibility in the biological assembly of holoenzyme since the order of binding of the enzyme's chromophores is apparently unimportant, the binding of FAD is unaffected by its redox state, and enzyme preparations containing only one chromophore are reasonably stable. The same catalytic properties are observed with dithionite-reduced native enzyme or EFADH2. These preparations do not exhibit a lag in catalytic assays whereas lags are observed with preparations containing FADox or FADH. in the presence or absence of pterin. Photochemical studies show that these lags can be attributed to enzyme activation under assay conditions in a reaction involving photoreduction of enzyme-bound FADox or FADH. to FADH2. EPte is catalytically inactive, but catalytic activity is restored upon reconstitution of EPte with FADox. The results show that pterin is not required for dimer repair when FADH2 acts as the sensitizer but that FADH2 is required when dimer repair is initiated by excitation of the pterin chromophore. The relative intensity of pterin fluorescence in EPte, EPteFADH., EPteFADox, or EPteFADH2 has been used to estimate the efficiency of pterin singlet quenching by FADH. (93%), FADox (90%), or FADH2 (58%). Energy transfer from the excited pterin to flavin is energetically feasible and may account for the observed quenching of pterin fluorescence and also explain why photoreduction of FADox or FADH. is accelerated by the pterin chromophore. An irreversible photobleaching of the pterin chromophore is accelerated by FADH2 in a reaction that is accompanied by a transient oxidation of FADH2 to FADH.. Both pterin bl

Topics: Apoenzymes; Apoproteins; Catalysis; Deoxyribodipyrimidine Photo-Lyase; Dithionite; Dithiothreitol; DNA Repair; Escherichia coli; Flavin-Adenine Dinucleotide; Kinetics; Lyases; Oxidation-Reduction; Photochemistry; Pterins; Spectrometry, Fluorescence; Spectrophotometry; Tetrahydrofolates

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)

Topics: Catalysis; Deoxyribodipyrimidine Photo-Lyase; Escherichia coli; Flavin-Adenine Dinucleotide; Lyases; Molecular Weight; Oligodeoxyribonucleotides; Oxidation-Reduction; Photochemistry; Pyrimidine Dimers; Spectrometry, Fluorescence; Spectrophotometry; Tetrahydrofolates; Ultraviolet Rays