Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:4.1.99.3 (PRE)
 1,923 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A DNA fragment including most of the tyrA gene from E. coli B/r strain WU (Tyr-, Leu-) was amplified in vitro by polymerase chain reaction. The sequence was determined, first, for essentially all of the fragment to locate an ochre nonsense defect, and second, repeatedly for a region of the fragment from several independent isolates containing backmutations at the ochre codon (spontaneous and UV-induced). There were 20 single base differences in the tyrA gene region from the analogous wild-type E. coli K12 sequence: an ochre codon at amino acid position 161, 18 silent changes (1 at the first codon base and 17 at the third) and one replacement of valine by alanine. Different backmutations at the ochre codon encoded lysine, glutamine, glutamic acid, leucine, cysteine, phenylalanine, serine or tyrosine. The diversities of base substitutions at the ochre codon after UV mutagenesis or after mutagenesis where targeting by dimers was reduced or eliminated (after photoreversal of irradiated cells treated with nalidixic acid to induce SOS functions or after UV mutagenesis of cells containing amplified DNA photolyase) were similar (with two notable exceptions). The overall differences between the gene sequences for E. coli K12 or B/r seemed consistent with the neutral theory of molecular evolution.
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PMID:Diverse backmutations at an ochre defect in the tyrA gene sequence of E. coli B/r. 198 58

Escherichia coli DNA photolyase is a flavoprotein that when purified is blue in color and contains a stable neutral radical FAD (E-FADH). In the presence of a suitable electron donor (i.e., thiols, tyrosine, or NADH) the radical FAD adsorbs visible light and undergoes photoreduction to the fully reduced FAD (E-FADH2). The in vitro quantum yield of dimer repair for E-FADH is 0.07 while that of E-FADH2 approaches the in vivo value of 1. Electron paramagnetic resonance studies on whole cells indicate that the in vivo form of photolyase is E-FADH2 with enzyme containing radical FAD generated predominantly during the ammonium sulfate precipitation step of the purification. Activity measurements of E-FADH using long-wavelength photoreactivating light indicate that enzyme containing FAD in the radical form is not active in dimer repair. Dimer repair observed with E-FADH at shorter wavelengths is probably photoreduction of E-FADH followed by dimer repair by E-FADH2.
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PMID:The active form of Escherichia coli DNA photolyase contains a fully reduced flavin and not a flavin radical, both in vivo and in vitro. 282 44

Escherichia coli DNA photolyase contains a stable flavin radical and a second chromophore (SC) of unknown structure. The effects of flash (both conventional and laser) excitation of either the radical alone or both the radical and the second chromophore have been investigated by variation of the excitation wavelengths. Radical excitation leads to an electron abstraction by the lowest excited doublet state of the radical from an amino acid residue, probably a cysteine or tyrosine. On a longer time scale, a back-reaction occurs that can be prevented by the presence of certain electron donors, e.g., thiols, NADH, or tyrosine, but not pyrimidine dimers. Excitation of the second chromophore leads to electronic energy transfer from second chromophore excited states to the ground-state flavin radical doublet state, thus increasing the population of the lowest excited doublet state. Repetitive excitation of the enzyme with white light leads to photodecomposition of the second chromophore but not of the flavin adenine dinucleotide cofactor. Enzyme with photodecomposed SC retains full activity.
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PMID:Photochemical properties of Escherichia coli DNA photolyase: selective photodecomposition of the second chromophore. 331 Nov 50

Light-induced electron transfer reactions leading to the fully reduced, catalytically competent state of the flavin adenine dinucleotide (FAD) cofactor have been studied by flash absorption spectroscopy in DNA photolyase from Anacystis nidulans. The protein, overproduced in Escherichia coli, was devoid of the antenna cofactor, and the FAD chromophore was present in the semireduced form, FADH., which is inactive for DNA repair. We show that after selective excitation of FADH. by a 7-ns laser flash, fully reduced FAD (FADH-) is formed in less than 500 ns by electron abstraction from a tryptophan residue. Subsequently, a tyrosine residue is oxidized by the tryptophanyl radical with t(1)/(2) = 50 microseconds. The amino acid radicals were identified by their characteristic absorption spectra, with maxima at 520 nm for Trp. and 410 nm for TyrO. The newly discovered electron transfer between tyrosine and tryptophan occurred for approximately 40% of the tryptophanyl radicals, whereas 60% decayed by charge recombination with FADH- (t(1)/(2) = 1 ms). The tyrosyl radical can also recombine with FADH- but at a much slower rate (t(1)/(2) = 76 ms) than Trp. In the presence of an external electron donor, however, TyrO. is rereduced efficiently in a bimolecular reaction that leaves FAD in the fully reduced state FADH-. These results show that electron transfer from tyrosine to Trp. is an essential step in the process leading to the active form of photolyase. They provide direct evidence that electron transfer between tyrosine and tryptophan occurs in a native biological reaction.
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PMID:Intraprotein electron transfer between tyrosine and tryptophan in DNA photolyase from Anacystis nidulans. 1031 99

Tryptophan radicals, which are generated in the reconstitution reaction of mutants Y122F and Y177W of subunit R2 apoprotein of E. coli and mouse ribonucleotide reductase (RNR), respectively, with Fe(2+) and oxygen, are investigated by high-field EPR at 94 GHz and compared with the tyrosine radicals occurring in the respective wild-type proteins. For the first time, accurate g-values are obtained for protein-associated neutral tryptophan free radicals, which show only a small anisotropy. The apparent hyperfine patterns observed in frozen solutions are very similar for tryptophan and tyrosine radicals in mouse subunit R2 at conventional X-band EPR. The radicals can, however, be discriminated by their different g-tensors using high-field EPR. Tryptophan radicals were postulated as reaction intermediates in the proposed radical transfer pathway of RNR. Furthermore, the data obtained here for the electronic structure of protein-associated tryptophan neutral free radicals are important for identification and understanding of the functional important tryptophan radicals which occur in other enzymes, e.g., DNA photolyase and cytochrome c peroxidase, where they are magnetically coupled to other radicals or to a metal center.
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PMID:Tryptophan and tyrosine radicals in ribonucleotide reductase: a comparative high-field EPR study at 94 GHz. 1173 19

The light-induced electron transfer reaction of flavin cofactor photoactivation in Xenopus laevis (6-4) photolyase has been studied by continuous-wave and time-resolved electron paramagnetic resonance spectroscopy. When the photoactivation is initiated from the fully oxidized form of the flavin, a neutral flavin radical is observed as a long-lived paramagnetic intermediate of two consecutive single-electron reductions under participation of redox-active amino acid residues. By time-resolved electron paramagnetic resonance, a spin-polarized transient radical-pair signal was detected that shows remarkable differences to the signals observed in the related cyclobutane pyrimidine dimer photolyase enzyme. In (6-4) photolyase, a neutral tyrosine radical has been identified as the final electron donor, on the basis of the characteristic line width, hyperfine splitting pattern, and resonance magnetic field position of the tyrosine resonances of the transient radical pair.
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PMID:Photoactivation of the flavin cofactor in Xenopus laevis (6 - 4) photolyase: observation of a transient tyrosyl radical by time-resolved electron paramagnetic resonance. 1180 94

Charge separation and radical transfer in DNA photolyase from Escherichia coli is investigated by computing electrostatic free energies from a solution of the Poisson-Boltzmann equation. For the initial charge separation 450 meV are available. According to recent experiments [Aubert et al. Nature 2000, 405, 586-590] the flavin receives an electron from the proximal tryptophan W382, which consequently forms a cationic radical WH(*)(+)382. The radical state is subsequently transferred along the triad W382-W359-W306 of conserved tryptophans. The radical transfer to the intermediate tryptophan W359 is nearly isoenergetic (58 meV uphill); the radical transfer from the intermediate W359 to the distal W306 is 200 meV downhill in energy, funneling and stabilizing the radical state at W306. The resulting cationic radical WH(*)(+)306 is further stabilized by deprotonation, yielding the neutral radical W(*)306, which is 214 meV below WH(*)(+)306. The time scale of the charge recombination process yielding back the resting enzyme with FADH(*) is governed by reprotonation of W306, with a calculated lifetime of 1.2 ms that correlates well with the measured lifetime of 17 ms. In photolyase from Anacystis nidulans the radical state is partially transferred to a tyrosine [Aubert et al. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 5423-5427]. In photolyase from Escherichia coli, there is a tyrosine (Y464) close to the distal tryptophan W306 that could play this role. We show that this tyrosine cannot be involved in radical transfer, because the electron transfer from tyrosine to W306 is much too endergonic (750 meV) and a direct hydrogen transfer is likely too slow. Coupling of specific charge states of the tryptophan triad with protonation patterns of titratable residues of photolyase is small.
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PMID:Energetics of radical transfer in DNA photolyase. 1192 68

The coupling of electron and proton transfer is an important controlling factor in radical proteins, such as photosystem II, ribinucleotide reductase, cytochrome oxidases, and DNA photolyase. This was investigated in model complexes in which a tyrosine or tryptophan residue was oxidized by a laser-flash generated trisbipyridine-Ru(III) moiety in an intramolecular, proton-coupled electron transfer (PCET) reaction. The PCET was found to proceed in a competition between a stepwise reaction, in which electron transfer is followed by deprotonation of the amino acid radical (ETPT), and a concerted reaction, in which both the electron and proton are transferred in a single reaction step (CEP). Moreover, we found that we could analyze the kinetic data for PCET by Marcus' theory for electron transfer. By altering the solution pH, the strength of the Ru(III) oxidant, or the identity of the amino acid, we could induce a switch between the two mechanisms and obtain quantitative data for the parameters that control which one will dominate. The characteristic pH-dependence of the CEP rate (M. Sjodin et al. J. Am. Chem. Soc. 2000, 122, 3932) reflects the pH-dependence of the driving force caused by proton release to the bulk. For the pH-independent ETPT on the other hand, the driving force of the rate-determining ET step is pH-independent and smaller. On the other hand, temperature-dependent data showed that the reorganization energy was higher for CEP, while the pre-exponential factors showed no significant difference between the mechanisms. Thus, the opposing effect of the differences in driving force and reorganization energy determines which of the mechanisms will dominate. Our results show that a concerted mechanism is in general quite likely and provides a low-barrier reaction pathway for weakly exoergonic reactions. In addition, the kinetic isotope effect was much higher for CEP (kH/kD > 10) than for ETPT (kH/kD = 2), consistent with significant changes along the proton reaction coordinate in the rate-determining step of CEP.
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PMID:Switching the redox mechanism: models for proton-coupled electron transfer from tyrosine and tryptophan. 1577 21

Cryptochromes are blue light-activated photoreceptors found in multiple organisms with significant similarity to photolyases, a class of light-dependent DNA repair enzymes. Unlike photolyases, cryptochromes do not repair DNA and instead mediate blue light-dependent developmental, growth, and/or circadian responses by an as yet unknown mechanism of action. It has recently been shown that Arabidopsis cryptochrome-1 retains photolyase-like photoreduction of its flavin cofactor FAD by intraprotein electron transfer from tryptophan and tyrosine residues. Here we demonstrate that substitution of two conserved tryptophans that are constituents of the flavin-reducing electron transfer chain in Escherichia coli photolyase impairs light-induced electron transfer in the Arabidopsis cryptochrome-1 photoreceptor in vitro. Furthermore, we show that these substitutions result in marked reduction of light-activated autophosphorylation of cryptochrome-1 in vitro and of its photoreceptor function in vivo, consistent with biological relevance of the electron transfer reaction. These data support the possibility that light-induced flavin reduction via the tryptophan chain is the primary step in the signaling pathway of plant cryptochrome.
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PMID:Light-induced electron transfer in Arabidopsis cryptochrome-1 correlates with in vivo function. 1577 75

The Collared peccary (Pecari tajacu) is one of the three extant recognised species of the family Tayassuidae, living in the Americas. To understand phylogenetic relationships among Collared peccaries, the entire mitochondrial DNA control region and cytochrome b as well as partial nuclear GPIP and PRE-1 P27, PRE-1 P642 and TYR sequences from specimens from Colombia, Argentina, Bolivia, Mexico, United States and Australian zoo animals of unknown origin were analysed. Separate and combined analyses of the mitochondrial sequences provided good resolution of Collared peccary relationships. Nuclear sequences were partially informative when combined sequence analyses were performed. Maximum Likelihood analyses of mitochondrial sequences showed that Collared peccaries clustered in two major clades, representing North-Central American and South American specimens. Collared peccaries from Colombia are paraphyletic. Statistical Parsimony analysis of combined nuclear sequences showed a distribution of DNA variants consistent with mitochondrial sequence analyses. However, there is an uncoupling of nuclear and mitochondrial sequence variation in two specimens from Colombia. The present study suggests the recent contact of isolated populations within Colombia and possible mitochondrial introgression between the North/Central clade and the South clade. Pairwise genetic distances comparison of mitochondrial sequences show that divergence between the two major clades of the Collared peccary was higher and comparable respectively with that within and between the other two recognised peccary species. Divergence between the two major clades of the Collared peccary was also higher than that observed within and even between recognised species of the Suidae family. The divergence within the major clades of the Collared peccary showed comparable values with those observed within the other two species of Tayassuidae and within six species of Suidae. The results show that the geographically widespread and phenotypically diverse Collared peccary consists of two species or at least subspecies with implications for management of wild, zoo and captive populations.
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PMID:Phylogenetic divisions among Collared peccaries (Pecari tajacu) detected using mitochondrial and nuclear sequences. 1683 18


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