Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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

DNA photolyases repair cyclobutadipyrimidines (Pyr()Pyr) in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains methenyltetrahydrofolate (MTHF), which functions as photoantenna, and FADH2, which is the redox-active cofactor. During purification, FADH2 is oxidized to the blue neutral radical form, FADH., which has greatly diminished activity. Previous nanosecond flash photolysis studies [Heelis, P.F., Okamura, T., & Sancar, A. (1990) Biochemistry 29, 5694-5698] indicated that excitation of FADH. either directly by absorbing a photon or indirectly by electronic energy transfer from MTHF excited singlet state yielded an FADH. quartet which abstracted a hydrogen atom from a nearby tryptophan to generate the catalytically competent FADH2 from of the enzyme. Using site-directed mutagenesis, we replaced all 15 photolyase tryptophan residues by phenylalanine, individually, in order to identify the internal hydrogen atom donor responsible for photoreduction. We found that W306F mutation abolished photoreduction of FADH. without affecting the excited-state properties of FADH. or the substrate binding (KA approximately 10(9) M-1) of the enzyme. The specificity constant (kcat/km) was approximately 0 for the mutant enzyme in the absence of reducing agents in the reaction mixture, indicating that photoreduction of FADH. is an essential step for photorepair by photolyase in vitro. Chemical reduction of FADH. of the mutant enzyme restored the specificity constant to the wild-type level.
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PMID:Active site of DNA photolyase: tryptophan-306 is the intrinsic hydrogen atom donor essential for flavin radical photoreduction and DNA repair in vitro. 205 37

Escherichia coli DNA photolyase repairs pyrimidine dimers by a photoinduced electron-transfer reaction. The enzyme binds to UV-damaged DNA independent of light (the dark reaction) and upon absorbing a 300-500-nm photon breaks the cyclobutane ring of the dimer (the light reaction) and thus restores the DNA. No structural information on the enzyme is available at present. However, comparison of the sequences of photolyases from five different organisms has identified highly conserved regions of homology. These regions are presumably involved in chromophore (flavin and folate) and substrate binding or catalysis. Trp277 (W277) in E. coli photolyase is conserved in all photolyases sequenced to date. We replaced this residue with Arg, Glu, Gln, His, and Phe by site-specific mutagenesis. Properties of the mutant proteins indicate that W277 is involved in binding to DNA but not in chromophore binding or catalysis. Of particular significance is the finding that compared to wild type W277R and W277E mutants have about 300- and 1000-fold lower affinity, respectively, for substrate but were indistinguishable from wild-type enzyme in their photochemical and photocatalytic properties.
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PMID:Active site of Escherichia coli DNA photolyase: mutations at Trp277 alter the selectivity of the enzyme without affecting the quantum yield of photorepair. 220 May 11

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.
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PMID:Time-resolved EPR studies with DNA photolyase: excited-state FADH0 abstracts an electron from Trp-306 to generate FADH-, the catalytically active form of the cofactor. 839 57

The present study was designed to determine whether consumption of an oral essential amino acid-carbohydrate supplement (EAC) before exercise results in a greater anabolic response than supplementation after resistance exercise. Six healthy human subjects participated in two trials in random order, PRE (EAC consumed immediately before exercise), and POST (EAC consumed immediately after exercise). A primed, continuous infusion of L-[ring-(2)H(5)]phenylalanine, femoral arteriovenous catheterization, and muscle biopsies from the vastus lateralis were used to determine phenylalanine concentrations, enrichments, and net uptake across the leg. Blood and muscle phenylalanine concentrations were increased by approximately 130% after drink consumption in both trials. Amino acid delivery to the leg was increased during exercise and remained elevated for the 2 h after exercise in both trials. Delivery of amino acids (amino acid concentration times blood flow) was significantly greater in PRE than in POST during the exercise bout and in the 1st h after exercise (P < 0.05). Total net phenylalanine uptake across the leg was greater (P = 0.0002) during PRE (209 +/- 42 mg) than during POST (81 +/- 19). Phenylalanine disappearance rate, an indicator of muscle protein synthesis from blood amino acids, increased after EAC consumption in both trials. These results indicate that the response of net muscle protein synthesis to consumption of an EAC solution immediately before resistance exercise is greater than that when the solution is consumed after exercise, primarily because of an increase in muscle protein synthesis as a result of increased delivery of amino acids to the leg.
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PMID:Timing of amino acid-carbohydrate ingestion alters anabolic response of muscle to resistance exercise. 1144 Aug 94

In Escherichia coli photolyase, excitation of the FAD cofactor in its semireduced radical state (FADH*) induces an electron transfer over approximately 15 A from tryptophan W306 to the flavin. It has been suggested that two additional tryptophans are involved in an electron transfer chain FADH* <-- W382 <-- W359 <-- W306. To test this hypothesis, we have mutated W382 into redox inert phenylalanine. Ultrafast transient absorption studies showed that, in WT photolyase, excited FADH* decayed with a time constant tau approximately 26 ps to fully reduced flavin and a tryptophan cation radical. In W382F mutant photolyase, the excited flavin was much longer lived (tau approximately 80 ps), and no significant amount of product was detected. We conclude that, in WT photolyase, excited FADH* is quenched by electron transfer from W382. On a millisecond scale, a product state with extremely low yield ( approximately 0.5% of WT) was detected in W382F mutant photolyase. Its spectral and kinetic features were similar to the fully reduced flavin/neutral tryptophan radical state in WT photolyase. We suggest that, in W382F mutant photolyase, excited FADH* is reduced by W359 at a rate that competes only poorly with the intrinsic decay of excited FADH* (tau approximately 80 ps), explaining the low product yield. Subsequently, the W359 cation radical is reduced by W306. The rate constants of electron transfer from W382 to excited FADH* in WT and from W359 to excited FADH* in W382F mutant photolyase were estimated and related to the donor-acceptor distances.
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PMID:Dissection of the triple tryptophan electron transfer chain in Escherichia coli DNA photolyase: Trp382 is the primary donor in photoactivation. 1283 19

Timing of nutrient ingestion has been demonstrated to influence the anabolic response of muscle following exercise. Previously, we demonstrated that net amino acid uptake was greater when free essential amino acids plus carbohydrates were ingested before resistance exercise rather than following exercise. However, it is unclear if ingestion of whole proteins before exercise would stimulate a superior response compared with following exercise. This study was designed to examine the response of muscle protein balance to ingestion of whey proteins both before and following resistance exercise. Healthy volunteers were randomly assigned to one of two groups. A solution of whey proteins was consumed either immediately before exercise (PRE; n = 8) or immediately following exercise (POST; n = 9). Each subject performed 10 sets of 8 repetitions of leg extension exercise. Phenylalanine concentrations were measured in femoral arteriovenous samples to determine balance across the leg. Arterial amino acid concentrations were elevated by approximately 50%, and net amino acid balance switched from negative to positive following ingestion of proteins at either time. Amino acid uptake was not significantly different between PRE and POST when calculated from the beginning of exercise (67 +/- 22 and 27 +/- 10 for PRE and POST, respectively) or from the ingestion of each drink (60 +/- 17 and 63 +/- 15 for PRE and POST, respectively). Thus the response of net muscle protein balance to timing of intact protein ingestion does not respond as does that of the combination of free amino acids and carbohydrate.
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PMID:Stimulation of net muscle protein synthesis by whey protein ingestion before and after exercise. 1689 66

Photoreduction of the semi-reduced flavin adenine dinucleotide cofactor FADH* in DNA photolyase from Escherichia coli into FADH- involves three tryptophan (W) residues that form a closely spaced electron-transfer chain FADH*-W382-W359-W306. To investigate this process, we have constructed a mutant photolyase in which W359 is replaced by phenylalanine (F). Monitoring its photoproducts by femtosecond spectroscopy, the excited-state FADH* was found to decay in approximately 30 ps, similar as in wild type (WT) photolyase. In contrast to WT, however, in W359F mutant photolyase the ground-state FADH* fully recovered virtually concomitantly with the decay of its excited state and, despite the presence of the primary electron donor W382, no measurable flavin reduction was observed at any time. Thus, W359F photolyase appears to behave like many other flavoproteins, where flavin excited states are quenched by very short-lived oxidation of aromatic residues. Our analysis indicates that both charge recombination of the primary charge separation state FADH-W382*+ and (in WT) electron transfer from W359 to W382*+ occur with time constants <4 ps, considerably faster than the initial W382-->FADH* electron-transfer step. Our results provide a first experimental indication that electron transfer between aromatic residues can take place on the time scale of approximately 10(-12) s.
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PMID:Role of the middle residue in the triple tryptophan electron transfer chain of DNA photolyase: ultrafast spectroscopy of a Trp-->Phe mutant. 1689 6

DNA photolyases (EC 4.1.99.3) are enzymes that catalyze photoreversion of cis,syn-thymine photodimer (T[c,s]T), which is one of major photolesion products in DNA, by utilizing UV light. In this work, we have designed and synthesized Zn2+ -1,4,7,10-tetraazacyclododecane complexes bearing a lumiflavin and L: -tryptophan (ZnL3) or L: -phenylalanine (ZnL4) as artificial DNA photolyases. We have found that (ZnL3)red, whose flavin unit was reduced in situ by Na2S2O4, accelerates the photoreversion of T[c,s]T utilizing near-UV light in aqueous solution at pH 7.6 and 11. Interestingly, more efficient photoreversion of T[c,s]T was achieved by UV irradiation of an oxidized form of ZnL3 [(ZnL3)ox] in the presence of an excess amount of Et3N at pH 11. UV-vis and fluorescence measurements and action spectra showed that an oxidized form of flavin of (ZnL3)ox was photoreduced by Et3N into its reduced form (ZnL3)red, which promoted the photoreduction of T[c,s]T. Comparison of the photochemical properties of ZnL3 with those of ZnL4 suggested that a tryptophan unit in ZnL3 contributed to the stabilities of the flavin through intramolecular photoinduced electron transfer.
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PMID:Efficient cycloreversion of cis,syn-thymine photodimer by a Zn2+ -1,4,7,10-tetraazacyclododecane complex bearing a lumiflavin and tryptophan by chemical reduction and photoreduction of a lumiflavin unit. 1694 31

DNA photolyases repair UV-induced cyclobutane pyrimidine dimers in DNA by photoinduced electron transfer. The redox-active cofactor is FAD in its doubly reduced state FADH-. Typically, during enzyme purification, the flavin is oxidized to its singly reduced semiquinone state FADH degrees . The catalytically potent state FADH- can be reestablished by so-called photoactivation. Upon photoexcitation, the FADH degrees is reduced by an intrinsic amino acid, the tryptophan W306 in Escherichia coli photolyase, which is 15 A distant. Initially, it has been believed that the electron passes directly from W306 to excited FADH degrees , in line with a report that replacement of W306 with redox-inactive phenylalanine (W306F mutant) suppressed the electron transfer to the flavin [Li, Y. F., et al. (1991) Biochemistry 30, 6322-6329]. Later it was realized that two more tryptophans (W382 and W359) are located between the flavin and W306; they may mediate the electron transfer from W306 to the flavin either by the superexchange mechanism (where they would enhance the electronic coupling between the flavin and W306 without being oxidized at any time) or as real redox intermediates in a three-step electron hopping process (FADH degrees * <-- W382 <-- W359 <-- W306). Here we reinvestigate the W306F mutant photolyase by transient absorption spectroscopy. We demonstrate that electron transfer does occur upon excitation of FADH degrees and leads to the formation of FADH- and a deprotonated tryptophanyl radical, most likely W359 degrees. These photoproducts are formed in less than 10 ns and recombine to the dark state in approximately 1 micros. These results support the electron hopping mechanism.
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PMID:Observation of an intermediate tryptophanyl radical in W306F mutant DNA photolyase from Escherichia coli supports electron hopping along the triple tryptophan chain. 1769 63


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