flavin-adenine-dinucleotide and 5-10-methylenetetrahydrofolic-acid

This brief review gives an overview of the gene family of photolyases and cryptochromes, followed by a description of the main features of the three-dimensional structures of photolyases known to date. It then discusses recent biophysical studies of photolyase function, and modelling studies on the interaction between the enzyme and its substrate.

Topics: Animals; Arabidopsis; Arabidopsis Proteins; Bacterial Proteins; Cryptochromes; Cyanobacteria; Deoxyribodipyrimidine Photo-Lyase; DNA Damage; DNA Repair; Drosophila melanogaster; Drosophila Proteins; Escherichia coli; Eye Proteins; Flavin-Adenine Dinucleotide; Flavoproteins; Insect Proteins; Macromolecular Substances; Models, Molecular; Multigene Family; Photoreceptor Cells, Invertebrate; Plant Proteins; Protein Conformation; Protein Structure, Tertiary; Pyrimidine Dimers; Receptors, G-Protein-Coupled; Structure-Activity Relationship; Substrate Specificity; Tetrahydrofolates

The synthesis of thymine for DNA is catalyzed by the enzyme thymidylate synthase (TS). A family of flavin-dependent TSs encoded by the thyX gene has been discovered recently. These newly discovered TSs require a reducing substrate in addition to 2'-deoxyuridine monophosphate (dUMP) and 5,10-methylenetetrahydrofolate (CH2THF), suggesting that the enzyme-bound flavin is a redox intermediary in catalysis. The oxidation of the reduced flavin of the TS from Campylobacter jejuni has been observed directly upon mixing with dUMP and CH2THF under anaerobic conditions, thus providing the first direct demonstration of its redox role in catalysis. Product analysis showed that the one mole of 2'-deoxythymidine monophosphate is formed along with one mole of tetrahydrofolate for each mole of reduced enzyme-bound flavin. The classic TS inactivator 5-fluoro-2'-deoxyuridine monophosphate (FdUMP) was able to bind to the reduced enzyme but was unable to oxidize the flavin, even in the presence of CH2THF. Furthermore, the nucleotide binding site of the enzyme treated with FdUMP and CH2THF was irreversibly blocked, suggesting the formation of a stable substrate adduct analogous to that formed by the well-studied thyA-encoded TS. The formation of inactivated enzyme without flavin oxidation indicates that methylene transfer from the folate to the nucleotide occurs prior to flavin redox chemistry.

Topics: Campylobacter jejuni; Deoxyuracil Nucleotides; Flavin-Adenine Dinucleotide; Oxidation-Reduction; Spectrophotometry; Tetrahydrofolates; Thymidine Monophosphate; Thymidylate Synthase

The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using NADH as the source of reducing equivalents. The enzyme also catalyzes the transfer of reducing equivalents from NADH or CH(3)-H(4)folate to menadione, an artificial electron acceptor. Here, we have determined the midpoint potential of the enzyme-bound flavin to be -237 mV. We have examined the individual reductive and oxidative half-reactions constituting the enzyme's activities. In an anaerobic stopped-flow spectrophotometer, we have measured the rate constants of flavin reduction and oxidation occurring in each half-reaction and have compared these with the observed catalytic turnover numbers measured under steady-state conditions. We have shown that, in all cases, the half-reactions proceed at rates sufficiently fast to account for overall turnover, establishing that the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by CH(2)-H(4)folate is substantially rate limiting in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH(3)-H(4)folate in the CH(3)-H(4)folate-menadione oxidoreductase reaction. We conclude that studies of individual half-reactions catalyzed by E. coli MTHFR may be used to probe mechanistic questions relevant to the overall oxidoreductase reactions.

Topics: Catalysis; Escherichia coli; Flavin-Adenine Dinucleotide; Kinetics; Methylenetetrahydrofolate Reductase (NADPH2); Models, Chemical; NAD; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Oxidoreductases Acting on CH-NH Group Donors; Spectrophotometry; Tetrahydrofolates; Vitamin K

Methylenetetrahydrofolate reductase catalyzes the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate. Because this substrate is unstable and dissociates spontaneously into formaldehyde and tetrahydrofolate, the customary method to assay the catalytic activity of this enzyme has been to measure the oxidation of [14C]N(5)-methyltetrahydrofolate to N(5), N(10)-methylenetetrahydrofolate and quantify the [14C]formaldehyde that dissociates from this product. This report describes a very sensitive radioenzymatic assay that measures directly the reductive catalysis of N(5),N(10)-methylenetetrahydrofolate. The radio-labeled substrate, [14C]N(5),N(10)-methylenetetrahydrofolate, is prepared by condensation of [C(14)]formaldehyde with tetrahydrofolate and the stability of this substrate is maintained for several months by storage at -80 degrees C in a pH 9.5 buffer. Partially purified methylenetetrahydrofolate reductase from rat liver, incubated with the radio-labeled substrate and the cofactors, NADPH and FAD at pH 7. 5, generates [14C]N(5)-methyltetrahydrofolate, which is stable and partitions into the aqueous phase after the assay is terminated with dimedone and toluene. A K(m) value of 8.2 microM was obtained under conditions of increasing substrate concentration to ensure saturation kinetics. This method is simple, very sensitive and measures directly the reduction of N(5), N(10)-methylenetetrahydrofolate to N(5)-methyltetrahydrofolate, which is the physiologic catalytic pathway for methylenetetrahydrofolate reductase.

Topics: Animals; Carbon Radioisotopes; Catalysis; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Flavin-Adenine Dinucleotide; Humans; Kinetics; Liver; Methylenetetrahydrofolate Reductase (NADPH2); NADP; Oxidation-Reduction; Oxidoreductases Acting on CH-NH Group Donors; Rats; S-Adenosylhomocysteine; S-Adenosylmethionine; Sensitivity and Specificity; Tetrahydrofolates; Tumor Cells, Cultured

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.

Topics: Apoenzymes; Deoxyribodipyrimidine Photo-Lyase; Escherichia coli; Flavin-Adenine Dinucleotide; Folic Acid; Stereoisomerism; Substrate Specificity; Tetrahydrofolates

Escherichia coli DNA photolyase, which photorepairs cyclobutane pyrimidine dimers, contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and 5,10-methenyltetrahydrofolate (MTHF). Previous work has shown that MTHF is the primary photoreceptor which transfers energy to the FADH2 cofactor; the FADH2 singlet excited state then repairs the photodimer by electron transfer. In this study, we have determined the rate constants for these photophysical processes by time-resolved fluorescence and absorption spectroscopy. From time-resolved fluorescence, we find that energy transfer from MTHF to FADH2 and FADH degrees occurs at rates of 4.6 x 10(9) and 3.0 x 10(10) s-1, respectively, and electron transfer from FADH2 to a pyrimidine dimer occurs at a rate of 5.5 x 10(9) s-1. Using Förster theory for long-range energy transfer and assuming K2 = 2/3, the interchromophore distances were estimated to be 22 A in the case of the MTHF-FADH2 pair and 21 A for the MTHF-FADH degrees pair. Picosecond absorption spectroscopy identified an MTHF single state which decays to yield the first excited singlet state of FADH2. The lifetimes of MTHF and FADH2 singlets and the rates of interchromophore energy transfer, as well as the rate of electron transfer from FADH2 to DNA measured by time-resolved fluorescence, were in excellent agreement with the values obtained by picosecond laser flash photolysis. Similarly, fluorescence or absorption lifetime studies of the folate-depleted enzyme with and without photodimer suggest that FADH2, in its singlet excited state, transfers an electron to the dimer with 89% efficiency. The distance between FADH2 and the photodimer was calculated to be ca. 14 A.

Topics: Apoenzymes; Deoxyribodipyrimidine Photo-Lyase; Electron Transport; Energy Transfer; Escherichia coli; Flavin-Adenine Dinucleotide; Flavins; Folic Acid; Kinetics; Mathematics; Models, Theoretical; Spectrometry, Fluorescence; Tetrahydrofolates

The methyl carbon of ribothymidine in Loop IV of the tRNA of Streptococcus faecalis, Bacillus subtilis, and some other microorganisms is derived directly from 5,10-methylenetetrahydrofolate, not S-adenosylmethionine. The pure enzyme from S. faecalis also requires FADH2. We have obtained evidence that tetrahydrofolate is a product of the reaction and demonstrated that label from [5-3H]5-deazaFMNH2 is incorporated into the methyl moiety of ribothymidine. These data indicate that the enzyme uses methylenetetrahydrofolate solely as a 1-carbon donor and employs FADH2 as a reducing agent in vitro according to the following reaction: tRNA(U psi C) + CH2 = THF + FADH2 leads to tRNA(T psi C) + THF + FAD.

Topics: Enterococcus faecalis; Flavin-Adenine Dinucleotide; Methylation; Methyltransferases; Oxidation-Reduction; Ribonucleosides; RNA, Transfer; Species Specificity; Tetrahydrofolates; Uridine

Topics: Alkylation; Amino Acids; Anemia; Anemia, Pernicious; Biochemical Phenomena; Biochemistry; Chemistry Techniques, Analytical; Chromatography; Enzyme Inhibitors; Enzyme Repression; Escherichia coli; Feedback; Flavin-Adenine Dinucleotide; Folic Acid; Methionine; Methylenetetrahydrofolate Reductase (NADPH2); Mutation; Oxidoreductases; Research; Spectrophotometry; Tetrahydrofolates; Vitamin B 12