Direct evidence for singlet-singlet energy transfer in Escherichia coli DNA photolyase.

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)     

Effect of 5-deazaflavin on energy transduction during catalysis by Escherichia coli DNA photolyase.

DNA photolyase from Escherichia coli contains 1,5-dihydroFAD (FADH2) plus 5,10-methenyltetrahydropteroylpolyglutamate. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 (EdFADH2) matched its absorption spectrum after correction for the presence of a small amount of inactive 5-deazaFADox. The quantum yield for dimer repair with EdFADH2 (phi EdFADH2 = 0.110) was 6-fold lower than that observed with apoenzyme reconstituted with FADH2. Excited-state redox potential calculations indicate that 5-deazaFADH2 singlet is a better one-electron donor (E = -3.5 V) than FADH2 singlet (E = -2.7 V). Other studies indicate that the quantum yield for electron transfer from reduced flavin singlet to pyrimidine dimer (0.88) is unaffected when FADH2 is replaced by 5-deazaFADH2. Enhanced back electron transfer from pyrimidine dimer radical to flavin radical may account for the decreased quantum yield observed with EdFADH2 since, in the ground state, 5-deazaFADH. is a better oxidant than FADH.. The action spectrum observed for apoenzyme reconstituted with 5-deazaFADH2 plus 5,10-CH(+)-H4folate (EPtedFADH2) matched the absorption spectrum determined for enzyme-bound 5-deazaFADH2, indicating that the pterin chromophore was inactive as a sensitizer. This differs from results obtained with native enzyme, where pterin acts as a sensitizer via efficient singlet-singlet energy transfer to FADH2. The quantum yield for dimer repair by 5-deazaFADH2 bound to EPtedFADH2 (phi EPtedFADH2 = 0.0318) was 28.9% of that observed for EdFADH2. Spectroscopic studies indicate that singlet-singlet energy transfer in EPtedFADH2 is very efficient but only occurs in the "wrong" direction, i.e., from excited 5-deazaFADH2 to pterin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Absolute action spectrum of E-FADH2 and E-FADH2-MTHF forms of Escherichia coli DNA photolyase

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.     

Chromophore function and interaction in Escherichia coli DNA photolyase: reconstitution of the apoenzyme with pterin and/or flavin derivatives

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 bleaching and FADH2 oxidation are inhibited by substrate.     

Effect of flavin structure and redox state on catalysis by and flavin-pterin energy transfer in Escherichia coli DNA photolyase

5-DeazaFAD bound to a hydrophobic site in apophotolyase and formed a stable reconstituted enzyme, similar to that observed with FAD. Although stoichiometric incorporation was observed, the flavin ring modification in 1-deazaFAD interfered with normal binding, decreased protein stability, and prevented formation of a stable flavin radical, unlike that observed with FAD. The results suggest that an important hydrogen bond is formed between the protein and N (1) in FAD, but not N (5), and that there is sufficient space at the normal flavin binding site near N (5) to accommodate an additional hydrogen but not near N (1). Catalytic activity was observed with enzyme containing 5-deazaFADH2 (42% of native enzyme) or 1-deazaFADH2 (11% of native enzyme) as its only chromophore, but no activity was observed with the corresponding oxidized flavins, similar to that observed with FAD and consistent with a mechanism where dimer cleavage is initiated by electron donation from excited reduced flavin to substrate. The protein environment in photolyase selectively enhanced photochemical reactivity in the fully reduced state, as evidenced by comparison with results obtained in model studies with the corresponding free flavins. Phosphorescence was observed with free or photolyase-bound 5-deazaFADH2, providing the first example of a flavin that exhibits phosphorescence in the fully reduced state. Formation of an enzyme-substrate complex resulted in a nearly identical extent of quenching of 5-deazaFADH2 phosphorescence (85.1%) and fluorescence (87.5%). The data are consistent with a mechanism involving exclusive reaction of substrate with the excited singlet state of 5-deazaFADH2, analogous to that proposed for FADH2 in native enzyme. Direct evidence for singlet-singlet energy transfer from enzyme-bound 5-deazaFADH2 to 5,10-CH(+)-H4folate was provided by the fact that pterin fluorescence was observed upon excitation of 5-deazaFADH2, accompanied by a decrease in 5-deazaFADH2 fluorescence. On the other hand, the fluorescence of enzyme-bound pterin was quenched by 5-deazaFADox, consistent with energy transfer from pterin to 5-deazaFADox. In each case, the spectral properties of the chromophores were consistent with the observed direction of energy transfer and indicated that transfer in the opposite direction was energetically unlikely. Unlike 5-deazaFAD, energy transfer from pterin to FAD is energetically feasible with FADH2 or FADox. The results indicate that the direction of flavin-pterin energy transfer at the active site of photolyase can be manipulated by changes in the flavin ring or redox state which alter the energy level of the flavin singlet.     

DNA photorepair: chromophore composition and function in two classes of DNA photolyases

DNA photolyase catalyzes the repair of pyrimidine dimers in UV-damaged DNA in a reaction which requires visible light. Class I photolyases (Escherichia coli, yeast) contain 1,5-dihydroFAD (FADH2) plus a pterin derivative (5,10-methenyltetrahydropteroylpolyglutamate). In class II photolyases (Streptomyces griseus, Scenedesmus acutus, Anacystis nidulans, Methanobacterium thermoautotrophicum) the pterin chromophore is replaced by an 8-hydroxy-5-deazaflavin derivative. The two classes of enzymes exhibit a high degree of amino acid sequence homology, suggesting similarities in protein structure. Action spectra studies show that both chromophores in each enzyme tested act as sensitizers in catalysis. Studies with E. coli photolyase show that the pterin chromophore is not required when FADH2 acts as the sensitizer but that FADH2 is required when the pterin chromophore acts as sensitizer. FADH2 is probably the chromophore that directly interacts with substrate in a reaction which may be initiated by electron transfer from the excited singlet state (1FADH2*) to form a flavin radical plus an unstable pyrimidine dimer radical. Pterin, the major chromophore in E. coli photolyase, may act as an antenna to harvest light energy which is then transferred to FADH2.     

Identification of the folate binding sites on the Escherichia coli T-protein of the glycine cleavage system.

T-protein is a component of the glycine cleavage system and catalyzes the tetrahydrofolate-dependent reaction. To determine the folate-binding site on the enzyme, 14C-labeled methylenetetrahydropteroyltetraglutamate (5,10-CH2-H4PteGlu4) was enzymatically synthesized from methylenetetrahydrofolate (5, 10-CH2-H4folate) and [U-14C]glutamic acid and subjected to cross-linking with the recombinant Escherichia coli T-protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a zero-length cross-linker between amino and carboxyl groups. The cross-linked product was digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase high performance liquid chromatography. Amino acid sequencing of the labeled peptides revealed that three lysine residues at positions 78, 81, and 352 were involved in the cross-linking with polyglutamate moiety of 5, 10-CH2-H4PteGlu4. The comparable experiment with 5,10-CH2-H4folate revealed that Lys-81 and Lys-352 were also involved in cross-linking with the monoglutamate form. Mutants with single or multiple replacement(s) of these lysine residues to glutamic acid were constructed by site-directed mutagenesis and subjected to kinetic analysis. The single mutation of Lys-352 caused similar increase (2-fold) in Km values for both folate substrates, but that of Lys-81 affected greatly the Km value for 5,10-CH2-H4PteGlu4 rather than for 5,10-CH2-H4folate. It is postulated that Lys-352 may serve as the primary binding site to alpha-carboxyl group of the first glutamate residue nearest the p-aminobenzoic acid ring of 5,10-CH2-H4folate and 5,10-CH2-H4PteGlu4, whereas Lys-81 may play a key role to hold the second glutamate residue through binding to alpha-carboxyl group of the second glutamate residue.     

Reconstitution of Escherichia coli DNA photolyase with various folate derivatives

DNA photolyase from Escherichia coli contains both flavin and pterin. However, the isolated enzyme is depleted with respect to the pterin chromophore (0.5 mol of pterin/mol of flavin). The extinction coefficient of the pterin chromophore at 360 nm is underestimated by a method used in earlier studies which assumes stoichiometric amounts of pterin and flavin. The extinction coefficient of the pterin chromophore, determined on the basis of its (p-aminobenzoyl)polyglutamate content (epsilon 360 = 25.7 x 10(3) M-1 cm-1), is in good agreement with that expected for a 5,10-methenyltetrahydrofolate derivative. Also consistent with this structure, the pterin chromophore could be reversibly hydrolyzed to yield a 10-formyltetrahydrofolate derivative or reduced to yield a 5-methyltetrahydrofolate derivative. The isolated enzyme could be reconstituted with various folate derivatives to yield enzyme that contained equimolar amounts of pterin and flavin. Similar results were obtained in reconstitution studies with the natural pterin chromophore, with 5,10-methenyltetrahydrofolate, and with 10-formyltetrahydrofolate. The results show that the polyglutamate moiety, previously identified in the natural chromophore, is not critical for binding. Reconstitution with the natural pterin chromophore did not affect catalytic activity. The latter is consistent with our previous studies which show that, although the pterin chromophore acts as a sensitizer in native enzyme, it is not essential for dimer repair which can occur at the same rate under saturating light with flavin (1,5-dihydro-FAD) as the only chromophore.     

Determination of rates and yields of interchromophore (folate----flavin) energy transfer and intermolecular (flavin----DNA) electron transfer in Escherichia coli photolyase by time-resolved fluorescence and absorption spectroscopy

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 F?rster 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.     

Binding of (6R,S)-methyltetrahydrofolate to methyltransferase from Clostridium thermoaceticum: role of protonation of methyltetrahydrofolate in the mechanism of methyl transfer

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoacetium catalyzes transfer of the N5-methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate) to the cob(I)amide center of a corrinoid/iron-sulfur protein (CFeSP), forming H4folate and methylcob(III)amide. We have investigated binding of 13C-enriched (6R,S)-CH3-H4folate and (6R)-CH3-H4folate to MeTr by 13C NMR, equilibrium dialysis, fluorescence quenching, and proton uptake experiments. The results described here and in the accompanying paper [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5728-5735] constitute the first evidence for protonation of the pterin ring of CH3-H4folate. The pH dependence of the chemical shift in the 13C NMR spectrum for the N5-methyl resonance indicates that MeTr decreases the acidity of the N5 tertiary amine of CH3-H4folate by 1 pK unit in both water and deuterium oxide. Binding of (6R,S)-CH3H4folate is accompanied by the uptake of one proton. These results are consistent with a mechanism of activation of CH3-H4folate by protonation to make the methyl group more electrophilic and the product H4folate a better leaving group toward nucleophilic attack by cob(I)amide. When MeTr is present in excess over (6R,S)-13CH3-H4folate, the 13C NMR signal is split into two broad signals that reflect the bound states of the two diastereomers. This unexpected ability of MeTr to bind both isomers was confirmed by the observation of MeTr-bound (6R)-13CH3-H4folate by NMR and by the measurement of similar dissociation constants for (6R)- and (6S)-CH3-H4folate diastereomers by fluorescence quenching experiments. The transversal relaxation time (T2) of 13CH3-H4folate bound to MeTr is pH independent between pH 5.50 and 7.0, indicating that neither changes in the protonation state of bound CH3-H4folate nor the previously observed pH-dependent MeTr conformational change contribute to broadening of the 13C resonance signal. The dissociation constant for (6R,S)-CH3-H4folate is also pH independent, indicating that the role of the pH-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H4folate.     

Structures of NADH and CH3-H4folate complexes of Escherichia coli methylenetetrahydrofolate reductase reveal a spartan strategy for a ping-pong reaction

Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a (betaalpha)(8) barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH(3)-H(4)folate have now been determined at resolutions of 1.95 and 1.85 A, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH(3)-H(4)folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops beta2-alpha2 (L2), beta3-alpha3 (L3), and beta4-alpha4 (L4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformation that allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open" conformation to allow NADH to bind.     

Folate metabolism in the rat liver during regeneration after partial hepatectomy

1. Folate metabolism was studied during the early phases of liver regeneration after partial hepatectomy in rats accustomed to eating during the first 8h of a daily 12h dark period. 2. The content of 5-CH(3)-H(4)folate was drastically decreased during the first hours of regeneration. 3. The total HCO-H(4)folate coenzymes showed a constant decrease during the first 3 days of regeneration, and a continuous interconversion between 5-HCO-H(4)folate and 10-HCO-H(4)folate. 4. 10-HCO-H(4)folate synthetase, serine hydroxymethyl-transferase and 5,10-CH(2)-H(4)folate dehydrogenase activities were relatively low during the first hours after the operation, and increased only several hours later. 5. The increase in enzyme activities showed a stepwise pattern, apparently due to an interaction between the regeneration process and the controlled feeding schedules.     

Evidence from chemical degradation studies for a covalent bond from 5-fluoro-2'-deoxyuridylate to N-5 of tetrahydrofolate in the ternary complex of thymidylate synthetase-5-fluoro-2'-deoxyuridylate-5,10-methylenetetrahydrofolate

In the ternary complex of thymidylate synthetase, 5-fluoro-2'-deoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (5,10-CH2H4folate), the 5-fluorouracil moiety is covalently bound to the enzyme by a sulfide linkage from C-6 and to either N-5 or N-10 of H4folate by a methylene bridge from C-5. In an effort to establish the site by which H4folate is attached to FdUMP, the ternary complex was subjected to reagents that cleave the C-9, N-10 bond of folate derivatives. The complex was stable to zinc dust in hydrochloric acid, a reagent that cleaves N-10-substituted but not N-5-substituted folates. The conditions of the Bratton-Marshall reaction, which involve the use of nitrous acid, were found to cleave N-5-substituted folates in yields ranging from 5 to 50%. Exposure of the double-labeled thymidylate synthetase-FdUMP-[2-14C,7,9,3',5'-3H]5,10-CH2H4folate complex to the Bratton-Marshall reaction resulted in 16% cleavage of the C-9, N-10 bond with release solely of p-aminobenzoylglutamate, whereas all of the carbon-14-labeled pterin residue remained covalently bound to the protein. These results demonstrate that in the ternary complex, the 5-fluorouracil residue is connected by a covalent bond to N-5 of H4folate.     

The folate cofactor of Escherichia coli DNA photolyase acts catalytically

Escherichia coli DNA photolyase catalyzes the light-driven (300-500 nm) repair of pyrimidine dimers formed between adjacent pyrimidine bases in DNA exposed to UV light (200-300 nm). The light-driven repair process is facilitated by two enzyme-bound cofactors, FADH2 and 5,10-methenyltetrahydrofolate. The function of the folate has been characterized in greater detail in this series of experiments. Investigations of the relative binding affinities of photolyase for the monoglutamate and polyglutamate forms of 5,10-methenyltetrahydrofolate show that the enzyme has a greater affinity for the naturally occurring polyglutamate forms of the folate and that the exogenously added monoglutamate derivative is less tightly associated with the protein. Multiple turnover experiments reveal that the folate remains bound to photolyase even after 10 turnovers of the enzyme. Examination of the rates of repair by photolyase containing stoichiometric folate in the presence or absence of free folate under multiple turnover conditions and at micromolar concentrations of enzyme also demonstrates that the folate acts catalytically. The stimulation of turnover by exogenous folate seen at low concentrations of photolyase is shown to be due to the lower affinity of photolyase for the monoglutamate derivative used in reconstitution procedures. These results demonstrate that the folate of E. coli DNA photolyase is a bona fide cofactor and does not decompose or dissociate during multiple turnovers of the enzyme.     

Mouse folylpoly-gamma-glutamate synthetase isoforms respond differently to feedback inhibition by folylpolyglutamate cofactors.

Folylpoly-gamma-glutamate synthetase (FPGS) is the enzyme responsible for metabolic trapping of reduced folate cofactors in cells for use in nucleotide and amino acid biosynthesis. There are two isoforms of FPGS expressed in mouse tissues, one is expressed in differentiated tissue, principally liver and kidney, and the other in all rapidly proliferating cell types. The present study sought the functional difference that would explain the evolution of two mouse FPGS species. Recombinant cytosolic mouse isozymes were compared with respect to steady state kinetics, chain length of polyglutamate derivatives formed, and end-product inhibition by the major reduced folylpentaglutamate cofactors. Both isoforms were equally effective in catalyzing the addition of a mole of glutamic acid to reduced folate monoglutamate substrates. Each isoform was also capable of forming long chain polyglutamate derivatives of the model folate, 5,10-dideazatetrahydrofolate. In contrast, the FPGS isoform derived from rapidly proliferating tissue was much more sensitive to inhibition by (6R)-5,10-CH(2)-H(4)PteGlu(5) and (6S)-H(4)PteGlu(5) than the isoform expressed in differentiated tissues, as demonstrated by 13- and 6-fold lower inhibition constants (K(i)), respectively. Interestingly, each isozyme was equally sensitive to inhibition by (6R)-10-CHO-H(4)PteGlu(5). We drew the conclusion that the decreased sensitivity of the FPGS expressed in mouse liver and kidney to feedback inhibition by 5,10-CH(2)-H(4)PteGlu(5-6) and H(4)PteGlu(5-6) may have evolved to permit accumulation of a larger folate cofactor pool than that found within rapidly proliferating tissue.     

Action mechanism of Escherichia coli DNA photolyase. III. Photolysis of the enzyme-substrate complex and the absolute action spectrum

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.+     

Roles of FAD and 8-hydroxy-5-deazaflavin chromophores in photoreactivation by Anacystis nidulans DNA photolyase.

DNA photolyase from the cyanobacterium Anacystis nidulans contains two chromophores, flavin adenine dinucleotide (FADH2) and 8-hydroxy-5-deazaflavin (8-HDF) (Eker, A. P. M., Kooiman, P., Hessels, J. K. C., and Yasui, A. (1990) J. Biol. Chem. 265, 8009-8015). While evidence exists that the flavin chromophore (in FADH2 form) can catalyze photorepair directly and that the 8-HDF chromophore is the major photosensitizer in photoreactivation it was not known whether 8-HDF splits pyrimidine dimer directly or indirectly through energy transfer to FADH2 at the catalytic center. We constructed a plasmid which over-produces the A. nidulans photolyase in Escherichia coli and purified the enzyme from this organism. Apoenzyme was prepared and enzyme containing stoichiometric amounts of either or both chromophores was reconstituted. The substrate binding and catalytic activities of the apoenzyme (apoE), E-FADH2, E-8-HDF, E-FAD(ox)-8-HDF, and E-FADH2-8-HDF were investigated. We found that FAD is required for substrate binding and catalysis and that 8-HDF is not essential for binding DNA, and participates in catalysis only through energy transfer to FADH2. The quantum yields of energy transfer from 8-HDF to FADH2 and of electron transfer from FADH2 to thymine dimer are near unity.     

Methylenetetrahydrofolate reductase from Escherichia coli: elucidation of the kinetic mechanism by steady-state and rapid-reaction studies

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.     

Mechanism of transfer of the methyl group from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein catalyzed by the methyltransferase from Clostridium thermoaceticum: a key step in the Wood-Ljungdahl pathway of acetyl-CoA synthesis

The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate. This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood-Ljungdahl or reductive acetyl-CoA pathway. The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction. These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods [Seravalli, J., Shoemaker, R. K., Sudbeck, M. J., and Ragsdale, S. W. (1999) Biochemistry 38, 5736-5745]. Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting. CH3-H4folate and the CFeSP [in the cob(I)amide state] bind randomly and independently to form a ternary complex. Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation. This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pKa at N5 of CH3-H4folate. Next, the Co(I)-CFeSP attacks the methyl group in a rate-limiting SN2 reaction to form methylcob(III)amide. Finally, the products randomly dissociate. The following steady-state constants were obtained: kcat = 14.7 +/- 1.7 s-1, Km of the CFeSP = 12 +/- 4 microM, and Km of (6S)-CH3-H4folate = 2.0 +/- 0.3 microM. We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.