Isolation, characterization, and structural organization of 10-formyltetrahydrofolate synthetase from spinach leaves

One-carbon metabolism mediated by folate coenzymes plays an essential role in several major cellular processes. In the prokaryotes studied, three folate-dependent enzymes, 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) generally exist as monofunctional or bifunctional proteins, whereas in eukaryotes the three activities are present on one polypeptide. The structural organization of these enzymes in plants had not previously been examined. We have purified the 10-formyltetrahydrofolate synthetase activity from spinach leaves to homogeneity and raised antibodies to it. The protein was a dimer with a subunit molecular weight of Mr = 67,000. The Km values for the three substrates, (6R)-tetrahydrofolate, ATP, and formate were 0.94, 0.043, and 21.9 mM, respectively. The enzyme required both monovalent and divalent cations for maximum activity. The 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase activities of spinach coeluted separately from the 10-formyltetrahydrofolate synthetase activity on a Matrex Green-A column. On the same column, the activities of the yeast trifunctional C1-tetrahydrofolate synthase coeluted. In addition, antibodies raised to the purified spinach protein immunoinactivated and immunoprecipitated only the 10-formyltetrahydrofolate synthetase activity in a crude extract of spinach leaves. These results suggest that unlike the trifunctional form of C1-tetrahydrofolate synthase in the other eukaryotes examined, 10-formyltetrahydrofolate synthetase in spinach leaves is monofunctional and 5,10-methyl-enetetrahydrofolate dehydrogenase and 5,10-methenyltetrahydrofolate cyclohydrolase appear to be bifunctional. Although structurally dissimilar to the other eukaryotic trifunctional enzymes, the 35 amino-terminal residues of spinach 10-formyltetrahydrofolate synthetase showed 35% identity with six other tetrahydrofolate synthetases.     

Cloning, expression, and purification of 5,10-methenyltetrahydrofolate synthetase from Mus musculus

Folate metabolism is necessary for the biosyntheses of purine nucleotides and thymidylate and for the synthesis of S-adenosylmethionine, a cofactor required for cellular methylation reactions and a precursor of spermidine and spermine syntheses. Disruption of folate metabolism is associated with several pathologies and developmental anomalies including cancer and neural tube defects. The enzyme 5,10-methenyltetrahydrofolate synthetase (MTHFS, EC 6.3.3.2) catalyzes the ATP-dependent conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate, and has been shown to affect intracellular folate concentrations by accelerating folate degradation. Mammalian MTHFS proteins described to date are not stable and no recombinant mammalian MTHFS protein has been successfully expressed in Escherichia coli. The three-dimensional structure of MTHFS has not been solved. The cDNA coding for Mus musculus MTHFS was isolated and expressed in E. coli with a hexa-histidine tag. Milligram quantities of recombinant mouse MTHFS were purified using metal affinity chromatography and the protein was stabilized with Tween 20. Mouse MTHFS has a molecular mass of 23 kDa and is 84% identical in amino acid sequence to the human enzyme. Activity assays confirmed the functionality of the recombinant protein, with K_m=5 μM for (6S)-5-formyltetrahydrofolate and K_m=769 μM for Mg–ATP. This is the first example of a mammalian form of MTHFS expressed in E. coli that yielded sufficient quantities of stable purified protein to allow for detailed characterization of its three-dimensional structure and kinetic properties.     

Serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate

The combined activities of rabbit liver cytosolic serine hydroxymethyltransferase and C1-tetrahydrofolate synthase convert tetrahydrofolate and formate to 5-formyltetrahydrofolate. In this reaction C1-tetrahydrofolate synthase converts tetrahydrofolate and formate to 5,10-methenyltetrahydrofolate, which is hydrolyzed to 5-formyltetrahydrofolate by a serine hydroxymethyltransferase-glycine complex. Serine hydroxymethyltransferase, in the presence of glycine, catalyzes the conversion of chemically synthesized 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate with biphasic kinetics. There is a rapid burst of product that has a half-life of formation of 0.4 s followed by a slower phase with a completion time of about 1 h. The substrate for the burst phase of the reaction was shown not to be 5,10-methenyltetrahydrofolate but rather a one-carbon derivative of tetrahydrofolate which exists in the presence of 5,10-methenyltetrahydrofolate. This derivative is stable at pH 7 and is not an intermediate in the hydrolysis of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate by C1-tetrahydrofolate synthase. Cytosolic serine hydroxymethyltransferase catalyzes the hydrolysis of 5,10-methenyltetrahydrofolate pentaglutamate to 5-formyltetrahydrofolate pentaglutamate 15-fold faster than the hydrolysis of the monoglutamate derivative. The pentaglutamate derivative of 5-formyltetrahydrofolate binds tightly to serine hydroxymethyltransferase and dissociates slowly with a half-life of 16 s. Both rabbit liver mitochondrial and Escherichia coli serine hydroxymethyltransferase catalyze the conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at rates similar to those observed for the cytosolic enzyme. Evidence that this reaction accounts for the in vivo presence of 5-formyltetrahydrofolate is suggested by the observation that mutant strains of E. coli, which lack serine hydroxymethyltransferase activity, do not contain 5-formyltetrahydrofolate, but both these cells, containing an overproducing plasmid of serine hydroxymethyltransferase, and wild-type cells do have measurable amounts of this form of the coenzyme.     

Mammalian folyl polyglutamate synthetase: partial purification and properties of the mouse liver enzyme

Folyl polyglutamate synthetase has been partially purified from mouse liver, and the general features of this enzyme have been characterized. The purification procedure utilized fractionation with ammonium sulfate, gel filtration, and affinity chromatography on ATP-agarose and resulted in a 350-fold increase in specific activity with 8-20% recovery of enzyme activity. Enzyme could be stabilized by glycerol or by ATP, but stability was not appreciably enhanced by folate. The enzymatic reaction was completely dependent on folate, ATP, and Mg2+ while partial reaction rates were observed in the absence of KCl or beta-mercaptoethanol. Highest reaction rates were observed at pH 8.2-9.5 at 37 degrees C. Chromatography of purified enzyme on calibrated gel filtration columns suggested a molecular weight of 65 000. Mouse liver folyl polyglutamate synthetase coupled [3H]glutamic acid to all of the naturally occurring folates studied. Analysis of the reaction products by high-performance liquid chromatography demonstrated that several folyl oligoglutamates were formed at low substrate concentrations but that only folyl diglutamate was formed at substrate concentrations approaching saturation. Dihydrofolate, tetrahydrofolate, 5,10-methylenetetrahydrofolate, 10-formyltetrahydrofolate, and 5-formyltetrahydrofolate were the best substrates. Folic acid and 5-methyltetrahydrofolate were also substrates for this reaction, but much higher concentrations of these compounds were required to saturate the enzyme. These data suggest that all of the tetrahydrofolyl compounds (except 5-methyltetrahydrofolate) are the monoglutamyl substrates for polyglutamation in vivo and that 5-methyltetrahydrofolate is not likely to be a direct precursor for folate polyglutamates in mouse liver.     

Human red blood cell-mediated metabolism of leucovorin [(R,S)5-formyltetrahydrofolate

The ability of human blood in vitro, and partially purified red blood cells, to metabolize leucovorin, or 5-formyltetrahydrofolate, has been examined. A radioenzymatic assay based upon entrapment of 5,10-methylenetetrahydrofolate, and other reduced folates after cycling to this form, into a stable ternary complex with thymidylate synthase and tritiated 5-fluoro-2'-deoxyuridine-5'-monophosphate was used to estimate reduced folate metabolites. Incubation of whole blood samples with (R,S)5-formyltetrahydrofolate resulted in a time- and concentration-dependent extracellular accumulation of the reduced folates, 5-methyltetrahydrofolate, tetrahydrofolate, 10-formyltetrahydrofolate, and 5,10-methylenetetrahydrofolate. While accumulation with time was nonlinear, the tetrahydrofolate pool showed the greatest overall increase in concentration. 5-Methyltetrahydrofolate, which was the only reduced folate detected in plasma prior to introduction of (R,S)5-formyltetrahydrofolate, accumulated more slowly than tetrahydrofolate. 10-Formyltetrahydrofolate and 5,10-methylenetetrahydrofolate accumulated even more slowly but exhibited nonlinear kinetic patterns similar to those of tetrahydrofolate and 5-methyltetrahydrofolate. When blood cells were removed by centrifugation, a complete loss of metabolic activity was observed. Exposure of purified red blood cells to (R,S)5-formyltetrahydrofolate resulted in accumulation of extracellular reduced folates that was similar to that in whole blood samples while partially purified white blood cells exhibited little activity. Metabolism of the (S) diastereomer of 5-formyltetrahydrofolate accounted for essentially all of the observed extracellular accumulation of reduced folates. We propose that red blood cell-mediated metabolism of 5-formyltetrahydrofolate could, in part at least, account for reduced folate accumulation in plasma when leucovorin is administered to humans.     

Oxidation of 10-formyltetrahydrofolate to 10-formyldihydrofolate by complex IV of rat mitochondria

We hypothesized that the unanticipated bioactivity of orally administered unnatural carbon-6 isomers, (6R)-5-formyltetrahydrofolate (5-HCO-THF) and (6S)-5,10-methenyltetrahydrofolate (5,10-CH-THF), in humans [Baggott, J. E., and Tamura, T. (1999) Biochim. Biophys. Acta 1472, 323-32] is explained by the rapid oxidation of (6S)-10-formyltetrahydrofolate (10-HCO-THF), which is produced by in vivo chemical processes from the above folates. An oxidation of 10-HCO-THF produces 10-formyldihydrofolate (10-HCO-DHF), which no longer has the asymmetric center at carbon-6 and is metabolized by aminoimidazole carboxamide ribotide (AICAR) transformylase forming bioactive dihydrofolate. Since cytochrome c (Fe(3+)) rapidly oxidizes both (6R)- and (6S)-10-HCO-THF [Baggott et al. (2001) Biochem. J. 354, 115-22], we investigated the metabolism of 10-HCO-THF by isolated rat liver mitochondria. We found that 10-HCO-THF supported the respiration of mitochondria without uncoupling ATP synthesis. The site of electron donation was identified as complex IV, which contains cytochrome c; the folate product was 10-HCO-DHF, and the reaction was saturable with respect to 10-HCO-THF. Both (6S)- (unnatural) and (6R)-10-HCO-THF supported the respiration of mitochondria, whereas (6S)-5-formyltetrahydrofolate (5-HCO-THF) was inactive. To our knowledge, this cytochrome c oxidation of 10-HCO-THF to 10-HCO-DHF in the mitochondrial intermembrane space represents a possible folate metabolic pathway previously unidentified and would explain the bioactivity of unnatural carbon-6 isomers, (6R)-5-HCO-THF and (6S)-5,10-CH-THF, in humans.     

Hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate at pH 2.5 to 4.5

At pH 4.0 to 4.5, 5,10-methenyltetrahydrofolate is hydrolyzed to only 5-formyltetrahydrofolate if reducing agents are present or iron-redox cycling is suppressed. At pH 4.0, the equilibrium position for this hydrolysis is approximately equal concentrations of both folates. If no reducing agents are used or iron-redox cycling is promoted, considerable amounts of 10-formyldihydrofolate are also formed. It is likely that 10-formyldihydrofolate has been misidentified as 5,10-hydroxymethylenetetrahydrofolate, which was reported to accumulate during the hydrolysis of 5, 10-methenyltetrahydrofolate to 5-formyltetrahydrofolate [Stover, P. and Schirch, V. (1992) Biochemistry 31, 2148-2155 and 2155-2164; (1990) J. Biol. Chem. 265, 14227-14233]. Since 5, 10-hydroxymethylenetetrahydrofolate is reported to be the viable in vivo substrate for serine hydroxymethyltransferase-catalyzed formation of 5-formyltetrahydrofolate, and 5, 10-hydroxymethylenetetrahydrofolate probably does not accumulate, the above folate metabolism is now doubtful. It is hypothesized that mildly acidic subcellular organelles provide an environment for the hydrolysis of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate in vivo, and there is no requirement for enzyme catalysis. Finally, 10-formyltetrahydrofolate is susceptible to iron-catalyzed oxidation to 10-formyldihydrofolate at pH 4 to 4.5.     

Role of enzyme-bound 5,10-methenyltetrahydropteroylpolyglutamate in catalysis by Escherichia coli DNA photolyase

DNA photolyase catalyzes the photoreversal of pyrimidine dimers. The enzymes from Escherichia coli and yeast contain a flavin chromophore and a folate cofactor, 5,10-methenyltetrahydropteroylpolyglutamate. E. coli DNA photolyase contains about 0.3 mol of folate/mol flavin, whereas the yeast photolyase contains the full complement of folate. E. coli DNA photolyase is reconstituted to a full complement of the folate by addition of 5,10-methenyltetrahydrofolate to cell lysates or purified enzyme samples. The reconstituted enzyme displays a higher photolytic cross section under limiting light. Treatment of photolyase with sodium borohydride or repeated camera flashing results in the disappearance of the absorption band at 384 nm and is correlated with the formation of modified products from the enzyme-bound 5,10-methenyltetrahydrofolate. Photolyase modified in this manner has a decreased photolytic cross section under limiting light. Borohydride reduction results in the formation of 5,10-methylenetetrahydrofolate and 5-methyltetrahydrofolate, both of which are released from the enzyme. Repeated camera flashing results in photodecomposition of the enzyme-bound 5,10-methenyltetrahydrofolate and release of the decomposition products. Finally, it is observed that photolyase binds 10-formyltetrahydrofolate and appears to cyclize it to form the 5,10-methenyltetrahydrofolate chromophore.     

Human liver methenyltetrahydrofolate synthetase: improved purification and increased affinity for folate polyglutamate substrates

Methenyltetrahydrofolate synthetase (5-formyltetrahydrofolate cyclodehydrase (cyclo-ligase) (ADP-forming) EC 6.3.3.2) catalyzes the ATP- and Mg2+-dependent transformation of 5-formyltetrahydrofolate (leucovorin) to 5,10-methenyltetrahydrofolate. The enzyme has been purified 49,000-fold from human liver by a two-column procedure with Blue Sepharose followed by folinate-Sepharose chromatography. It appears as a single band both on SDS-polyacrylamide gel electrophoresis (Mr 27,000) and on isoelectric focusing (pI = 7.0) and is monomeric, with a molecular weight of 27,000 on gel filtration. Initial-velocity studies suggest that the enzyme catalyzes a sequential mechanism and at 30 degrees C and pH 6.0 the turnover number is 1000 min-1. The enzyme has a higher affinity for its pentaglutamate substrate (Km = 0.6 microM) than for the monoglutamate (Km = 2 microM). The antifolate methotrexate has no inhibitory effect at concentrations up to 350 microM, while methotrexate pentaglutamate is a competitive inhibitor with a Ki = 15 microM. Similarly, dihydrofolate monoglutamate is a weak inhibitor with a Ki = 50 microM, while the pentaglutamate is a potent competitive inhibitor with a Ki of 3.8 microM. Thus, dihydrofolate and methotrexate pentaglutamates could regulate enzyme activity and help explain why leucovorin fails to rescue cells from high concentrations of methotrexate.     

Investigations of the Roles of Arginine 115 and Lysine 120 in the Active Site of 5,10-Methenyltetrahydrofolate Synthetase from <Emphasis Type="Italic">Mycoplasma pneumoniae</Emphasis>

5,10-Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate coupled to the hydrolysis of ATP. A co-crystal structure of MTHFS bound to its substrates has been published (Chen et al., Proteins 56:839-843, 2005) that provides insights into the mechanism of this reaction. To further investigate this mechanism, we have replaced the arginine at position 115 and the lysine at position 120 with alanine (R115A and K120A, respectively). Circular dichroism spectra for both mutants are consistent with folded proteins. R115A shows no activity, suggesting that R115 plays a critical role in the activity of the enzyme. The K120A mutation increases the Michaelis constant (K(m)) for ATP from 76 to 1,200 muM and the K(m) for 5-formylTHF from 2.5 to 7.1 muM. The weaker binding of substrates by K120A may be due to movement of a loop consisting of residues 117 though 120, which makes several hydrogen bonds to ATP and may be held in position by K120.     

Isolation and sequencing of the cDNA coding for spinach 10-formyltetrahydrofolate synthetase. Comparisons with the yeast, mammalian, and bacterial proteins.

The one-carbon metabolism enzymes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) can be found on a single trifunctional protein in the eukaryotes examined. The one exception is in spinach leaves where 10-formyltetrahydrofolate synthetase is monofunctional (Nour, J. M., and Rabinowitz, J. C. (1991) J. Biol. Chem. 266, 18363-18369). In the prokaryotes examined, 10-formyltetrahydrofolate synthetase is either absent or is monofunctional. A cDNA clone encoding spinach leaf 10-formyltetrahydrofolate synthetase was isolated through the use of antibodies to the purified enzyme. This clone had an open reading frame of 1914 base pairs and encoded for a protein containing 636 amino acids with a calculated M(r) of 67,727. The percentage identity between spinach 10-formyltetrahydrofolate synthetase and the synthetase domains in the four trifunctional eukaryotic enzymes and the two monofunctional prokaryotic enzymes that have been cloned and sequenced was: 64.9% human, 63.8% rat, 55.6% yeast cytoplasm, 53.8% yeast mitochondria, 47.8% Clostridium acidi-urici, and 47.9% Clostridium thermoaceticum. Clearly the spinach monofunctional protein had greatest homology with the mammalian proteins. The spinach protein is longer than the two other monofunctional prokaryotic proteins. Possible reasons for this are presented. The codon usage and the putative translation initiation sites are examined and compared with other spinach proteins.     

C1-Tetrahydrofolate synthase from rabbit liver. Structural and kinetic properties of the enzyme and its two domains

C1-Tetrahydrofolate synthase is a multifunctional enzyme which catalyzes three reactions in 1-carbon metabolism: 10-formyltetrahydrofolate synthetase; 5,10-methenyltetrahydrofolate cyclohydrolase; 5,10-methylenetetrahydrofolate dehydrogenase. A rapid 1-day purification procedure has been developed which gives 40 mg of pure enzyme from 10 rabbit livers. The 10-formyltetrahydrofolate synthetase activity of this trifunctional enzyme has a specific activity that is 4-fold higher than the enzyme previously purified from rabbit liver. Conditions have been developed for the rapid isolation of a tryptic fragment of the enzyme which contains the methylenetetrahydrofolate dehydrogenase and methenyltetrahydrofolate cyclohydrolase activities. This fragment is a monomer exhibiting a subunit and native molecular weight of 36,000 in most buffers. However, in phosphate buffers the native molecular weight suggests that the fragment is a dimer. Conditions are also given whereby chymotryptic digestion allows the simultaneous isolation from the native enzyme of a large fragment containing the 10-formyltetrahydrofolate synthetase activity and a smaller fragment containing the dehydrogenase and cyclohydrolase activities. The large fragment is a dimer with a subunit molecular weight of 66,000. The small fragment retains all of the dehydrogenase and cyclohydrolase activities of the native enzyme. The large fragment is unstable but retains most of the 10-formyltetrahydrofolate synthetase activity. Km values of substrates for the two fragments are the same as the values for the native enzyme. The 10-formyltetrahydrofolate synthetase activity of the native enzyme requires ammonium or potassium ions for expression of full catalytic activity. The effect of these two ions on the catalytic activity of the large chymotryptic fragment is the same as with the native enzyme. We have shown by differential scanning calorimetry that the native enzyme contains two protein domains which show thermal transitions at 47 and 60 degrees C. Evidence is presented that the two domains are related to the two protein fragments generated by proteolysis of the native enzyme. The larger of the two domains contains the active site for the 10-formyltetrahydrofolate synthetase activity while the smaller domain contains the active site which catalyzes the dehydrogenase and cyclohydrolase reactions. Replacement of sodium ion buffers with either ammonium or potassium ions results in an increase in stability of the large domain of the native enzyme. This change in stability is not accompanied by a change in the quaternary structure of the enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bioactivity of orally administered unnatural isomers, [6R]-5-formyltetrahydrofolate and [6S]-5,10-methenyltetrahydrofolate, in humans

It has been assumed that humans cannot utilize 5,6,7,8-tetrahydrofolates with the unnatural configuration at carbon 6, since these folates are enzymatically and microbiologically inactive. We hypothesized that orally administered unnatural [6R]-5-formyltetrahydrofolate or [6S]-5,10-methenyltetrahydrofolate is bioactive in humans. Subjects were given independent oral doses of these unnatural folates and of a natural [6S]-5-formyltetrahydrofolate. Plasma, before and after the dose for 4 h, and 2 h urine were collected. Areas under the curve for the change in plasma folate concentrations were measured microbiologically and urinary folates were measured using HPLC. Based on findings of plasma and urinary folates, the unnatural folates were estimated to be 14-50% active as compared to [6S]-5-formyltetrahydrofolate. The major plasma and urinary folate was [6S]-5-methyltetrahydrofolate in all experiments. In urine, a [6S]-5-formyltetrahydrofolate peak was observed only after a [6S]-5-HCO-H4folate dose and peaks of unnatural [6S]-10-formyltetrahydrofolate and 5-formyltetrahydrofolate were identified after a [6R]-5-formyltetrahydrofolate dose. A possible pathway that explains our findings is discussed. This pathway includes the oxidation of the unnatural [6S]-10-formyltetrahydrofolate to 10-formyl-7,8-dihydrofolate which can be further metabolized by 5-amino-4-imidazolecarboxamide-ribotide transformylase producing dihydrofolate. Dihydrofolate can then be metabolized to [6S]-5-methyltetrahydrofolate by well-established metabolism.     

Formyl-methenyl-methylenetetrahydrofolate synthetase from rabbit liver (combined). Evidence for a single site in the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate.

The rabbit liver enzymes 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase, and 10-formyltetrahydrofolate synthetase have been purified to apparent homogeneity. Polyacrylamide gel electrophoresis patterns suggest a single protein is responsible for all three catalytic activities. The properties of the dehydrogenase and cyclohydrolase activities suggest that a single active site may catalyze these two reactions. This conclusion is based on spectral changes observed in the conversion of 5,10-methylenetetrahydrofolate to 10-formyltetrahydrofolate, the similarity of dissociation constants determined from initial velocity studies for the two reactions, and the similarity of the pH-activity curves for the two reactions. NADP⁺ and NADPH lower the K_m for 5,10-methenyltetrahydrofolate 2- to 3-fold above pH 7 in the cyclohydrolase reaction but below pH 7 they act as partial inhibitors.     

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.     

Methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase from porcine liver. Isolation of a dehydrogenase-cyclohydrolase fragment from the multifunctional enzyme

Tryptic digestion of a multifunctional enzyme from porcine liver containing methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5), methenyltetrahydrofolate cyclohydrolase (5,10-methenyltetrahydrofolate 5-hydrolase, EC 3.5.4.9) and formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase, EC 6.3.4.3) activities destroys the synthetase. A fragment containing both dehydrogenase and cyclohydrolase activities has been isolated by affinity chromatography on an NADP+-Sepharose affinity column. The purified fragment is homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis where its molecular weight was determined as 33 000 +/- 1200 compared with 100 000 for the undigested protein. The cyclohydrolase activity retains sensitivity to inhibition by NADP+, MgATP and ATP.     

Methenyltetrahydrofolate synthetase prevents the inhibition of phosphoribosyl 5-aminoimidazole 4-carboxamide ribonucleotide formyltransferase by 5-formyltetrahydrofolate polyglutamates

Methenyltetrahydrofolate synthetase (EC 6.3.3.2) catalyzes the irreversible ATP and Mg2+-dependent transformation of 5-formyltetrahydrofolate (N5-HCO-H4-pteroylglutamic acid (PteGlu] to 5,10-methenyltetrahydrofolate. The physiological function of this reaction remains unknown even though it is potentially involved in the intracellular metabolism of the large doses of N5-HCO-H4-PteGlu (leucovorin) administered to cancer patients. We have tried to elucidate methenyltetrahydrofolate synthetase's physiological role by examining the consequences of its inhibition in MCF-7 human breast cancer cells by the folate analog 5-formyltetrahydrohomofolate (fTHHF), a potent competitive inhibitor with a Ki of 1.4 microM. fTHHF inhibited MCF-7 cell growth with an IC50 of 2.0 microM during 72-h exposures, and this effect was fully reversible by hypoxanthine but not thymidine, indicating specific inhibition of de novo purine synthesis. A correlation was observed between increases in intracellular N5-HCO-H4-PteGlu concentrations following fTHHF and cell growth inhibition. De novo purine synthesis was inhibited at the second folate-dependent enzyme, phosphoribosyl aminoimidazole-carboxamide formyltransferase (AICAR transferase; EC 2.1.2.3), as determined by aminoimidazole carboxamide rescue and azaserine inhibition studies. N5-HCO-H4-PteGlu pentaglutamate was a potent inhibitor of purified MCF-7 cell AICAR transferase with a Ki of 3.0 microM while the monoglutamate was not an inhibitor up to 10 microM and fTHHF was only weakly inhibitory with a Ki of 16 microM. These findings suggest that methenyltetrahydrofolate synthetase activity is needed to prevent de novo purine synthesis inhibition by N5-HCO-H4-PteGlu polyglutamates.     

Distribution of Folate Derivatives and Enzymes for Synthesis of 10-Formyltetrahydrofolate in Cytosolic and Mitochondrial Fractions of Pea Leaves

Leaf extracts of 14-d-old pea (Pisum sativum L. cv Homesteader) seedlings were examined for folate derivatives and for 10-formyltetrahydrofolate synthetase (SYN), 5,10-methenyltetrahydrofolate cyclohydrolase (CYC), and 5,10-methylenetetrahydrofolate dehydrogenase (DHY) activities. Microbiological and enzyme assays showed that leaf folates SYN, CYC, and DHY were predominantly cytosolic. Extracts of Percoll gradient-purified mitochondria contained less than 1% of total leaf folate and less that 1% of each enzyme activity. Fractionation of whole-leaf homogenates resulted in the copurification of DHY and CYC (subunit 38 kD) and the isolation of a SYN protein (subunit 66 kD). Polyclonal antibodies were raised against purified cytosolic DHY-CYC (DHY-CYC-Ab) and cytosolic SYN (SYN-Ab), respectively. Immunoblots showed that DHY-CYC-Ab cross-reacted with a mitochondrial protein band (38 kD). Two mitochondrial protein bands (subunit Mr = 40,000 and 44,000) cross-reacted with SYN-Ab. Immunoaffinity chromatography (DHY-CYC-Ab as the immobile ligand) indicated that the bulk of mitochondrial SYN activity was not associated with mitochondrial DHY or CYC. When 9-d-old etiolated pea seedlings were exposed to light for up to 3 d, the specific enzyme activities of DHY-CYC in whole-leaf extracts rose 2-fold and more DHY-CYC-Ab cross-reacting protein was detected. In contrast, the specific activity of SYN fell from 5 to 1 [mu]mol min-1 mg-1 protein and less SYN-Ab cross-reacting protein was detected. The data suggest that in pea leaves, the bulk of one-carbon-substituted tetrahydrofolates and enzymes for the generation of 10-formyltetrahydrofolate are extra-mitochondrial.     

Fermentation of Fructose and Synthesis of Acetate from Carbon Dioxide by Clostridium formicoaceticum

Clostridium formicoaceticum ferments fructose labeled with (14)C in carbon 1, 4, 5, or 6 via the Embden Meyerhof pathway. In fermentations of fructose in the presence of (14)CO(2), acetate is formed labeled equally in both carbons. Extracts convert the methyl groups of 5-methyltetrahydrofolate and methyl-B(12) to the methyl group of acetate in the presence of pyruvate. Formate dehydrogenase, 10-formyltetrahydrofolate synthetase, 5,10-methenyltetrahydrofolate cyclohydrolase, 5,10-methylenetetrahydrofolate dehydrogenase, and 5,10-methylenetetrahydrofolate reductase are present in extracts of C. formicoaceticum. These enzymes are needed for the conversion of CO(2) to 5-methyltetrahydrofolate. It is proposed that acetate is totally synthesized from CO(2) via the reactions catalyzed by the enzymes listed above and that 5-methyltetra-hydrofolate and a methylcorrinoid are intermediates in this synthesis.