The uridylyltransferase GlnD and tRNA modification GTPase MnmE allosterically control Escherichia coli folylpoly-γ-glutamate synthase FolC.

Folate derivatives are important cofactors for enzymes in several metabolic processes. Folate-related inhibition and resistance mechanisms in bacteria are potential targets for antimicrobial therapies and therefore a significant focus of current research. Here, we report that the activity of Escherichia coli poly-γ-glutamyl tetrahydrofolate/dihydrofolate synthase (FolC) is regulated by glutamate/glutamine-sensing uridylyltransferase (GlnD), THF-dependent tRNA modification enzyme (MnmE), and UDP-glucose dehydrogenase (Ugd) as shown by direct in vitro protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the Khalf of FolC for its substrate l-glutamate. Moreover, FolC inhibited the GTPase activity of MnmE at low GTP concentrations. The growth phenotypes associated with these proteins are discussed. These results, obtained using direct in vitro enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in E. coli and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.

Improved methods for the preparation of [3H]folate polyglutamates: biosynthesis with Lactobacillus casei and enzymatic synthesis with Escherichia coli folylpolyglutamate synthetase.

Tritiated forms of polyglutamyl folates are not commercially available but are often needed for experimental uses in folate biochemistry. Thus, considerable interest exists in the preparation of polyglutamyl [(3)H]folates from the commercial monoglutamyl [(3)H]folates. However, refinement of established enzymatic and biological synthesis methods is still needed. To address this need we developed improved procedures for the conversion of monoglutamyl [(3)H]folates to various polyglutamyl forms. In the bacterial synthesis, Lactobacillus casei was grown in the presence of 1 ng/ml (2.27 nM) [(3)H]folic acid in Folic Acid Casei Medium. Washed cells were resuspended in 2% sodium ascorbate containing 10mM beta-mercaptoethanol and heated to release the folates. The extracted [(3)H]folates were purified on a folate-binding protein affinity column and then applied to a Sephadex G-10 column to separate the eluted poly- from the monoglutamyl folate species. High performance liquid chromatography with multichannel electrochemical detection indicated that the bacterial synthesis yielded predominantly polyglutamates of [(3)H]5-methyltetrahydrofolate and [(3)H]5-formyltetrahydrofolate (di- through heptaglutamates). The alternative method consisted of enzymatic polyglutamylation of [(3)H]folic acid catalyzed by recombinant Escherichia coli folylpolyglutamate synthetase. This enzymatic synthesis yielded predominantly tri-, tetra-, and pentaglutamyl species for the [(3)H]folate substrate.

Purification and characteristics of recombinant human folylpoly-gamma-glutamate synthetase expressed at high levels in insect cells.

Folylpoly-gamma-glutamate synthetase activity is central to the operation of folate metabolism and is essential for the survival of mammalian stem cell populations but the very low levels of endogenous expression of this enzyme have greatly limited its study. We now report the expression of cytosolic folylpoly-gamma-glutamate synthetase (FPGS) cloned from human leukemic cells in baculovirus-infected insect cells at levels of 4-5% of the total soluble protein of the cells. As was the case with endogenously expressed mammalian FPGS, recombinant enzyme was quantitatively blocked at the amino terminus in spite of the large-scale production in insect cells. A three-step purification procedure resulted in an overall yield of 7-35 mg per liter of culture with a recovery of about 50% and purity approximately 95%; pure enzyme was stable to storage for extended periods. Pure protein had a specific activity of 25 micromol h(-1)mg(-1) with aminopterin as a substrate and used a broad spectrum of folates as substrates. The pure enzyme also carried out ATP hydrolysis in the absence of a folate substrate or glutamic acid; this partial reaction occurred at a k(cat) about 0.4% that of the full reaction. In vitro, this single protein added several (1-8) moles of glutamic acid per mole of folate analog, the same spectrum of folate polyglutamates as seen in vivo. The quantities of pure enzyme achievable in insect cells should allow functional and structural studies on this enzyme.

Folylpolyglutamate synthesis in Neurospora crassa: primary structure of the folylpolyglutamate synthetase gene and elucidation of the met-6 mutation.

In Neurospora crassa, the met-6+ gene encodes folylpoly-gamma-glutamate synthetase (FPGS) which catalyzes the formation of polyglutamate forms of folate. Methionine auxotrophy of the Neurospora crassa met-6 mutant is related to a lesion affecting this enzyme. Functional complementation of the mutant strain was achieved by introducing copies of the wild-type met-6+ gene into mutant spheroplasts. The complementing sequences were found to be contained on a 3.5 kb EcoRI-BamHI restriction fragment. The nucleotide sequence of the met-6+ gene was determined and an open reading frame of 1587 bp was identified, interrupted by two introns. This open reading frame contained several AUG codons but translation beginning from either of the first two would theoretically produce a protein of appropriate size and with similarity to five other FPGS proteins. Northern blot analyses of met-6+ transcripts revealed a 2.0 kb product. The position of the transcription stop site and an intron were identified by sequencing partial cDNA clones which were truncated at the 5' end. DNA sequence analysis of the met-6 mutant allele revealed a T to C transition which would result in replacement of a highly conserved serine with a proline.

Pteroylpolyglutamate synthesis by lung- and culture-derived Pneumocystis carinii.

Pneumocystis carinii synthesizes folates de novo from exogenous p-aminobenzoic acid (pABA). Lung-derived organisms take up [3H]pABA in vitro except in the presence of sulfamethoxazole. Supernatants from spinner-flask cultures take up [3H]pABA if they were inoculated with lungs from infected rats, but not if they were inoculated with lungs from uninfected rats. P. carinii folates consist primarily of pteroylpentaglutamates. Plasmodium falciparum, in contrast, contains primarily pteroyltetraglutamates. Culture-derived organisms synthesize folates at a four-fold higher specific activity than lung-derived organisms, possibly because they contain less contaminating lung debris. These data suggest that P. carinii remains metabolically active in culture for at least 4 days.

DL-beta,beta-difluoroglutamic acid mediates position-dependent enhancement or termination of pteroylpoly(gamma-glutamate) synthesis catalyzed by folylpolyglutamate synthetase.

Poly(gamma-glutamylation) of glutamate (L-Glu)-containing antifolates and natural folates is important in pharmacological mechanisms and in physiological processes. Based on previous work from our laboratories, we hypothesized that replacement of the L-Glu moiety in parent molecules with DL-beta,beta-difluoroglutamic acid (DL-beta,beta-F2Glu) might be a generic means of increasing polyglutamylation by both increasing the synthesis rate and decreasing the degradation rate (J. J. McGuire et al., J. Biol. Chem. 265, 14073-14079 (1990)); thus biological potency might be increased without other biochemical properties being altered. DL-beta,beta-F2Glu, synthesized by an improved route (B. P. Hart and J. K. Coward, Tetrahedron Lett. 34, 4917-4920 (1993)), has been incorporated into a methotrexate (MTX) homolog, beta,beta-difluoromethotrexate (beta,beta-F2MTX), and a folic acid (PteGlu) homolog, beta,beta-difluorofolic acid (beta,beta-PteF2Glu). Biochemical properties of beta,beta-F2MTX (e.g., inhibition of isolated dihydrofolate reductase, transport in whole cells) are similar to those of MTX except that, in accord with our hypothesis, apparent substrate efficiency for rat and human folylpolyglutamate synthetase (FPGS) is 4- to 7.5-fold higher, respectively, for beta,beta-F2MTX than for MTX. Analysis of the products synthesized by purified FPGS, however, suggests that while addition of the first gamma-Glu to beta,beta-F2MTX is highly efficient, subsequent additions occur at a negligible rate; this premise was confirmed by directly comparing the in vitro FPGS substrate activity of MTX-gamma-Glu and beta,beta-F2MTX-gamma-Glu. Furthermore, the dramatically diminished in vitro growth inhibitory potency of beta,beta-F2MTX as compared to MTX when exposure time to drug is decreased (despite otherwise similar biochemical properties) suggests that polyglutamylation is also impaired in intact cells. Similar results with FPGS have been obtained with oxidized and reduced forms of beta,beta-PteF2Glu. These data suggest that the effect of beta,beta-F2Glu on polyglutamylation by FPGS is dependent on its position relative to the point of L-Glu ligation. When beta,beta-F2Glu is the acceptor amino acid (i.e., point of attachment), ligation of Glu is enhanced; however, if beta,beta-F2Glu is one residue distal to the acceptor amino acid, further elongation is blocked.

Folylpolyglutamate synthetase in Lactobacillus leichmannii: in vitro synthesis and characterization of polyglutamylfolates.

In vitro synthesis of folylpolyglutamates by folylpolyglutamate synthetase from Lactobacillus leichmannii has been studied and optimal conditions for enzyme activity determined. It is found that while ATP (5 mM) is essential for the synthesis of folylpolyglutamates homocysteine augments the same. Replacement of vitamin B12 (2 ng/ml) with deoxyuridine (20 micrograms/ml) in growth medium does not alter the enzymatic parameters studied. DEAE-cellulose column chromatography of in vitro synthesised folylpolyglutamates indicates that folylpolyglutamate synthetase of L. leichmannii can synthesize polyglutamates up to a chain length of four glutamate residues.

Enzymatic synthesis of folate and antifolate polyglutamates with Escherichia coli folylpolyglutamate synthetase.

Escherichia coli folylpolyglutamate synthetase was used to synthesize micromole quantities of polyglutamyl conjugates of folic acid, methotrexate, and other analogs of folic acid. The products of the enzymatic reactions were purified by semipreparative C18 HPLC. The position of each amide linkage (gamma or alpha carboxyl) in the polyglutamated products was determined by limited and exhaustive hydrolyses with hog kidney folylpolyglutamate hydrolase and with yeast carboxypeptidase Y. Under standard reaction conditions, the E. coli enzyme added up to five glutamyl residues to each monoglutamated substrate, primarily at the gamma carboxyl position. Thus, an enzyme which naturally adds only two glutamates to naturally occurring folates can be used synthetically to make higher polyglutamates of a wide range of synthetic substrates. The products of the reactions are valuable tools for the study of the metabolism of antifolate drugs as well as metabolic reactions involving folate cofactors.

DL-3,3-difluoroglutamate: an enhancer of folylpolyglutamate elongation.

The effect of 3,3-difluoroglutamate (F2Glu) on the reaction catalyzed by rat liver folypolyglutamate synthetase was investigated. F2Glu was a potent, concentration-dependent inhibitor of poly(gamma-glutamylation) using [3H]Glu and either methotrexate (4-NH2-10-CH3PteGlu) or tetrahydrofolate as substrates. It was determined that F2Glu acted as an alternate substrate, but in contrast to the previously characterized alternate substrate 4-fluoroglutamate (McGuire, J.J., and Coward, J.K. (1985) J. Biol. Chem. 260, 6747-6754), it did not terminate polyglutamate chain elongation. Instead, F2Glu promoted chain elongation. Thus, synthesis of products from [3H]methotrexate containing 1 and 2 additional amino acid residues occurred at a substantially higher rate in the presence of F2Glu when compared to identical reactions in the presence of Glu; this was more pronounced for the product containing 2 additional residues. Identities of the products were established by their respective chromatographic elution positions and by limit digestion with gamma-glutamyl hydrolases. Ligation of Glu to 4-NH2-10-CH3PteGlu-gamma-(3,3-difluoroglutamate) was also enhanced. These results are consistent with F2Glu enhancing the synthesis of poly(gamma-glutamate) metabolites at the level of either the incoming amino acid (glutamate analog) or the gamma-glutamyl acceptor species. F2Glu is thus the first glutamate analog which enhances chain elongation catalyzed by folypolyglutamate synthetase.

De novo and salvage biosynthesis of pteroylpentaglutamates in the human malaria parasite, Plasmodium falciparum.

Plasmodium falciparum was shown to synthesize pteroylpolyglutamate de novo from guanosine 5'-triphosphate (GTP), p-aminobenzoate (PABA), and L-glutamate (L-Glu). The parasite also had the capacity to synthesize pteroylpolyglutamate from both intact and degradation moieties (p-aminobenzoylglutamate and pterin-aldehyde) of exogenous folate added into the growth medium. The major product was identified as 5-methyl-tetrahydroteroylpentaglutamate following exposure to pteroylpolyglutamate hydrolase and oxidative degradation of the C9-N10 bond in the molecule and identification of products by reversed-phase high performance liquid chromatography. Inhibition of pteroylpentaglutamate synthesis from the radiolabelled metabolic precursors (GTP, PABA, L-Glu) and folate by the antifolate antimalarials, pyrimethamine and sulfadoxine at therapeutic concentrations, may suggest the existence of a unique biosynthetic pathway in the malaria parasite.

Comparison of the conversion of 5-formyltetrahydrofolate and 5-methyltetrahydrofolate to 5,10-methylenetetrahydrofolates and tetrahydrofolates in human colon tumors.

Four hr infusions i.v. of [6RS]5-formyltetrahydrofolate ([6RS]5-CHO-H4PteGlu; 500 mg/m2) and [6RS]5-methyltetrahydrofolate ([6RS]5-CH3-H4PteGlu; 500 mg/m2) were compared for their relative effects on expansion of pools of 5,10-methylenetetrahydrofolates (CH2-H4PteGlun) and tetrahydrofolates (H4PteGlun) in two human colon adenocarcinoma xenografts in mice. Expansion of these pools by 253-661% of control and increase in predominance of di-, tri-, and tetra-glutamate species were observed during [6RS]5-CHO-H4PteGlu infusion. In contrast, only modest pool size expansion (148-164% of control) and limited modulation of polyglutamate species were detected in four tumor lines during infusion with [6RS]5-CH3-H4PteGlu. The data suggest that [6RS]5-CH3-H4PteGlu is less effective than [6RS]5-CHO-H4PteGlu as a precursor for pools of CH2-H4PteGlun and H4PteGlun in colon tumors.

Folylpolyglutamates in Leishmania major.

The intracellular folates of the protozoan parasite Leishmania major have been examined. About 95% of the exogenous [3H]folate accumulated by the protozoan is metabolized to polyglutamate conjugates within 65 hr, and the intracellular folates are about forty-fold concentrated over the folate in the medium. The predominant metabolite of folic acid is the pentaglutamate conjugate (85%), with lessor amounts of the tetraglutamate (approximately 9%) and hexaglutamate (approximately 3%), and trace (less than 2.5%) amounts of di-, tri- and hepta-glutamate conjugates. Chromatographic properties of the products indicate that the conjugates are linked through the gamma-carboxyl groups. The folylpolyglutamate distribution in Leishmania is similar to that found in mammalian tissues.

In vitro synthesis of alpha-carboxyl-linked folylpolyglutamates by an enzyme preparation from Escherichia coli.

Extracts of Escherichia coli contained an enzymatic activity which catalyzed the addition of L-glutamate to the alpha-carboxyl of various polyglutamate substrates, including folylpolyglutamates. Much of the enzyme activity was separated by DE52 chromatography and gel filtration from the enzyme which adds the first three glutamates in the biosynthesis of folylpolyglutamates, dihydrofolate synthetase-folylpolyglutamate synthetase. The two enzyme activities differed in many properties. Whereas dihydrofolate synthetase-folylpolyglutamate synthetase preferred pteroate or pteroylmonoglutamate substrates, the folylpoly-alpha-glutamate synthetase preparations effectively utilized tetrahydropteroylpolyglutamates, pteroylpolyglutamates, p-aminobenzoylpolyglutamates (pAB(Glu)n), and even a polyglutamate tripeptide. Several di- and triglutamyl peptides were inhibitory to folylpoly-alpha-glutamate synthetase activity, but not to dihydrofolate synthetase-folylpolyglutamate synthetase. Conversely, dihydropteroate noncompetitively inhibits the folylpolyglutamate synthetase reaction of the dihydrofolate synthetase-folylpolyglutamate synthetase protein, but did not inhibit the folylpoly-alpha-glutamate synthetase reaction. Potassium chloride was inhibitory to folylpoly-alpha-glutamate synthetase activity (as were other salts and several polyanions), in contrast to the absolute requirement of dihydrofolate synthetase-folylpolyglutamate synthetase activity for a monovalent cation such as K+. Incubation of a folylpoly-alpha-glutamate synthetase preparation with (6S)-tetrahydropteroyltri(gamma)glutamate generated products which after chemical cleavage to pAB(Glu)n were identical to those from growing E. coli, in high performance liquid chromatography retention times and in pattern of digestion by alpha-COOH bond-specific carboxypeptidase Y. High performance liquid chromatography and mass spectral analysis of the products of the in vitro reactions of folylpoly-alpha-glutamate synthetase with several substrates also demonstrated the addition of glutamate residues via alpha-COOH linkages. Thus, there appear to be two folylpolyglutamate synthetase activities in E. coli, dihydrofolate synthetase-folylpolyglutamate synthetase which adds the first three glutamate residues by gamma-COOH linkages and the folylpoly-alpha-glutamate synthetase activity which extends the folylpolyglutamate chain via gamma-COOH peptide bonds.

Biosynthesis of polyglutamates of folates.

Folylpolyglutamate synthetase (FPGS) catalyzes the synthesis of the poly-gamma-glutamate forms of tetrahydrofolate and its co-enzyme adducts, as well as of the folate-analogue drugs. This paper reviews current knowledge of the preparations of FPGS from mammalian sources (rat, hog, mouse, and beef liver). Kinetic constants for the substrates and activators of FPGS are compared. Tetrahydrofolate and 5-formyltetrahydrofolate are excellent substrates for the enzyme. The Km values for the antifolates and their 7-hydroxy metabolites are much higher than those for the tetrahydrofolates. Aminopterin has higher activity with FPGS than does methotrexate, which partially explains its greater toxicity. 5-Formyltetrahydrofolate, which is used as a rescue agent in high-dose methotrexate-rescue chemotherapy, is a better alternate substrate of FPGS than is methotrexate and therefore is a potent competitive inhibitor of the glutamylation of methotrexate. Thus, low concentrations of the rescue agent prevent formation of cytotoxic polyglutamates of methotrexate. The pathway of the reaction is the addition of a glutamate residue to the terminal gamma-carboxyl of the pteridine substrate. That longer folylpolyglutamates are poorer substrates possibly is a result of this addition pathway. Pteroic acid activates FPGS by lowering the Km value of the pteridine substrate. It also greatly increases the activity of the synthetase at physiological pH values.

The effect of ethanol consumption on folate polyglutamate biosynthesis in the rat.

Contrary to previous studies, the folate polyglutamate chain length in the rat liver appears to be unaffected by ethanol ingestion for periods of 2-13 weeks. The appearance of short chain length folates after 13 weeks has been shown to arise as a result of increased in vitro folate polyglutamate breakdown during extraction due to a greater release of lysosomal conjugase in these animals.

Folate polyglutamate synthetase activity in the cobalamin-inactivated rat.

Exposure to N2O inactivates cob[I]alamin and interferes with the activity of methionine synthetase, of which cob[I]alamin is a coenzyme. Less directly, it stops the formation of folate polyglutamate from tetrahydrofolates. Studies on the activity of folate polyglutamate synthetase in rat liver in vivo were carried out. The synthetase activity increased after exposure to N2O for up to 48 h, but longer exposure was accompanied by a return of activity to baseline values. The rise in synthetase activity was prevented by supplying methionine, 5'-methylthioadenosine or 5-formyltetrahydrofolate. The fall in folate polyglutamate synthetase activity after 48 h was accompanied by a restoration of hepatic synthesis of folate polyglutamate despite continuation of N2O exposure.