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.

Chronic cobalamin inactivation impairs folate polyglutamate synthesis in the rat.

Nitrous oxide, by inactivating cobalamin in vivo, produces a suitable animal model for cobalamin 'deficiency.' The synthesis of folate polyglutamate with tetrahydrofolate as substrate is severely impaired in the N2O-treated rat, but is normal with formyltetrahydrofolate as substrate. Methionine restores the capacity of the N2O-treated rat to utilize tetrahydrofolate the minimum effective dose being 16 mumol. S-Adenosylmethionine was somewhat less effective than methionine but 5'methylthioadenosine, a product of S-adenosylmethionine metabolism, was significantly more effective than methionine in correcting the defect in folate polyglutamate synthesis. 5'Methylthioadenosine is metabolised to yield formate. It is suggested that these compounds have their effect in correcting folate polyglutamate synthesis by supplying formate for the formylation of tetrahydrofolate. Formyltetrahydrofolate, at least in the cobalamin-inactivated animal, is the required substrate for folate polyglutamate synthesis. Cobalamin is concerned with the maintenance of normal levels of methionine and this in turn is a major source of formate through S-adenosylmethionine and 5'methylthioadenosine.

Utilization of [2-14C]tetrahydropteroylglutamic acid and 5-[G-3H]methyltetrahydropteroylglutamic acid as substrates for folate polyglutamate synthesis in fruit bats: effect of vitamin B-12-deficiency.

[2-14C]Tetrahydropteroylglutamic acid and 5-[G-3H]methyltetrahydropteroylglutamic acid were given intraperitoneally to fruit bats. Folate polyglutamates were formed in the liver from both substrates in different amounts and at different rates. The methylfolate pool appeared to remain separate from the tetrahydrofolate pool. More polyglutamate was formed from tetrahydropteroylglutamic acid than from 5-methyltetrahydropteroylglutamic acid. There was a fall in the folate content of the liver in the vitamin B-12-deficient bat and a more rapid incorporation of folates into polyglutamates but thereafter a more rapid loss of the labelled folate from liver.

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.

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.

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.

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.

Comparison of folylderivative biosynthesis in Ehrlich ascites carcinoma cells and in some organs of healthy and tumor-bearing mice.

Biosynthesis of folylderivatives derived from subcutaneously injected 2-[14C]folate was studied in Ehrlich ascites carcinoma (EAC) cells and in liver and kidneys of healthy and tumor-bearing mice. Retention of exogenous folate was followed by measurements of the total radioactivity of folylderivatives present in the EAC cells and organs examined. Identification of unconjugated and conjugated folylderivatives was done by means of column chromatography on Sephadex G-25, G-15 and cellulose sheets. The level of retained radioactivity in folyderivatives, being 5% in the liver and 1% in the kidneys of the radioactivity administered to mice, was similar in healthy and tumor-bearing animals. Moreover, no quantitative and qualitative differences were found in folylmono-and polyglutamates originated from the organs of healthy or tumor-bearing mice, although the content of folylpolyglutamates arose faster in liver and kidneys of EAC cells-bearing mice as well as in the tumor cells than in the organs of healthy mice.

Uptake and metabolism of [3H] pteroylglutamic acid by developing chick embryo.

Chick embryos at 9 and 12 days of development were injected into the air space with [3', 5', 7, 9 -3H]pteroylglutamic acid. After various time periods the incorporation of radioactivity into folate derivatives was measured. In the 9-day embryo the label incorporation into longer chain polyglutamates, expressed per gram, was higher than in the 12-day embryo. Since the endogenous content of these compounds was lower at 9 days, the results suggest that, at this stage of development, the turnover of longer pteroylpolyglutamates is more rapid.

Synthesis of pteroylpolyglutamates in rat liver during regeneration after partial hepatectomy.

The liver content of higher polyglutamate forms of folic acid, which in partially hepatectomized rats is markedly lower as compared with sham operated animals, is normalized by the vitamin administration. The greater availability of folic acid and consequently of the tetrahydrofolates, promotes the synthesis of actual coenzymic forms; so their content, in regenerating liver, is maintained at normal values in spite of a higher requirement caused by the exaltation of biosynthetic processes.

The metabolism of [3',5',9(n)-3H] pteroylglutamic acid by chicken liver in vivo. Effects of folate deficiency and vitamin B12 supplementation.

1. Control, folate-deficient and vitamin B12-supplemented chickens were given [3', 5', 9 (n)-3H] pteroylglutamic acid by intraperitoneal injection. After various time intervals (1.5 h-8d) the incorporation of radioactivity into hepatic folate derivatives was measured. 2. The results confirm that avian species have a faster assimilation of folate than most mammalian species. 3. Folate deficiency increased the incorporation and retention of [3H] pteroylglutamic acid. The degree of folate conjugation was unaffected by folate deficiency but the formyl to methyl folate ratio was decreased. 4. Vitamin B12 supplementation had no effect on the uptake or conjugation of [3H] pteroylglutamic acid, but increased the formyl to methyl folate ratio.

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.