Polyglutamate Biosynthesis Research Articles

FolC2-mediated folate metabolism contributes to suppression of inflammation by probiotic Lactobacillus reuteri.

Bacterial‐derived compounds from the intestinal microbiome modulate host mucosal immunity. Identification and mechanistic studies of these compounds provide insights into host–microbial mutualism. Specific Lactobacillus reuteri strains suppress production of the proinflammatory cytokine, tumor necrosis factor (TNF), and are protective in a mouse model of colitis. Human‐derived L. reuteri strain ATCC PTA 6475 suppresses intestinal inflammation and produces 5,10‐methenyltetrahydrofolic acid polyglutamates. Insertional mutagenesis identified the bifunctional dihydrofolate synthase/folylpolyglutamate synthase type 2 (folC2) gene as essential for 5,10‐methenyltetrahydrofolic acid polyglutamate biosynthesis, as well as for suppression of TNF production by activated human monocytes, and for the anti‐inflammatory effect of L. reuteri 6475 in a trinitrobenzene sulfonic acid‐induced mouse model of acute colitis. In contrast, folC encodes the enzyme responsible for folate polyglutamylation but does not impact TNF suppression by L. reuteri. Comparative transcriptomics between wild‐type and mutant L. reuteri strains revealed additional genes involved in immunomodulation, including previously identified hdc genes involved in histidine to histamine conversion. The folC2 mutant yielded diminished hdc gene cluster expression and diminished histamine production, suggesting a link between folate and histadine/histamine metabolism. The identification of genes and gene networks regulating production of bacterial‐derived immunoregulatory molecules may lead to improved anti‐inflammatory strategies for digestive diseases.

Genetic design of conditional d-glutamate auxotrophy for Bacillus subtilis: Use of a vector-borne poly-γ-glutamate synthetic system

Bacillus subtilis possesses two glutamate racemase isozymes, RacE and YrpC. For the first time, we succeeded in constructing glutamate racemase-gene disruptants of B. subtilis. Phenotypic analysis of their d-glutamate auxotrophy indicated that the RacE-type glutamate racemase is important for ensuring maximum growth rate but dispensable. The YrpC-type glutamate racemase probably operates as an anaplerotic enzyme for RacE, especially under liquid culture conditions. We found novel applicability of RacE-less mutants inheriting only a marginal activity for endogenous d-glutamate supply, viz. the employment for the in vivo identification of d-glutamate-consuming systems. In fact, the genetic induction of a poly-γ-glutamate synthetic system led a RacE-less mutant to severe growth suppression, which was overcome in the presence of a high concentration of exogenous d-glutamate. The results indicate that a significant amount of d-glutamate is consumed during poly-glutamate biosynthesis. To our knowledge, this is the first report of conditional d-glutamate auxotrophy for B. subtilis.

Horizontal transfer of a bacterial gene involved in polyglutamate biosynthesis to the plant-parasitic nematode Meloidogyne artiellia

Analysis of a genomic fragment from the plant parasitic nematode Meloidogyne artiellia revealed the presence of a gene which, in bacteria, is involved in the formation of polyglutamate capsule. Searching of various databases, including the Caenorhabditis elegans genome sequence and the large EST datasets from a variety of parasitic nematodes, showed that no similar genes have been identified in other nematodes or in any other eukaryotic organisms. The M. artiellia gene has a typical eukaryotic structure and its mRNA is present in the intestine. The gene is expressed in all life cycle stages tested. These findings demonstrate horizontal gene transfer may be important in catalyzing the diversification of nematode lineages.

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.

ChemInform Abstract: SYNTHESIS AND ANALYSIS OF THE PTEROYLPOLYGLUTAMATES

Publisher Summary This chapter discusses the synthesis and analysis of the Pteroylpolyglutamates. The biological importance of the polyglutamyl chain was recognized on the basis of its universal occurrence and by the fact that, during evolution, the ability to synthesize the peptide chain has been retained, even by organisms that have lost the pathways for folic acid synthesis. The polyglutamyl chain of the folates plays a role in the regulation of one-carbon metabolism. The polyglutamates are the best cosubstrates of the folate-requiring enzymes, and some of these enzymes show a clear preference for certain specific polyglutamyl chain lengths. The part played by the poly-γ-glutamyl chain in affecting the translocation of folates across cellular and subcellular membranes is established. This role acquires special practical significance when considering the pharmacodynamics of polyglutamyl derivatives of folic acid analogs possessing antitumor activity. Changes in the patterns of distribution of polyglutamates of different chain lengths occur under conditions, which alter the steady-state of one-carbon metabolism, a necessary effect when the polyglutamyl chain is involved in regulation. The properties of the enzymes of poly-glutamate biosynthesis and degradation are reviewed in the chapter.

Synthesis and Analysis of the Pteroylpolyglutamates

Publisher Summary This chapter discusses the synthesis and analysis of the Pteroylpolyglutamates. The biological importance of the polyglutamyl chain was recognized on the basis of its universal occurrence and by the fact that, during evolution, the ability to synthesize the peptide chain has been retained, even by organisms that have lost the pathways for folic acid synthesis. The polyglutamyl chain of the folates plays a role in the regulation of one-carbon metabolism. The polyglutamates are the best cosubstrates of the folate-requiring enzymes, and some of these enzymes show a clear preference for certain specific polyglutamyl chain lengths. The part played by the poly-γ-glutamyl chain in affecting the translocation of folates across cellular and subcellular membranes is established. This role acquires special practical significance when considering the pharmacodynamics of polyglutamyl derivatives of folic acid analogs possessing antitumor activity. Changes in the patterns of distribution of polyglutamates of different chain lengths occur under conditions, which alter the steady-state of one-carbon metabolism, a necessary effect when the polyglutamyl chain is involved in regulation. The properties of the enzymes of poly-glutamate biosynthesis and degradation are reviewed in the chapter.

Folate polyglutamate biosynthesis in the liver, tumour and intestine of rats bearing the Walker 256 carcosarcinoma.

Folate polyglutamate biosynthesis in the liver, tumour and intestine of rats bearing the Walker 256 carcosarcinoma.

The substrate for folate polyglutamate biosynthesis in the vitamin B 12- inactivated rat

Rats were maintained for 24 hours in a chamber containing 50% N 2O in order to inactivate cobIalamin. Control animals breathed air. Folate analogues [0.01μmol] labelled with either 3H or 14C, were given IP and their hepatic uptake and conversion into folate polyglutamate measured. There was impaired hepatic uptake of folate by N 2O-treated animals varying from 21% of that in controls given H 4PteGlu to 69% with 10-CHO-H 4PteGlu. There was no detectable formation of folate polyglutamate in the N 2O-treated animal with H 4PteGlu and 5-CH 3-H 4PteGlu but ‘normal’ polyglutamate synthesis with 5-CHO-H 4PteGlu, 10-CHO-H 4PteGlu and 5,10-CH-H 4PteGlu.

The effect of vitamin B 12 inhibition [formula omitted]: Impaired folate polyglutamate biosynthesis indicating that 5-methyltetrahydropteroylglutamate is not its usual substrate

Using 5-[ 14C] methyltetrahydropteroylglutamate and [ 3H] pteroylglutamate it was found that vitamin B 12 dependent methyltransferase can be inhibited in vivo in mice by nitrous oxide. Examination of the liver [ 3H] folates showed as expected that both controls and nitrous oxide treated mice had synthesised the reduced 5-methyl form. However, the controls had converted [ 3H] almost exclusively into a polyglutamate with virtually no monoglutamate present, while nitrous oxide treated mice had failed to add glutamates to over half of the incorporated folate. Thus in vivo demethylation may be necessary prior to polyglutamate biosynthesis and act as a method of controlling folate accumulation, or be used to create a concentration gradient with the circulating folate.

Biosynthesis of folate polyglutamate in the rat with different tracers.

1. The recent suggestions that folate polyglutamate biosynthesis demonstrated in vivo with [3'5'9(n)-3H]folic acid is due to exchange and cannot be achieved with [2(-14)C]folic acid has been demonstrated to be untrue. 2. The compounds formed with both types of radioactive tracer have been shown to be folate polyglutamates from their elution position from precalibrated ion-exchange columns and their susceptibility to hydrolysis by a gamma-carboxypeptidase (conjugase) from human sera. 3. By the use of the methods involving [2(-14)C]folic acid, reported by others to give only folate monoglutamates, we have been able to demonstrate clearly the presence of folate polyglutamates.

Specificity of folate-γ- l-glutamate ligase in rat liver and kidney. biosynthesis of poly-γ- l-glutamates of unreduced methotrexate and the effect of methotrexate on folate polyglutamate biosynthesis

1. 1. Prior administration of methotrexate reduces the 24-hour incorporation ofparenteral [ 3H]pteroylglutamate by rat liver from 30% to less than 2%. 2. 2. The folate compounds synthesized showed the same ratio of reduced 5-methyl- and 5-formyl-derivatives as normal but the number of glutamyl residues to which these were attached was reduced. No polyglutarnyl forms of unreduced pteroylglutamate were found. 3. 3. Under similar conditions, 10% of a parenteral dose of [ 3H] methotrexate was incorporated into the liver and a small proportion of this was converted to conjugated forms of unreduced methotrexate. Kidney also demonstrated the ability to conjugate methotrexate.

Formation of 10-formylfolic acid, a potent inhibitor of dihydrofolate reductase, in rat liver slices incubated with folic acid.

During investigation of folate polyglutamate biosynthesis in rat liver slices utilizing [2-(14)C]folic acid, a folate compound that behaved like a polyglutamate form in the Sephadex G-15 gel filtration system was found to accumulate. Subsequent chromatographic, spectral, chemical, and enzymic studies have indicated that the compound formed in liver slices incubated with [(14)C]folic acid with and without methotrexate was 10-formyl folate. This folate is of interest in that it is the most potent natural inhibitor of dihydrofolate reductase known and may be capable of serving a regulatory function within the cell.

The identification of the forms of folate found in the liver, kidney and intestine of the monkey and their biosynthesis from exogenous pteroylglutamate (folic acid)

The distribution of the endogenous folates and the forms biosynthesized from parenterally administered [3′5′9(n)- 3 H] pteroylglutamate (PteGlu) were examined in monkey tissues by column chromatography and microbiological assay. Tissue extracts were either subjected to “conjugase” (γ-glutamylcarboxy-peptidase) treatment yielding pteroylglutamates or to KMnO 4H 2 O 2 oxidation yielding p-aminobenzoyl-poly-γ- l-glutamates. Components of these mixtures were then separated by column chromatography with Sephadex QAE-A25 and Whatman DE52 anion exchangers respectively. The liver incorporated the largest amount (26%) of tritium activity. A greater proportion of labelled polyglutamate compounds were found at three days following administration than at one day. The predominant forms observed at three days contained five and six glutamyl residues and substitutions were primarily methyltetrahydro (i.e. 5-CH 3-H 4 PteGlu 5 ,6) with small quantities of 10 and/or 5 formyltetrahydro (10-CHO, 5-HCO-H 4 PteGlu 5 ,6). Estimation of endogenous distribution by microbiological assay for pteroyloligoglutamates ( Lactobacillus casei) and p-aminobenzoate ( Escherichia coli) indicates that exogenous tritiated compounds are not fully equilibrated with the endogenous pools at periods of up to three days. Polyglutamate biosynthesis occurred more slowly in kidney with unmetabolized [ 3 H] PteGlu still present after one day, but closely resembled liver distribution at three days. The biosynthesis of a complex mixture of labelled polyglutamates has also been demonstrated in intestinal in intestinal mucosal cells.