Tetrahydrofolate Pathway Research Articles

Tailored extraction and ion mobility-mass spectrometry enables isotopologue analysis of tetrahydrofolate vitamers

Climate change directs the focus in biotechnology increasingly on one-carbon metabolism for fixation of CO2 and CO2-derived chemicals (e.g. methanol, formate) to reduce our reliance on both fossil and food-competing carbon sources. The tetrahydrofolate pathway is involved in several one-carbon fixation pathways. To study such pathways, stable isotope-labelled tracer analysis performed with mass spectrometry is state of the art. However, no such method is currently available for tetrahydrofolate vitamers. In the present work, we established a fit-for-purpose extraction method for the methylotrophic yeast Komagataella phaffii that allows access to intracellular methyl- and methenyl-tetrahydrofolate (THF) with demonstrated stability over several hours. To determine isotopologue distributions of methyl-THF, LC-QTOFMS provides a selective fragment ion with suitable intensity of at least two isotopologues in all samples, but not for methenyl-THF. However, the addition of ion mobility separation provided a critical selectivity improvement allowing accurate isotopologue distribution analysis of methenyl-THF with LC-IM-TOFMS. Application of these new methods for 13C-tracer experiments revealed a decrease from 83 ± 4 to 64 ± 5% in the M + 0 carbon isotopologue fraction in methyl-THF after 1 h of labelling with formate, and to 54 ± 5% with methanol. The M + 0 carbon isotopologue fraction of methenyl-THF was reduced from 83 ± 2 to 78 ± 1% over the same time when using 13C-methanol labelling. The labelling results of multiple strains evidenced the involvement of the THF pathway in the oxygen-tolerant reductive glycine pathway, the presence of the in vivo reduction of formate to formaldehyde, and the activity of the spontaneous condensation reaction of formaldehyde with THF in K. phaffii.

Metagenome Assembled Genome of a Novel Verrucomicrobial Methanotroph From Pantelleria Island.

Verrucomicrobial methanotrophs are a group of aerobic bacteria isolated from volcanic environments. They are acidophiles, characterized by the presence of a particulate methane monooxygenase (pMMO) and a XoxF-type methanol dehydrogenase (MDH). Metagenomic analysis of DNA extracted from the soil of Favara Grande, a geothermal area on Pantelleria Island, Italy, revealed the presence of two verrucomicrobial Metagenome Assembled Genomes (MAGs). One of these MAGs did not phylogenetically classify within any existing genus. After extensive analysis of the MAG, we propose the name of “Candidatus Methylacidithermus pantelleriae” PQ17 gen. nov. sp. nov. The MAG consisted of 2,466,655 bp, 71 contigs and 3,127 predicted coding sequences. Completeness was found at 98.6% and contamination at 1.3%. Genes encoding the pMMO and XoxF-MDH were identified. Inorganic carbon fixation might use the Calvin-Benson-Bassham cycle since all genes were identified. The serine and ribulose monophosphate pathways were incomplete. The detoxification of formaldehyde could follow the tetrahydrofolate pathway. Furthermore, “Ca. Methylacidithermus pantelleriae” might be capable of nitric oxide reduction but genes for dissimilatory nitrate reduction and nitrogen fixation were not identified. Unlike other verrucomicrobial methanotrophs, genes encoding for enzymes involved in hydrogen oxidation could not be found. In conclusion, the discovery of this new MAG expands the diversity and metabolism of verrucomicrobial methanotrophs.

Biochemical properties and crystal structure of formate-tetrahydrofolate ligase from Methylobacterium extorquens CM4

Methylobacterium extorquens is a methylotroph model organism that has the ability to assimilate formate using the tetrahydrofolate (THF) pathway. The formate-tetrahydrofolate ligase from M. extorquens (MeFtfL) is an enzyme involved in the THF pathway that catalyzes the conversion of formate, THF, and ATP into formyltetrahydrofolate and ADP. To investigate the biochemical properties of MeFtfL, we evaluated the metal usage and enzyme kinetics of the enzyme. MeFtfL uses the Mg ion for catalytic activity, but also has activity for Mn and Ca ions. The enzyme kinetics analysis revealed that Km value of farmate was much higher than THF and ATP, which shows that the ligation activity of MeFtfL is highly dependent on formation concentration. We also determined the crystal structure of MeFtfL at 2.8 Å resolution. MeFtfL functions as a tetramer, and each monomer consists of three domains. The structural superposition of MeFtfL with FtfL from Moorella thermoacetica allowed us to predict the substrate binding site of the enzyme.

Promiscuous and adaptable enzymes fill "holes" in the tetrahydrofolate pathway in Chlamydia species.

ABSTRACTFolates are tripartite molecules comprising pterin, para-aminobenzoate (PABA), and glutamate moieties, which are essential cofactors involved in DNA and amino acid synthesis. The obligately intracellular Chlamydia species have lost several biosynthetic pathways for essential nutrients which they can obtain from their host but have retained the capacity to synthesize folate. In most bacteria, synthesis of the pterin moiety of folate requires the FolEQBK enzymes, while synthesis of the PABA moiety is carried out by the PabABC enzymes. Bioinformatic analyses reveal that while members of Chlamydia are missing the genes for FolE (GTP cyclohydrolase) and FolQ, which catalyze the initial steps in de novo synthesis of the pterin moiety, they have genes for the rest of the pterin pathway. We screened a chlamydial genomic library in deletion mutants of Escherichia coli to identify the “missing genes” and identified a novel enzyme, TrpFCtL2, which has broad substrate specificity. TrpFCtL2, in combination with GTP cyclohydrolase II (RibA), the first enzyme of riboflavin synthesis, provides a bypass of the first two canonical steps in folate synthesis catalyzed by FolE and FolQ. Notably, TrpFCtL2 retains the phosphoribosyl anthranilate isomerase activity of the original annotation. Additionally, we independently confirmed the recent discovery of a novel enzyme, CT610, which uses an unknown precursor to synthesize PABA and complements E. coli mutants with deletions of pabA, pabB, or pabC. Thus, Chlamydia species have evolved a variant folate synthesis pathway that employs a patchwork of promiscuous and adaptable enzymes recruited from other biosynthetic pathways.

Oxalyl-Coenzyme A Reduction to Glyoxylate Is the Preferred Route of Oxalate Assimilation in Methylobacterium extorquens AM1

Oxalate catabolism is conducted by phylogenetically diverse organisms, including Methylobacterium extorquens AM1. Here, we investigate the central metabolism of this alphaproteobacterium during growth on oxalate by using proteomics, mutant characterization, and (13)C-labeling experiments. Our results confirm that energy conservation proceeds as previously described for M. extorquens AM1 and other characterized oxalotrophic bacteria via oxalyl-coenzyme A (oxalyl-CoA) decarboxylase and formyl-CoA transferase and subsequent oxidation to carbon dioxide via formate dehydrogenase. However, in contrast to other oxalate-degrading organisms, the assimilation of this carbon compound in M. extorquens AM1 occurs via the operation of a variant of the serine cycle as follows: oxalyl-CoA reduction to glyoxylate and conversion to glycine and its condensation with methylene-tetrahydrofolate derived from formate, resulting in the formation of C3 units. The recently discovered ethylmalonyl-CoA pathway operates during growth on oxalate but is nevertheless dispensable, indicating that oxalyl-CoA reductase is sufficient to provide the glyoxylate required for biosynthesis. Analysis of an oxalyl-CoA synthetase- and oxalyl-CoA-reductase-deficient double mutant revealed an alternative, although less efficient, strategy for oxalate assimilation via one-carbon intermediates. The alternative process consists of formate assimilation via the tetrahydrofolate pathway to fuel the serine cycle, and the ethylmalonyl-CoA pathway is used for glyoxylate regeneration. Our results support the notion that M. extorquens AM1 has a plastic central metabolism featuring multiple assimilation routes for C1 and C2 substrates, which may contribute to the rapid adaptation of this organism to new substrates and the eventual coconsumption of substrates under environmental conditions.

Functional Promiscuity of the COG0720 Family

The biosynthesis of GTP derived metabolites such as tetrahydrofolate (THF), biopterin (BH(4)), and the modified tRNA nucleosides queuosine (Q) and archaeosine (G(+)) relies on several enzymes of the Tunnel-fold superfamily. A subset of these proteins includes the 6-pyruvoyltetrahydropterin (PTPS-II), PTPS-III, and PTPS-I homologues, all members of the COG0720 family that have been previously shown to transform 7,8-dihydroneopterin triphosphate (H(2)NTP) into different products. PTPS-II catalyzes the formation of 6-pyruvoyltetrahydropterin in the BH(4) pathway, PTPS-III catalyzes the formation of 6-hydroxylmethyl-7,8-dihydropterin in the THF pathway, and PTPS-I catalyzes the formation of 6-carboxy-5,6,7,8-tetrahydropterin in the Q pathway. Genes of these three enzyme families are often misannotated as they are difficult to differentiate by sequence similarity alone. Using a combination of physical clustering, signature motif, phylogenetic codistribution analyses, in vivo complementation studies, and in vitro enzymatic assays, a complete reannotation of the COG0720 family was performed in prokaryotes. Notably, this work identified and experimentally validated dual function PTPS-I/III enzymes involved in both THF and Q biosynthesis. Both in vivo and in vitro analyses showed that the PTPS-I family could tolerate a translation of the active site cysteine and was inherently promiscuous, catalyzing different reactions on the same substrate or the same reaction on different substrates. Finally, the analysis and experimental validation of several archaeal COG0720 members confirmed the role of PTPS-I in archaeosine biosynthesis and resulted in the identification of PTPS-III enzymes with variant signature sequences in Sulfolobus species. This study reveals an expanded versatility of the COG0720 family members and illustrates that for certain protein families extensive comparative genomic analysis beyond homology is required to correctly predict function.

Comparative mammalian choline metabolism with emphasis on the high-yielding dairy cow

The present review examines the importance of choline in dairy cow nutrition. Choline is an essential nutrient for mammals when excess methionine and folate are not available in the diet. The requirement for choline can be met by dietary choline and by transmethylation reactions. Two types of functions for choline are known: functions of choline per se; functions as a methyl donor. The two principal methyl donors in animal metabolism are betaine, a metabolite of choline, and S-adenosyl-methionine, a metabolite of methionine. Choline and methionine are interchangeable with regard to their methyl group-furnishing functions. In adult ruminants, choline is extensively degraded in the rumen; for this reason dietary choline contributes insignificantly to the choline body pool and methyl group metabolism is generally conservative with a relatively low rate of methyl catabolism and an elevated rate of de novo synthesis of methyl groups via the tetrahydrofolate system. In dairy ruminants, the dietary availability of choline is still low, but the output of methylated compounds in milk is high, and precursors from the tetrahydrofolate pathway are limiting, especially at the onset of lactation. Therefore choline may be a limiting nutrient for milk production in high-yielding dairy cows.

Biosynthesis and fate of compatible solutes in extremely halophilic phototrophic eubacteria

Most halophilic and halotolerant eubacteria are able to accumulate compatible solutes from their environment during salt stress. They are usually able to synthesize sugars and amino acids. These compatible solutes, however, are of less importance in extremely halophilic eubacteria, where usually glycinebetaine or ectoine is required. Extremely halophilic phototrophic sulfur bacteria of the genus Ectothiorhodospira are able to synthesize the three compatible solutes glycinebetaine, trehalose and ectoine. While glycinebetaine is the major compatible solute under all conditions the percentage of trehalose and ectoine varies depending on the availability of nitrogen sources. Using acetate plus bicarbonate as simultaneous substrates the three compatible solutes were analysed by 13C-NMR spectroscopy. The label found indicated the following biosynthesis pathways: glycine, derived from glyoxylate out of the Kornberg cycle, undergoes a three-fold methylation with S-adenosylmethionine as methyl donor. The latter is derived from the tetrahydrofolate pathway. Several enzymes of this pathway have been found and are under investigation. The labelling of trehalose indicates that the Calvin cycle is blocked in the presence of acetate. The two glucose moieties of trehalose are linked by trehalose-6-phosphate synthase. The enzyme was characterized. Ectoine is synthesized from aspartate via aspartophosphate, aspartate semialdehyde and α, γ-diaminobutyrate. Dilution stress leads to rapid excretion of betaine and ectoine, followed by immediate uptake to balance overshoot excretion. Trehalose is not excreted under dilution stress but is degraded by trehalase and subsequently metabolized.

Biosynthesis and fate of compatible solutes in extremely halophilic phototrophic eubacteria

Most halophilic and halotolerant eubacteria are able to accumulate compatible solutes from their environment during salt stress. They are usually able to synthesize sugars and amino acids. These compatible solutes, however, are of less importance in extremely halophilic eubacteria, where usually glycinebetaine or ectoine is required. Extremely halophilic phototrophic sulfur bacteria of the genus Ectothiorhodospira are able to synthesize the three compatible solutes glycinebetaine, trehalose and ectoine. While glycinebetaine is the major compatible solute under all conditions the percentage of trehalose and ectoine varies depending on the availability of nitrogen sources. Using acetate plus bicarbonate as simultaneous substrates the three compatible solutes were analysed by 13C-NMR spectroscopy. The label found indicated the following biosynthesis pathways: glycine, derived from glyoxylate out of the Kornberg cycle, undergoes a three-fold methylation with S-adenosylmethionine as methyl donor. The latter is derived from the tetrahydrofolate pathway. Several enzymes of this pathway have been found and are under investigation. The labelling of trehalose indicates that the Calvin cycle is blocked in the presence of acetate. The two glucose moieties of trehalose are linked by trehalose-6-phosphate synthase. The enzyme was characterized. Ectoine is synthesized from aspartate via aspartophosphate, aspartate semialdehyde and α, β-diaminobutyrate. Dilution stress leads to rapid excretion of betaine and ectoine, followed by immediate uptake to balance overshoot excretion. Trehalose is not excreted under dilution stress but is degraded by trehalase and subsequently metabolized.

Metabolism of One-Carbon Compounds by the Ruminal Acetogen Syntrophococcus sucromutans

Syntrophococcus sucromutans is the predominant species capable of O demethylation of methoxylated lignin monoaromatic derivatives in the rumen. The enzymatic characterization of this acetogen indicated that it uses the acetyl coenzyme A (Wood) pathway. Cell extracts possess all the enzymes of the tetrahydrofolate pathway, as well as carbon monoxide dehydrogenase, at levels similar to those of other acetogens using this pathway. However, formate dehydrogenase could not be detected in cell extracts, whether formate or a methoxyaromatic was used as electron acceptor for growth of the cells on cellobiose. Labeled bicarbonate, formate, [1-C] pyruvate, and chemically synthesized O-[methyl-C]vanillate were used to further investigate the catabolism of one-carbon (C(1)) compounds by using washed-cell preparations. The results were consistent with little or no contribution of formate dehydrogenase and pointed out some unique features. Conversion of formate to CO(2) was detected, but labeled formate predominantly labeled the methyl group of acetate. Labeled CO(2) readily exchanged with the carboxyl group of pyruvate but not with formate, and both labeled CO(2) and pyruvate predominantly labeled the carboxyl group of acetate. No CO(2) was formed from O demethylation of vanillate, and the acetate produced was position labeled in the methyl group. The fermentation pattern and specific activities of products indicated a complete synthesis of acetate from pyruvate and the methoxyl group of vanillate.

Tetrahydrofolate enzyme levels in Acetobacterium woodii and their implication in the synthesis of acetate from CO2.

Acetate synthesis from CO2 by Acetobacterium woodii may occur as in homoacetate-fermenting clostridia, as indicated by high levels of enzymes of the tetrahydrofolate pathway and by pyruvate-dependent formation of acetate from methyl-B12 and methyltetrahydrofolate.