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Recombinant 10-formyltetrahydrofolate dehydrogenase catalyses both dehydrogenase and hydrolase reactions utilizing the synthetic substrate 10-formyl-5,8-dideazafolate.

ABSTRACT: 10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6) is a bifunctional enzyme, displaying both NADP(+)-dependent dehydrogenase activity for the formation of tetrahydrofolate and CO2, and NADP(+)-independent hydrolase activity for the formation of tetrahydrofolate and formate. A previous report [Case, Kaisaki and Steele (1988) J. Biol. Chem. 263, 1024-1027] claimed that dehydrogenase and hydrolase activities were products of separate cytosolic and mitochondrial forms of this enzyme. Here we report that recombinant 10-formyltetrahydrofolate dehydrogenase carries out both enzymic reactions, proving that a product of a single gene, i.e. one protein, not two, has both activities. The stable synthetic analogue 10-formyl-5,8-dideazafolate can substitute for the labile natural substrate, 10-formyltetrahydrofolate, in both reactions. This was shown with both native and recombinant rat liver enzyme. The Km values for 10-formyl-5,8-dideazafolate were half of those for 10-formyltetrahydrofolate in both the dehydrogenase and hydrolytic reactions. The Vmax, values were similar for both substrates. Both dehydrogenase and hydrolase reactions were dependent on the presence of 2-mercaptoethanol. The pH optima were 7.8 and 5.6 for the dehydrogenase and hydrolase reactions respectively, consistent with the presence of two active sites in the enzyme.

SUBMITTER: Krupenko SA

PROVIDER: S-EPMC1136571 | biostudies-other | 1995 Mar

REPOSITORIES: biostudies-other

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ALDH1L2 is the mitochondrial homolog of 10-formyltetrahydrofolate dehydrogenase.

Project description:Cytosolic 10-formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an abundant enzyme of folate metabolism. It converts 10-formyltetrahydrofolate to tetrahydrofolate and CO(2) in an NADP(+)-dependent reaction. We have identified a gene at chromosome locus 12q24.11 of the human genome, the product of which has 74% sequence similarity with cytosolic FDH. This protein has an extra N-terminal sequence of 22 amino acid residues, predicted to be a mitochondrial translocation signal. Transfection of COS-7 or A549 cell lines with a construct in which green fluorescent protein was introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH) has demonstrated mitochondrial localization of the fusion protein, suggesting that the identified gene encodes a mitochondrial enzyme. Purified pig liver mtFDH displayed dehydrogenase/hydrolase activities similar to cytosolic FDH. Real-time PCR performed on an array of human tissues has shown that although cytosolic FDH mRNA is highest in liver, kidney, and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart, and brain. In contrast to the cytosolic enzyme, which is not detectable in cancer cells, the presence of mtFDH was demonstrated in several human cancer cell lines by conventional and real-time PCR and by Western blot. Analysis of genomes of different species indicates that the mitochondrial enzyme is a later evolutionary product when compared with the cytosolic enzyme. We propose that this novel mitochondrial enzyme is a likely source of CO(2) production from 10-formyltetrahydrofolate in mitochondria and plays an essential role in the distribution of one-carbon groups between the cytosolic and mitochondrial compartments of the cell.

| S-EPMC2906299 | biostudies-literature

Enzymatic properties of ALDH1L2, a mitochondrial 10-formyltetrahydrofolate dehydrogenase.

Project description:10-Formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1), an abundant cytosolic enzyme of folate metabolism, shares significant sequence similarity with enzymes of the aldehyde dehydrogenase (ALDH) family. The enzyme converts 10-formyltetrahydrofolate (10-fTHF) to tetrahydrofolate and CO(2) in an NADP(+)-dependent manner. The mechanism of this reaction includes three consecutive steps with the final occurring in an ALDH-homologous domain. We have recently identified a mitochondrial isoform of FDH (mtFDH), which is the product of a separate gene, ALDH1L2. Its overall identity to cytosolic FDH is about 74%, and the identity between the ALDH domains rises up to 79%. In the present study, human mtFDH was expressed in Escherichia coli, purified to homogeneity, and characterized. While the recombinant enzyme was capable of catalyzing the 10-fTHF hydrolase reaction, it did not produce detectable levels of ALDH activity. Despite the lack of typical ALDH catalysis, mtFDH was able to perform the characteristic 10-fTHF dehydrogenase reaction after reactivation by recombinant 4'-phosphopantetheinyl transferase (PPT) in the presence of coenzyme A. Using site-directed mutagenesis, it was determined that PPT modifies mtFDH specifically at Ser375. The C-terminal domain of mtFDH (residues 413-923) was also expressed in E. coli and characterized. This domain was found to exist as a tetramer and to catalyze an esterase reaction that is typical of other ALDH enzymes. Taken together, our studies suggest that ALDH1L2 has enzymatic properties similar to its cytosolic counterpart, although the inability to catalyze the ALDH reaction with short-chain aldehyde substrates remains an unresolved issue at present.

| S-EPMC3103650 | biostudies-literature

Cytosolic 10-formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver.

Project description:ALDH1L1 (10-formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism highly expressed in liver, metabolizes 10-formyltetrahydrofolate to produce tetrahydrofolate (THF). This reaction might have a regulatory function towards reduced folate pools, de novo purine biosynthesis, and the flux of folate-bound methyl groups. To understand the role of the enzyme in cellular metabolism, Aldh1l1-/- mice were generated using an ES cell clone (C57BL/6N background) from KOMP repository. Though Aldh1l1-/- mice were viable and did not have an apparent phenotype, metabolomic analysis indicated that they had metabolic signs of folate deficiency. Specifically, the intermediate of the histidine degradation pathway and a marker of folate deficiency, formiminoglutamate, was increased more than 15-fold in livers of Aldh1l1-/- mice. At the same time, blood folate levels were not changed and the total folate pool in the liver was decreased by only 20%. A two-fold decrease in glycine and a strong drop in glycine conjugates, a likely result of glycine shortage, were also observed in Aldh1l1-/- mice. Our study indicates that in the absence of ALDH1L1 enzyme, 10-formyl-THF cannot be efficiently metabolized in the liver. This leads to the decrease in THF causing reduced generation of glycine from serine and impaired histidine degradation, two pathways strictly dependent on THF.

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Ethanol-induced upregulation of 10-formyltetrahydrofolate dehydrogenase helps relieve ethanol-induced oxidative stress.

Project description:Alcoholism induces folate deficiency and increases the risk for embryonic anomalies. However, the interplay between ethanol exposure and embryonic folate status remains unclear. To investigate how ethanol exposure affects embryonic folate status and one-carbon homeostasis, we incubated zebrafish embryos in ethanol and analyzed embryonic folate content and folate enzyme expression. Exposure to 2% ethanol did not change embryonic total folate content but increased the tetrahydrofolate level approximately 1.5-fold. The expression of 10-formyltetrahydrofolate dehydrogenase (FDH), a potential intracellular tetrahydrofolate reservoir, was increased in both mRNA and protein levels. Overexpressing recombinant FDH in embryos alleviated the ethanol-induced oxidative stress in ethanol-exposed embryos. Further characterization of the zebrafish fdh promoter revealed that the -124/+40 promoter fragment was the minimal region required for transactivational activity. The results of site-directed mutagenesis and binding analysis revealed that Sp1 is involved in the basal level of expression of fdh but not in ethanol-induced upregulation of fdh. On the other hand, CEBP? was the protein that mediated the ethanol-induced upregulation of fdh, with an approximately 40-fold increase of fdh promoter activity when overexpressed in vitro. We concluded that upregulation of fdh involving CEBP? helps relieve embryonic oxidative stress induced by ethanol exposure.

| S-EPMC3911513 | biostudies-literature

Structures of the hydrolase domain of zebrafish 10-formyltetrahydrofolate dehydrogenase and its complexes reveal a complete set of key residues for hydrolysis and product inhibition.

Project description:10-Formyltetrahydrofolate dehydrogenase (FDH), which is composed of a small N-terminal domain (Nt-FDH) and a large C-terminal domain, is an abundant folate enzyme in the liver and converts 10-formyltetrahydrofolate (10-FTHF) to tetrahydrofolate (THF) and CO2. Nt-FDH alone possesses a hydrolase activity, which converts 10-FTHF to THF and formate in the presence of β-mercaptoethanol. To elucidate the catalytic mechanism of Nt-FDH, crystal structures of apo-form zNt-FDH from zebrafish and its complexes with the substrate analogue 10-formyl-5,8-dideazafolate (10-FDDF) and with the products THF and formate have been determined. The structures reveal that the conformations of three loops (residues 86-90, 135-143 and 200-203) are altered upon ligand (10-FDDF or THF) binding in the active site. The orientations and geometries of key residues, including Phe89, His106, Arg114, Asp142 and Tyr200, are adjusted for substrate binding and product release during catalysis. Among them, Tyr200 is especially crucial for product release. An additional potential THF binding site is identified in the cavity between two zNt-FDH molecules, which might contribute to the properties of product inhibition and THF storage reported for FDH. Together with mutagenesis studies and activity assays, the structures of zNt-FDH and its complexes provide a coherent picture of the active site and a potential THF binding site of zNt-FDH along with the substrate and product specificity, lending new insights into the molecular mechanism underlying the enzymatic properties of Nt-FDH.

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Cancer cells activate p53 in response to 10-formyltetrahydrofolate dehydrogenase expression.

Project description:A folate enzyme, FDH (10-formyltetrahydrofolate dehydrogenase; EC 1.5.1.6), is not a typical tumour suppressor, but it has two basic characteristics of one, i.e. it is down-regulated in tumours and its expression is selectively cytotoxic to cancer cells. We have recently shown that ectopic expression of FDH in A549 lung cancer cells induces G1 arrest and apoptosis that was accompanied by elevation of p53 and its downstream target, p21. It was not known, however, whether FDH-induced apoptosis is p53-dependent or not. In the present study, we report that FDH-induced suppressor effects are strictly p53-dependent in A549 cells. Both knockdown of p53 using an RNAi (RNA interference) approach and disabling of p53 function by dominant-negative inhibition with R175H mutant p53 prevented FDH-induced cytotoxicity in these cells. Ablation of the FDH-suppressor effect is associated with an inability to activate apoptosis in the absence of functional p53. We have also shown that FDH elevation results in p53 phosphorylation at Ser-6 and Ser-20 in the p53 transactivation domain, and Ser-392 in the C-terminal domain, but only Ser-6 is strictly required to mediate FDH effects. Also, translocation of p53 to the nuclei and expression of the pro-apoptotic protein PUMA (Bcl2 binding component 3) was observed after induction of FDH expression. Elevation of FDH in p53 functional HCT116 cells induced strong growth inhibition, while growth of p53-deficient HCT116 cells was unaffected. This implies that activation of p53-dependent pathways is a general downstream mechanism in response to induction of FDH expression in p53 functional cancer cells.

| S-EPMC1276951 | biostudies-other

Bile salt hydrolase catalyses formation of amine-conjugated bile acids.

Project description:Bacteria in the gastrointestinal tract produce amino acid bile acid amidates that can affect host-mediated metabolic processes1-6; however, the bacterial gene(s) responsible for their production remain unknown. Herein, we report that bile salt hydrolase (BSH) possesses dual functions in bile acid metabolism. Specifically, we identified a previously unknown role for BSH as an amine N-acyltransferase that conjugates amines to bile acids, thus forming bacterial bile acid amidates (BBAAs). To characterize this amine N-acyltransferase BSH activity, we used pharmacological inhibition of BSH, heterologous expression of bsh and mutants in Escherichia coli and bsh knockout and complementation in Bacteroides fragilis to demonstrate that BSH generates BBAAs. We further show in a human infant cohort that BBAA production is positively correlated with the colonization of bsh-expressing bacteria. Lastly, we report that in cell culture models, BBAAs activate host ligand-activated transcription factors including the pregnane X receptor and the aryl hydrocarbon receptor. These findings enhance our understanding of how gut bacteria, through the promiscuous actions of BSH, have a significant role in regulating the bile acid metabolic network.

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Abnormal downregulation of 10-formyltetrahydrofolate dehydrogenase promotes the progression of oral squamous cell carcinoma by activating PI3K/Akt/Rb pathway.

Project description:Background10-formyltetrahydrofolate dehydrogenase (ALDH1L1) is a major folate enzyme, which is usually underexpressed in malignant tumors and competes with tumors for the same folate substrate. However, the specific role and mechanisms of ALDH1L1 in oral squamous cell carcinoma (OSCC) remainsobscure.MethodsThe expression level of ALDH1L1 in paired OSCC tissues and adjacent noncancerous tissues were detected by quantitative realtime PCR, Western blot and immunohistochemistry. The relationship between ALDH1L1 expression and clinical characteristics was analyzed. Besides, CCK8, EdU staining, colony formation, wound healing, transwell invasion, apoptosis, cell cycle assays and nude mice tumor bearing experiments were employed to assess the role of ALDH1L1 in OSCC. To explore the underlying mechanisms of these effects, cell cycle-related markers were examined.ResultsIn this study, we revealed that ALDH1L1 expression was significantly reduced in OSCC, and its downregulation was associated with the malignancy of the tumor and poor prognosis of patients. In vivo and in vitro experiments, downregulation of ALDH1L1 in OSCC significantly inhibited the occurrence of NADP+ -dependent catalytic reactions and facilitated tumor cell growth, migration, invasion, survival, cell cycle progression, and xenograft tumor growth. On the contrary, re-expression of ALDH1L1 plays a similar role to anti-folate therapy, promoting NADPH production and suppressing the progression of OSCC. Furthermore, ALDH1L1 overexpressing obviously inhibited the expression of PI3K, p-Akt, CDK2, CDK6, Cyclin D1, Cyclin D3, and Rb in OSCC cells, and promoted the expression of p27. LY294002 and 740 Y-P were used to confirm the inhibitory effects of ALDH1L1 on OSCC progression through PI3K/Akt/Rb pathway.ConclusionOur findings highlight the clinical value of ALDH1L1 as a prognostic marker and the potential of a new target for anti-folate therapy.

| S-EPMC10028165 | biostudies-literature

A Detoxification Enzyme for <i>Apis mellifera</i> Newly Characterized by Recombinant Expression: 10-Formyl Tetrahydrofolate Dehydrogenase.

Project description:Honeybees are important managed pollinators that perform important ecological and economic functions. In recent decades, the obligate ectoparasite Varroa destructor severely affected survival of honeybees as it either feeds on hemolymph and fat bodies or acts as a vector for viruses. A common treatment against the varroa mite is formic acid, which has been used for many years by beekeepers. This treatment is known to be effective, but the therapeutic index is very narrow. Many beekeepers report negative effects of formic acid on bees, which include damage to brood, worker bee mortality, and queen loss. Little is yet known about the molecular mechanisms of formic acid detoxification in honeybees. Our previous study shows the upregulation of predicted 10-formyl tetrahydrofolate dehydrogenase (10-FTHFDH) transcripts in honeybees exposed to formic acid. Here, the predicted honeybee-specific 10-FTHFDH is recombinantly expressed, and its hydrolase and dehydrogenase activities are investigated. As a result, the enzyme shows similar dehydrogenase activity in comparison to known 10-FTHFDHs. This study provides further knowledge to better understand the detoxification mechanisms of formic acid in Apis mellifera.

| S-EPMC10926475 | biostudies-literature

A Cu(II)-ATP complex efficiently catalyses enantioselective Diels-Alder reactions.

Project description:Natural biomolecules have been used extensively as chiral scaffolds that bind/surround metal complexes to achieve stereoselectivity in catalytic reactions. ATP is ubiquitously found in nature as an energy-storing molecule and can complex diverse metal cations. However, in biotic reactions ATP-metal complexes are thought to function mostly as co-substrates undergoing phosphoanhydride bond cleavage reactions rather than participating in catalytic mechanisms. Here, we report that a specific Cu(II)-ATP complex (Cu2+·ATP) efficiently catalyses Diels-Alder reactions with high reactivity and enantioselectivity. We investigate the substrates and stereoselectivity of the reaction, characterise the catalyst by a range of physicochemical experiments and propose the reaction mechanism based on density functional theory (DFT) calculations. It is found that three key residues (N7, β-phosphate and γ-phosphate) in ATP are important for the efficient catalytic activity and stereocontrol via complexation of the Cu(II) ion. In addition to the potential technological uses, these findings could have general implications for the chemical selection of complex mixtures in prebiotic scenarios.

| S-EPMC7508818 | biostudies-literature

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