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Sergey A. Krupenko - One of the best experts on this subject based on the ideXlab platform.
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Cytosolic 10-Formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver
Scientific Reports, 2019Co-Authors: Natalia I. Krupenko, Jaspreet Sharma, Peter Pediaditakis, Baharan Fekry, Kristi L. Helke, Xiuxia Du, Susan Sumner, Sergey A. KrupenkoAbstract: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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Impact of Aldh1l1 Knockout On Metabolic Phenotype in Mouse Liver
The FASEB Journal, 2019Co-Authors: Jaspreet Sharma, Natalia I. Krupenko, Sergey A. KrupenkoAbstract:ALDH1L1 (10-Formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in liver. This enzyme metabolizes 10-Formyltetrahydrofolate to produce tetrahydrofolate (THF) ...
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Enzymatic properties of ALDH1L2, a mitochondrial 10-Formyltetrahydrofolate dehydrogenase.
Chemico-biological interactions, 2011Co-Authors: Kyle C. Strickland, Natalia I. Krupenko, Yaroslav Tsybovsky, Marianne E. Dubard, Sergey A. KrupenkoAbstract: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.
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ALDH1L2 Is the Mitochondrial Homolog of 10-Formyltetrahydrofolate Dehydrogenase
The Journal of biological chemistry, 2010Co-Authors: Natalia I. Krupenko, Natalia V. Oleinik, Marianne E. Dubard, Kyle C. Strickland, Kelly Moxley, Sergey A. KrupenkoAbstract:Cytosolic 10-Formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an abundant enzyme of folate metabolism. It converts 10-Formyltetrahydrofolate to tetrahydrofolate and CO2 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 CO2 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.
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Abstract 73: Mitochondrial 10-Formyltetrahydrofolate dehydrogenase: A novel enzyme in folate metabolism
Cellular and Molecular Biology, 2010Co-Authors: Marianne E. Dubard, Natalia I. Krupenko, Natalia V. Oleinik, Kyle C. Strickland, Sergey A. KrupenkoAbstract:Cytosolic 10-Formyltetrahydrofolate dehydrogenase (FDH, Aldh1L1) is an important regulator of intracellular folate pools, which displays antiproliferative effects in cancer cells. We have identified a gene at the chromosome locus 12q24.11 in the human genome, the product of which has 87% sequence similarity with cytosolic FDH. This protein has an extra amino-terminal sequence of 22 amino acid residues enriched in arginines, which is predicted to be a mitochondrial translocation signal. The mitochondrial targeting function of the leader has been confirmed in Cos7 cells: green fluorescent protein (GFP)-tagged at the amino-terminus with the leader, localizes to mitochondria. Transfection of Cos7 or A549 cell lines with a construct, in which GFP has been introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH), has also shown mitochondrial localization, suggesting that the identified gene encodes a mitochondrial enzyme. To evaluate the abundance of mtFDH, we have measured its mRNA levels in a wide array of human tissues by real-time PCR, and compared them to the levels of mRNA that encode cytosolic FDH. While cytosolic FDH mRNA is highest in liver, kidney and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart and brain, but not in liver or kidney. In contrast to the cytosolic enzyme, which is non detectable in human cancer cell lines, the presence of mtFDH mRNA was demonstrated in A549 and PC3 cells by conventional and real-time PCR. The presence of the endogenous enzyme in mitochondria of A549 cells has been further confirmed using specific polyclonal antibody generated against purified recombinant mtFDH. We have also shown that recombinant mtFDH, similar to the cytosolic enzyme, catalyzes NADP + -dependent oxidation of the 10-Formyltetrahydrofolate to tetrahydrofolate and CO 2 . Thus, the enzyme is a likely source of CO 2 production in mitochondria and we propose that it plays an essential role in distribution of one-carbon groups between cytosolic and mitochondrial compartments of the cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 73.
Robert J. Cook - One of the best experts on this subject based on the ideXlab platform.
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Identification of protein-arginine N-methyltransferase as 10-Formyltetrahydrofolate dehydrogenase
The Journal of biological chemistry, 1998Co-Authors: Sangduk Kim, Robert J. Cook, Gil Hong Park, Won A. Joo, Woon Ki Paik, Kenneth R. WilliamsAbstract:S-Adenosylmethionine:protein-arginine N-methyltransferase (EC 2.1.1. 23; protein methylase I) transfers the methyl group of S-adenosyl-L-methionine to an arginine residue of a protein substrate. The homogeneous liver protein methylase I was subjected to tryptic digestion followed by reverse phase high performance liquid chromatography (HPLC) separation and either "on-line" mass spectrometric fragmentation or "off-line" Edman sequencing of selected fractions. Data base searching of both the mass spectrometric and Edman sequencing data from several peptides identified the protein methylase as 10-Formyltetrahydrofolate dehydrogenase (EC 1.5.1.6; Cook, R. J., Lloyd, R. S., and Wagner, C. (1991) J. Biol. Chem. 266, 4965-4973; Swiss accession number). This identification was confirmed by comparative HPLC tryptic peptide mapping and affinity chromatography of the methylase on the 5-formyltetrahydrofolate-Sepharose affinity gel used to purify the dehydrogenase. The purified rat liver methylase had approximately 33% of the 10-Formyltetrahydrofolate dehydrogenase and 36% of the aldehyde dehydrogenase activity as compared with the recombinant dehydrogenase, which also had protein methylase I activity. Polyclonal antibodies against recombinant dehydrogenase reacted with protein methylase I purified either by polyacrylamide gel electrophoresis or 5-formyltetrahydrofolate affinity chromatography. In each instance there was only a single immunoreactive band at a molecular weight of approximately 106,000. Together, these results confirm the co-identity of protein-arginine methyltransferase and 10-Formyltetrahydrofolate dehydrogenase.
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DOMAIN STRUCTURE OF RAT 10-Formyltetrahydrofolate DEHYDROGENASE : RESOLUTION OF THE AMINO-TERMINAL DOMAIN AS 10-Formyltetrahydrofolate HYDROLASE
The Journal of biological chemistry, 1997Co-Authors: Sergey A. Krupenko, Conrad Wagner, Robert J. CookAbstract:We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-Formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-Formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-Formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-Formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.
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Expression, Purification, and Properties of the Aldehyde Dehydrogenase Homologous Carboxyl-terminal Domain of Rat 10-Formyltetrahydrofolate Dehydrogenase
The Journal of biological chemistry, 1997Co-Authors: Sergey A. Krupenko, Conrad Wagner, Robert J. CookAbstract:Abstract The liver cytosolic enzyme, 10-Formyltetrahydrofolate dehydrogenase (FDH) (EC 1.5.1.6) catalyzes two reactions: the NADP+-dependent oxidation of 10-Formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP+-independent hydrolysis of 10-Formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the enzyme (residues 420-902) is about 48% identical to a family of NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3), and FDH possesses aldehyde dehydrogenase activity. We expressed the COOH-terminal domain (residues 420-902) of FDH in insect cells using a baculovirus expression system. The recombinant protein was released from insect cells to the culture medium and was purified from the medium by a two-step procedure: precipitation with 35% saturated ammonium sulfate followed by chromatography on hydroxyapatite. The purified COOH-terminal domain displayed aldehyde dehydrogenase activity similar to that of native FDH but had neither dehydrogenase nor hydrolase activity toward folate substrates. Aldehyde dehydrogenase activity of the COOH-terminal domain and FDH was independent of the presence of 2-mercaptoethanol while 10-FDDF dehydrogenase activity of FDH occurred only in the presence of 2-mercaptoethanol. The COOH-terminal domain existed as a tetramer showing that the sites for oligomerization of subunits in native FDH resides in this domain. Using titration of tryptophan fluorescence, it was found that the COOH-terminal domain bound NADP+ to the same extent as FDH (Kd 0.2 and 0.3 μM, respectively) but did not bind folate. Both FDH and its COOH-terminal domain also bound NAD+ (Kd 11 and 16 μM, respectively) as measured by fluorescence titration. Both proteins were able to catalyze the aldehyde dehydrogenase reaction utilizing NADP+ or NAD+, but the Km for NAD+ was three orders higher than that for NADP+ (2 mM and 1.5-2.0 μM, respectively). The concentration of NAD+ required for the reaction was high compared with the physiological level of NAD+, suggesting that the reaction does not occur in vivo NAD+ at physiological concentrations stimulated the aldehyde dehydrogenase reaction performed by FDH or its COOH-terminal domain using NADP+.
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covalent binding of acetaminophen to n 10 formyl tetrahydrofolate dehydrogenase in mice
Journal of Pharmacology and Experimental Therapeutics, 1997Co-Authors: Neil R Pumford, Robert J. Cook, Conrad Wagner, Christine N Halmes, Brian M Martin, Jack A HinsonAbstract:The analgesic acetaminophen is frequently used as a model chemical to study hepatotoxicity; however, the critical mechanisms by which it produces toxicity within the cell are unknown. It has been postulated that covalent binding of a toxic metabolite to crucial proteins may inhibit vital cellular functions and may be responsible for, or contribute to, the hepatotoxicity. To further understand the importance of covalent binding in the toxicity, a major cytosolic acetaminophen-protein adduct of 100 kDa has been purified by a combination of anion exchange chromatography and preparative electrophoresis. N-Terminal and internal amino acid sequences of peptides from the purified 100-kDa acetaminophen-protein adduct were found to be homologous with the deduced amino acid sequence from the cDNA of N-10-Formyltetrahydrofolate dehydrogenase. Antiserum specific for N-10-Formyltetrahydrofolate dehydrogenase and acetaminophen react in a Western blot with the purified 100-kDa acetaminophen-protein adduct. Administration of a toxic dose of acetaminophen (400 mg/kg) to mice resulted in a 25% decrease in cytosolic N-10-Formyltetrahydrofolate dehydrogenase activity at 2 hr. The covalent binding of acetaminophen to proteins such as N-10-Formyltetrahydrofolate dehydrogenase and the subsequent decreases in their enzyme activity may play a role in acetaminophen hepatotoxicity.
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Use of 10-formyl-5,8-dideazafolate as substrate for rat 10-Formyltetrahydrofolate dehydrogenase.
Methods in enzymology, 1997Co-Authors: Robert J. CookAbstract:Publisher Summary This chapter discusses the use of 10-formyl-5,8-dideazafolate as substrate for rat 10-Formyltetrahydrofolate dehydrogenase. 10-Formyltetrahydrofolate (10-HCO-H 4 PteGlu) is susceptible to oxidative degradation and must be protected by reducing agents during synthesis and use in enzyme assays. The (6R,S)-10-HCO-H4PteGlu, while highly unstable, is easily generated from stable, commercially available (6R,S)-5-HCO-HaPteGlu in the presence of 2-mercaptoethanol (2-ME), using the method of Rabinowitz. The 10-formyl-5,8-dideazafolate was originally synthesized as a quinazoline analog of folic acid. It was found to be a modest inhibitor of rat liver dihydrofolate reductase and had activity against L1210 leukemia in mice. The oxidation of 10-formyl-5,8-dideazafolate is followed by the production of 5,8-dideazafolate at 295 nm 6 or NADPH at 340 nm. The absorption of 5,8-dideazafolate at 340 nm is reflected in the adjusted extinction coefficient for NADPH. The absorption of NADPH at 295 nm is approximately 2% of the extinction coefficient for 5,8-dideazafolate and is ignored. Hydrolase activity is followed by the production of 5,8-dideazafolate at 295 nm in the presence of 100 m M 2-ME. The assay may be run at room temperature. Assays of crude extracts require the removal of low molecular weight compounds by spin column desalting.
Conrad Wagner - One of the best experts on this subject based on the ideXlab platform.
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On the role of conserved histidine 106 in 10-Formyltetrahydrofolate dehydrogenase catalysis: connection between hydrolase and dehydrogenase mechanisms.
The Journal of biological chemistry, 2001Co-Authors: Sergey A. Krupenko, Alexander P. Vlasov, Conrad WagnerAbstract:The enzyme, 10-Formyltetrahydrofolate dehydrogenase (FDH), converts 10-Formyltetrahydrofolate (10-formyl-THF) to tetrahydrofolate in an NADP+-dependent dehydrogenase reaction or an NADP+-independent hydrolase reaction. The hydrolase reaction occurs in a 310-amino acid long amino-terminal domain of FDH (Nt-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. The amino-terminal domain of FDH shares some sequence identity with several other enzymes utilizing 10-formyl-THF as a substrate. These enzymes have two strictly conserved residues, aspartate and histidine, in the putative catalytic center. We have shown recently that the conserved aspartate is involved in FDH catalysis. In the present work we studied the role of the conserved histidine, His106, in FDH function. Site-directed mutagenesis experiments showed that replacement of the histidine with alanine, asparagine, aspartate, glutamate, glutamine, or arginine in Nt-FDH resulted in expression of insoluble proteins. Replacement of the histidine with another positively charged residue, lysine, produced a soluble mutant with no hydrolase activity. The insoluble mutants refolded from inclusion bodies adopted a conformation inherent to the wild-type Nt-FDH, but they did not exhibit any hydrolase activity. Substitution of alanine for three non-conserved histidines located close to the conserved one did not reveal any significant changes in the hydrolase activity of Nt-FDH. Expressed full-length FDH with the substitution of lysine for the His106 completely lost both the hydrolase and dehydrogenase activities. Thus, our study showed that His106, besides being an important structural residue, is also directly involved in both the hydrolase and dehydrogenase mechanisms of FDH. Modeling of the putative hydrolase catalytic center/folate-binding site suggested that the catalytic residues, aspartate and histidine, are unlikely to be adjacent to the catalytic cysteine in the aldehyde dehydrogenase catalytic center. We hypothesize that 10-formyl-THF dehydrogenase reaction is not an independent reaction but is a combination of hydrolase and aldehyde dehydrogenase reactions.
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Aspartate 142 Is Involved in Both Hydrolase and Dehydrogenase Catalytic Centers of 10-Formyltetrahydrofolate Dehydrogenase
The Journal of biological chemistry, 1999Co-Authors: Sergey A. Krupenko, Conrad WagnerAbstract:Abstract The enzyme 10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes conversion of 10-Formyltetrahydrofolate to tetrahydrofolate in either a dehydrogenase or hydrolase reaction. The hydrolase reaction occurs in a 310-residue amino-terminal domain of FDH (Nt-FDH), whereas the dehydrogenase reaction requires the full-length enzyme. Nt-FDH shares some sequence identity with several 10-Formyltetrahydrofolate-utilizing enzymes. All these enzymes have a strictly conserved aspartate, which is Asp142 in the case of Nt-FDH. Replacement of the aspartate with alanine, asparagine, glutamate, or glutamine in Nt-FDH resulted in complete loss of hydrolase activity. All the mutants, however, were able to bind folate, although with lower affinity than wild-type Nt-FDH. Six other aspartate residues located near the conserved Asp142 were substituted with an alanine, and these substitutions did not result in any significant changes in the hydrolase activity. The expressed D142A mutant of the full-length enzyme completely lost both hydrolase and dehydrogenase activities. This study shows that Asp142 is an essential residue in the enzyme mechanism for both the hydrolase and dehydrogenase reactions of FDH, suggesting that either the two catalytic centers of FDH are overlapped or the dehydrogenase reaction occurs within the hydrolase catalytic center.
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Overexpression of functional hydrolase domain of rat liver 10-Formyltetrahydrofolate dehydrogenase in Escherichia coli.
Protein expression and purification, 1998Co-Authors: Sergey A. Krupenko, Conrad WagnerAbstract:Rat liver 10-Formyltetrahydrofolate dehydrogenase (FDH) is a tetrameric enzyme composed of four identical 902-amino-acid-residue (99 kDa) monomers. We expressed the enzyme and its 310-amino-acid-residue amino-terminal domain, which is 10-Formyltetrahydrofolate hydrolase, in Escherichia coli BL21 (DE3) cells using the pRSET expression vector. We removed the entire translated region of the vector including the polyhistidyl tag and the recombinant proteins were expressed, not as a fusion constructs, but as unmodified sequences. The expressed full-length enzyme was found to be an insoluble protein and was not purified and characterized, while the amino-terminal domain was expressed as a soluble protein possessing hydrolase activity. The recombinant amino-terminal domain was purified in one step on a DEAE MemSep 1000 HP Ion-Exchange Membrane Chromatography Cartridge (Millipore) using a ConSep LC100 chromatographic system (Millipore). The chromatography gave a homogenous and active preparation of the recombinant protein with a yield of about 2 mg per 100 ml of bacterial culture. Kinetic parameters of the hydrolase reaction displayed by the amino-terminal domain expressed in E. coli were similar to those of the recombinant full-length enzyme and its amino-terminal domain previously expressed in insect cells. The purified recombinant enzyme remained active for at least 4 weeks at 4 degreesC. These results show that the hydrolase amino-terminal domain of FDH can be overexpressed as a functional enzyme in E. coli cells and purified in one step by a simple chromatographic procedure.
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DOMAIN STRUCTURE OF RAT 10-Formyltetrahydrofolate DEHYDROGENASE : RESOLUTION OF THE AMINO-TERMINAL DOMAIN AS 10-Formyltetrahydrofolate HYDROLASE
The Journal of biological chemistry, 1997Co-Authors: Sergey A. Krupenko, Conrad Wagner, Robert J. CookAbstract:We expressed the NH2-terminal domain of the multidomain, multifunctional enzyme, 10-Formyltetrahydrofolate dehydrogenase (FDH), using a baculovirus expression system in insect cells. Expression of the 203-amino acid NH2-terminal domain (residues 1-203), which is 24-30% identical to a group of glycinamide ribonucleotide transformylases (EC 2.1.2.2), resulted in the appearance of insoluble recombinant protein apparently due to incorrect folding. The longer NH2-terminal recombinant protein (residues 1-310), which shares 32% identity with Escherichia coli L-methionyl-tRNA formyltransferase (EC 2.1.2.9), was expressed as a soluble protein. During expression, this protein was released from cells to the culture medium and was purified from the culture medium by 5-formyltetrahydrofolate-Sepharose affinity chromatography followed by chromatography on a Mono-Q column. We found that the purified NH2-terminal domain bears a folate binding site, possesses 10-Formyltetrahydrofolate hydrolase activity, and exists as a monomer. Titration of tryptophan fluorescence showed that native FDH bound both the substrate of the reaction, 10-formyl-5, 8-dideazafolate, and the product of the reaction, 5,8-dideazafolate, with the same affinities as its NH2-terminal domain did and that both proteins bound the substrate with a 50-fold higher affinity than the product. Neither the NH2-terminal domain nor its mixture with the previously purified COOH-terminal domain had 10-Formyltetrahydrofolate dehydrogenase activity. Formation of complexes between the COOH- and NH2-terminal domains also was not observed. We conclude that the 10-Formyltetrahydrofolate dehydrogenase activity of FDH is a result of the action of the aldehyde dehydrogenase catalytic center residing in the COOH-terminal domain on the substrate bound in the NH2-terminal domain and that the intermediate domain is necessary to bring the two functional domains together in the correct orientation.
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Expression, Purification, and Properties of the Aldehyde Dehydrogenase Homologous Carboxyl-terminal Domain of Rat 10-Formyltetrahydrofolate Dehydrogenase
The Journal of biological chemistry, 1997Co-Authors: Sergey A. Krupenko, Conrad Wagner, Robert J. CookAbstract:Abstract The liver cytosolic enzyme, 10-Formyltetrahydrofolate dehydrogenase (FDH) (EC 1.5.1.6) catalyzes two reactions: the NADP+-dependent oxidation of 10-Formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP+-independent hydrolysis of 10-Formyltetrahydrofolate to tetrahydrofolate and formate. The COOH-terminal domain of the enzyme (residues 420-902) is about 48% identical to a family of NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3), and FDH possesses aldehyde dehydrogenase activity. We expressed the COOH-terminal domain (residues 420-902) of FDH in insect cells using a baculovirus expression system. The recombinant protein was released from insect cells to the culture medium and was purified from the medium by a two-step procedure: precipitation with 35% saturated ammonium sulfate followed by chromatography on hydroxyapatite. The purified COOH-terminal domain displayed aldehyde dehydrogenase activity similar to that of native FDH but had neither dehydrogenase nor hydrolase activity toward folate substrates. Aldehyde dehydrogenase activity of the COOH-terminal domain and FDH was independent of the presence of 2-mercaptoethanol while 10-FDDF dehydrogenase activity of FDH occurred only in the presence of 2-mercaptoethanol. The COOH-terminal domain existed as a tetramer showing that the sites for oligomerization of subunits in native FDH resides in this domain. Using titration of tryptophan fluorescence, it was found that the COOH-terminal domain bound NADP+ to the same extent as FDH (Kd 0.2 and 0.3 μM, respectively) but did not bind folate. Both FDH and its COOH-terminal domain also bound NAD+ (Kd 11 and 16 μM, respectively) as measured by fluorescence titration. Both proteins were able to catalyze the aldehyde dehydrogenase reaction utilizing NADP+ or NAD+, but the Km for NAD+ was three orders higher than that for NADP+ (2 mM and 1.5-2.0 μM, respectively). The concentration of NAD+ required for the reaction was high compared with the physiological level of NAD+, suggesting that the reaction does not occur in vivo NAD+ at physiological concentrations stimulated the aldehyde dehydrogenase reaction performed by FDH or its COOH-terminal domain using NADP+.
Natalia I. Krupenko - One of the best experts on this subject based on the ideXlab platform.
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Cytosolic 10-Formyltetrahydrofolate dehydrogenase regulates glycine metabolism in mouse liver
Scientific Reports, 2019Co-Authors: Natalia I. Krupenko, Jaspreet Sharma, Peter Pediaditakis, Baharan Fekry, Kristi L. Helke, Xiuxia Du, Susan Sumner, Sergey A. KrupenkoAbstract: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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Impact of Aldh1l1 Knockout On Metabolic Phenotype in Mouse Liver
The FASEB Journal, 2019Co-Authors: Jaspreet Sharma, Natalia I. Krupenko, Sergey A. KrupenkoAbstract:ALDH1L1 (10-Formyltetrahydrofolate dehydrogenase), an enzyme of folate metabolism, is highly expressed in liver. This enzyme metabolizes 10-Formyltetrahydrofolate to produce tetrahydrofolate (THF) ...
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Enzymatic properties of ALDH1L2, a mitochondrial 10-Formyltetrahydrofolate dehydrogenase.
Chemico-biological interactions, 2011Co-Authors: Kyle C. Strickland, Natalia I. Krupenko, Yaroslav Tsybovsky, Marianne E. Dubard, Sergey A. KrupenkoAbstract: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.
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ALDH1L2 Is the Mitochondrial Homolog of 10-Formyltetrahydrofolate Dehydrogenase
The Journal of biological chemistry, 2010Co-Authors: Natalia I. Krupenko, Natalia V. Oleinik, Marianne E. Dubard, Kyle C. Strickland, Kelly Moxley, Sergey A. KrupenkoAbstract:Cytosolic 10-Formyltetrahydrofolate dehydrogenase (FDH, ALDH1L1) is an abundant enzyme of folate metabolism. It converts 10-Formyltetrahydrofolate to tetrahydrofolate and CO2 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 CO2 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.
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Abstract 73: Mitochondrial 10-Formyltetrahydrofolate dehydrogenase: A novel enzyme in folate metabolism
Cellular and Molecular Biology, 2010Co-Authors: Marianne E. Dubard, Natalia I. Krupenko, Natalia V. Oleinik, Kyle C. Strickland, Sergey A. KrupenkoAbstract:Cytosolic 10-Formyltetrahydrofolate dehydrogenase (FDH, Aldh1L1) is an important regulator of intracellular folate pools, which displays antiproliferative effects in cancer cells. We have identified a gene at the chromosome locus 12q24.11 in the human genome, the product of which has 87% sequence similarity with cytosolic FDH. This protein has an extra amino-terminal sequence of 22 amino acid residues enriched in arginines, which is predicted to be a mitochondrial translocation signal. The mitochondrial targeting function of the leader has been confirmed in Cos7 cells: green fluorescent protein (GFP)-tagged at the amino-terminus with the leader, localizes to mitochondria. Transfection of Cos7 or A549 cell lines with a construct, in which GFP has been introduced between the leader sequence and the rest of the putative mitochondrial FDH (mtFDH), has also shown mitochondrial localization, suggesting that the identified gene encodes a mitochondrial enzyme. To evaluate the abundance of mtFDH, we have measured its mRNA levels in a wide array of human tissues by real-time PCR, and compared them to the levels of mRNA that encode cytosolic FDH. While cytosolic FDH mRNA is highest in liver, kidney and pancreas, mtFDH mRNA is most highly expressed in pancreas, heart and brain, but not in liver or kidney. In contrast to the cytosolic enzyme, which is non detectable in human cancer cell lines, the presence of mtFDH mRNA was demonstrated in A549 and PC3 cells by conventional and real-time PCR. The presence of the endogenous enzyme in mitochondria of A549 cells has been further confirmed using specific polyclonal antibody generated against purified recombinant mtFDH. We have also shown that recombinant mtFDH, similar to the cytosolic enzyme, catalyzes NADP + -dependent oxidation of the 10-Formyltetrahydrofolate to tetrahydrofolate and CO 2 . Thus, the enzyme is a likely source of CO 2 production in mitochondria and we propose that it plays an essential role in distribution of one-carbon groups between cytosolic and mitochondrial compartments of the cell. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 73.
Yaroslav Tsybovsky - One of the best experts on this subject based on the ideXlab platform.
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Enzymatic properties of ALDH1L2, a mitochondrial 10-Formyltetrahydrofolate dehydrogenase.
Chemico-biological interactions, 2011Co-Authors: Kyle C. Strickland, Natalia I. Krupenko, Yaroslav Tsybovsky, Marianne E. Dubard, Sergey A. KrupenkoAbstract: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.
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10 formyltetrahydrofolate dehydrogenase requires a 4 phosphopantetheine prosthetic group for catalysis
Journal of Biological Chemistry, 2007Co-Authors: Henry Donato, Natalia I. Krupenko, Yaroslav Tsybovsky, Sergey A. KrupenkoAbstract:Abstract 10-Formyltetrahydrofolate dehydrogenase (FDH) consists of two independent catalytic domains, N- and C-terminal, connected by a 100-amino acid residue linker (intermediate domain). Our previous studies on structural organization and enzymatic properties of rat FDH suggest that the overall enzyme reaction, i.e. NADP+-dependent conversion of 10-Formyltetrahydrofolate to tetrahydrofolate and CO2, consists of two steps: (i) hydrolytic cleavage of the formyl group in the N-terminal catalytic domain, followed by (ii) NADP+-dependent oxidation of the formyl group to CO2 in the C-terminal aldehyde dehydrogenase domain. In this mechanism, it was not clear how the formyl group is transferred between the two catalytic domains after the first step. This study demonstrates that the intermediate domain functions similarly to an acyl carrier protein. A 4′-phosphopantetheine swinging arm bound through a phosphoester bond to Ser354 of the intermediate domain transfers the formyl group between the catalytic domains of FDH. Thus, our study defines the intermediate domain of FDH as a novel carrier protein and provides the previously lacking component of the FDH catalytic mechanism.
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10-Formyltetrahydrofolate Dehydrogenase Requires a 4′-Phosphopantetheine Prosthetic Group for Catalysis
Journal of Biological Chemistry, 2007Co-Authors: Henry Donato, Natalia I. Krupenko, Yaroslav Tsybovsky, Sergey A. KrupenkoAbstract:Abstract 10-Formyltetrahydrofolate dehydrogenase (FDH) consists of two independent catalytic domains, N- and C-terminal, connected by a 100-amino acid residue linker (intermediate domain). Our previous studies on structural organization and enzymatic properties of rat FDH suggest that the overall enzyme reaction, i.e. NADP+-dependent conversion of 10-Formyltetrahydrofolate to tetrahydrofolate and CO2, consists of two steps: (i) hydrolytic cleavage of the formyl group in the N-terminal catalytic domain, followed by (ii) NADP+-dependent oxidation of the formyl group to CO2 in the C-terminal aldehyde dehydrogenase domain. In this mechanism, it was not clear how the formyl group is transferred between the two catalytic domains after the first step. This study demonstrates that the intermediate domain functions similarly to an acyl carrier protein. A 4′-phosphopantetheine swinging arm bound through a phosphoester bond to Ser354 of the intermediate domain transfers the formyl group between the catalytic domains of FDH. Thus, our study defines the intermediate domain of FDH as a novel carrier protein and provides the previously lacking component of the FDH catalytic mechanism.
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Crystal structures of the carboxyl terminal domain of rat 10-Formyltetrahydrofolate dehydrogenase : Implications for the catalytic mechanism of aldehyde dehydrogenases
Biochemistry, 2007Co-Authors: Yaroslav Tsybovsky, Henry Donato, Natalia I. Krupenko, Christopher Davies, Sergey A. KrupenkoAbstract:10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes an NADP+-dependent dehydrogenase reaction resulting in conversion of 10-Formyltetrahydrofolate to tetrahydrofolate and CO2. This reaction is a result of the concerted action of two catalytic domains of FDH, the amino-terminal hydrolase domain and the carboxyl-terminal aldehyde dehydrogenase domain. In addition to participation in the overall FDH mechanism, the C-terminal domain is capable of NADP+-dependent oxidation of short chain aldehydes to their corresponding acids. We have determined the crystal structure of the C-terminal domain of FDH and its complexes with oxidized and reduced forms of NADP. Compared to other members of the ALDH family, FDH demonstrates a new mode of binding of the 2‘-phosphate group of NADP via a water-mediated contact with Gln600 that may contribute to the specificity of the enzyme for NADP over NAD. The structures also suggest how Glu673 can act as a general base in both acylation and deacylation steps of the reaction. In...
