Regulation of de Novo Purine Biosynthesis by Methenyltetrahydrofolate Synthetase in Neuroblastoma
2005; Elsevier BV; Volume: 281; Issue: 7 Linguagem: Inglês
10.1074/jbc.m510624200
ISSN1083-351X
AutoresMartha S. Field, Doletha M. E. Szebenyi, Patrick J. Stover,
Tópico(s)Folate and B Vitamins Research
Resumo5-Formyltetrahydrofolate (5-formylTHF) is the only folate derivative that does not serve as a cofactor in folate-dependent one-carbon metabolism. Two metabolic roles have been ascribed to this folate derivative. It has been proposed to 1) serve as a storage form of folate because it is chemically stable and accumulates in seeds and spores and 2) regulate folate-dependent one-carbon metabolism by inhibiting folate-dependent enzymes, specifically targeting folate-dependent de novo purine biosynthesis. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme that metabolizes 5-formylTHF and catalyzes its ATP-dependent conversion to 5,10-methenylTHF. This reaction determines intracellular 5-formylTHF concentrations and converts 5-formylTHF into an enzyme cofactor. The regulation and metabolic role of MTHFS in one-carbon metabolism was investigated in vitro and in human neuroblastoma cells. Steady-state kinetic studies revealed that 10-formylTHF, which exists in chemical equilibrium with 5,10-methenylTHF, acts as a tight binding inhibitor of mouse MTHFS. [6R]-10-formylTHF inhibited MTHFS with a Ki of 150 nm, and [6R,S]-10-formylTHF triglutamate inhibited MTHFS with a Ki of 30 nm. MTHFS is the first identified 10-formylTHF tight-binding protein. Isotope tracer studies in neuroblastoma demonstrate that MTHFS enhances de novo purine biosynthesis, indicating that MTHFS-bound 10-formylTHF facilitates de novo purine biosynthesis. Feedback metabolic regulation of MTHFS by 10-formylTHF indicates that 5-formylTHF can only accumulate in the presence of 10-formylTHF, providing the first evidence that 5-formylTHF is a storage form of excess formylated folates in mammalian cells. The sequestration of 10-formylTHF by MTHFS may explain why de novo purine biosynthesis is protected from common disruptions in the folate-dependent one-carbon network. 5-Formyltetrahydrofolate (5-formylTHF) is the only folate derivative that does not serve as a cofactor in folate-dependent one-carbon metabolism. Two metabolic roles have been ascribed to this folate derivative. It has been proposed to 1) serve as a storage form of folate because it is chemically stable and accumulates in seeds and spores and 2) regulate folate-dependent one-carbon metabolism by inhibiting folate-dependent enzymes, specifically targeting folate-dependent de novo purine biosynthesis. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme that metabolizes 5-formylTHF and catalyzes its ATP-dependent conversion to 5,10-methenylTHF. This reaction determines intracellular 5-formylTHF concentrations and converts 5-formylTHF into an enzyme cofactor. The regulation and metabolic role of MTHFS in one-carbon metabolism was investigated in vitro and in human neuroblastoma cells. Steady-state kinetic studies revealed that 10-formylTHF, which exists in chemical equilibrium with 5,10-methenylTHF, acts as a tight binding inhibitor of mouse MTHFS. [6R]-10-formylTHF inhibited MTHFS with a Ki of 150 nm, and [6R,S]-10-formylTHF triglutamate inhibited MTHFS with a Ki of 30 nm. MTHFS is the first identified 10-formylTHF tight-binding protein. Isotope tracer studies in neuroblastoma demonstrate that MTHFS enhances de novo purine biosynthesis, indicating that MTHFS-bound 10-formylTHF facilitates de novo purine biosynthesis. Feedback metabolic regulation of MTHFS by 10-formylTHF indicates that 5-formylTHF can only accumulate in the presence of 10-formylTHF, providing the first evidence that 5-formylTHF is a storage form of excess formylated folates in mammalian cells. The sequestration of 10-formylTHF by MTHFS may explain why de novo purine biosynthesis is protected from common disruptions in the folate-dependent one-carbon network. Tetrahydrofolates (THF) 2The abbreviations used are: THF, tetrahydrofolate; MTHFS, methenyltetrahydrofolate synthetase; THHF, tetrahydrohomofolate; GARFT, glycinamide ribonucleotide formyltransferase; FPGS, folylpolyglutamate synthetase; pABA, p-aminobenzoate; pABG, p-aminobenzoylglutamate; HPLC, high pressure liquid chromatography; MES, 4-morpholineethanesulfonic acid. serve as a family of cofactors that carry and activate one-carbons for the synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine (Figs. 1 and 2) (1.Shane B. Bailey L.B. Folate in Health and Disease. Marcel Dekker, Inc., New York1995: 1-22Google Scholar). Methionine can be adenylated to form S-adenosylmethionine, which serves as a cofactor and methyl donor for numerous cellular reactions, including histone, DNA, RNA, phospholipid, and catecholamine methylation (2.Finkelstein J.D. Nutr. Rev. 2000; 58: 193-204Crossref PubMed Scopus (61) Google Scholar, 3.Finkelstein J.D. Semin. Thromb. Hemostasis. 2000; 26: 219-225Crossref PubMed Scopus (293) Google Scholar, 4.Clark S. Banfield K. Carmel R. Jacobsen D.W. Homocysteine in Health and Disease. Cambridge University Press, New York2001: 63-78Google Scholar). Disruptions in the folate-mediated one-carbon metabolic network are common and result from single nucleotide polymorphisms in genes that encode folate-dependent enzymes, from low intracellular folate concentrations, and/or from other environmental factors (5.Bailey L.B. Gregory III, J.F. J. Nutr. 1999; 129: 919-922Crossref PubMed Scopus (288) Google Scholar, 6.Suh J.R. Herbig A.K. Stover P.J. Annu. Rev. Nutr. 2001; 21: 255-282Crossref PubMed Scopus (224) Google Scholar). De novo thymidylate synthesis and homocysteine remethylation are highly sensitive to network disruptions, which result in increased incorporation of dUTP into DNA, elevated cellular homocysteine, and impaired S-adenosylmethionine-dependent methylation reactions. De novo purine biosynthesis is less sensitive to network disruptions, although the mechanisms for this protection are unknown. Impaired folate metabolism increases risk for pathologies and developmental anomalies, including epithelial cancers, cardiovascular disease, and neural tube defects (7.Stover P.J. Nutr. Rev. 2004; 62: S3-S13Crossref PubMed Google Scholar). Because of its role in nucleotide biosynthesis and cellular methylation reactions, this network continues to be an attractive target for the development of antiproliferative drugs (8.Kisliuk R.L. Pharmacol. Ther. 2000; 85: 183-190Crossref PubMed Scopus (19) Google Scholar, 9.Kisliuk R.L. Curr. Pharm. Des. 2003; 9: 2615-2625Crossref PubMed Scopus (42) Google Scholar, 10.Zhao R. Goldman I.D. Oncogene. 2003; 22: 7431-7457Crossref PubMed Scopus (223) Google Scholar).FIGURE 2The folate-dependent one-carbon metabolic network in the cytoplasm. The products of one-carbon metabolism are underlined. THF, tetrahydrofolate; MTHFS, methenyltetrahydrofolate synthetase; cSHMT, cytoplasmic serine hydroxymethyltransferase; AdoMet, S-adenosylmethionine; GARFT, glycinamide ribonucleotide formyltransferase; AICARFT, phosphoribosylaminoimidazole carboxamide formyltransferase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the cell, folates differ by the reduction state of the pteridine ring, one-carbon substitution at the N5 and/or N10 positions, and the length of the glutamate polypeptide, which can range from 1 to 9 glutamate residues linked through γ-peptide linkages (Fig. 1) (1.Shane B. Bailey L.B. Folate in Health and Disease. Marcel Dekker, Inc., New York1995: 1-22Google Scholar). Folates are transported across cell membranes as monoglutamate derivatives that are converted to folate polyglutamates by the enzyme folylpolyglutamate synthetase (1.Shane B. Bailey L.B. Folate in Health and Disease. Marcel Dekker, Inc., New York1995: 1-22Google Scholar, 11.Moran R.G. Semin. Oncol. 1999; 26: 24-32PubMed Google Scholar). The polyglutamate chain serves both to retain folates within the cell and to increase the affinity of folate derivatives for folate binding enzymes (1.Shane B. Bailey L.B. Folate in Health and Disease. Marcel Dekker, Inc., New York1995: 1-22Google Scholar). Typically, folate polyglutamates bind to proteins two to three orders of magnitude tighter than corresponding monoglutamate forms (12.Schirch V. Strong W.B. Arch. Biochem. Biophys. 1989; 269: 371-380Crossref PubMed Scopus (132) Google Scholar). There are five naturally occurring one-carbon-substituted forms of THF (Fig. 2). 5-MethylTHF carries the one-carbon unit at the oxidation level of methanol for the remethylation of homocysteine to methionine. 5,10-MethyleneTHF carries the one-carbon unit at the oxidation level of formaldehyde for thymidylate biosynthesis. Three THF derivatives, 10-formylTHF, 5-formylTHF, and 5,10-methenylTHF, carry the one-carbon unit at the oxidation level of formate. Of these forms, only 10-formylTHF is an enzyme cofactor and supplies the number 2 and number 8 carbons of the purine ring. Folate-dependent biosynthetic enzymes show absolute substrate specificity for a single folate cofactor. However, other one-carbon-substituted forms often function as potent enzyme inhibitors that regulate flux through the network (1.Shane B. Bailey L.B. Folate in Health and Disease. Marcel Dekker, Inc., New York1995: 1-22Google Scholar, 13.Wagner C. Bailey L.B. Folate in Health and Disease. Marcel Dekker, Inc., New York1995: 23-42Google Scholar). Although 5-formylTHF is the most stable natural folate, little is known about its physiological role and regulation in mammalian systems. 5-FormylTHF is synthesized from 5,10-methenylTHF in a reaction catalyzed by serine hydroxymethyltransferase (14.Stover P. Schirch V. J. Biol. Chem. 1990; 265: 14227-14233Abstract Full Text PDF PubMed Google Scholar, 15.Stover P. Schirch V. Biochemistry. 1992; 31: 2148-2155Crossref PubMed Scopus (19) Google Scholar, 16.Stover P. Schirch V. Biochemistry. 1992; 31: 2155-2164Crossref PubMed Scopus (44) Google Scholar, 17.Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 18.Roje S. Janave M.T. Ziemak M.J. Hanson A.D. J. Biol. Chem. 2002; 277: 42748-42754Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 5,10-Methenyltetrahydrofolate synthetase (MTHFS, EC 6.3.3.2) is the only enzyme that metabolizes 5-formylTHF and irreversibly catalyzesits ATP-dependent cyclization to 5,10-methenylTHF. In prokaryotes, there is evidence that 5-formylTHF is a storage form of folate and that its accumulation in dormant cells, including seeds and spores, is mediated by alterations in MTHFS expression (19.Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 20.Kruschwitz H.L. McDonald D. Cossins E.A. Schirch V. J. Biol. Chem. 1994; 269: 28757-28763Abstract Full Text PDF PubMed Google Scholar). However, 5-formylTHF is not known to account for more than 10–15% of total folate in mammalian cells (19.Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (112) Google Scholar). In mammalian systems, 5-formylTHF has been shown to inhibit several folate-dependent enzymes. 5-FormylTHF polyglutamates are tight binding inhibitors of serine hydroxymethyltransferase and phosphoribosylaminoimidazole carboxamide formyltransferase (21.Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 22.Bertrand R. Jolivet J. J. Biol. Chem. 1989; 264: 8843-8846Abstract Full Text PDF PubMed Google Scholar). In addition to catalyzing 5-formylTHF synthesis, serine hydroxymethyltransferase also catalyzes the interconversion of serine and THF to glycine and methyleneTHF, a reaction that generates one-carbon units for purine, thymidine, and methionine biosynthesis (21.Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Phosphoribosylaminoimidazole carboxamide formyltransferase catalyzes the incorporation of formate into the C2 position of the purine ring. Inhibition of MTHFS in human MCF-7 cells by exposure to the MTHFS antifolate inhibitor, 5-formyltetrahydrohomofolate (5-formyl-THHF), results in an accumulation of cellular folate as 5-formylTHF. This creates a purine auxotrophy because elevated levels of 5-formylTHF inhibit phosphoribosylaminoimidazole carboxamide formyltransferase (22.Bertrand R. Jolivet J. J. Biol. Chem. 1989; 264: 8843-8846Abstract Full Text PDF PubMed Google Scholar). Therefore, it has been proposed that alterations in cellular 5-formylTHF concentrations, mediated through changes in MTHFS activity, may regulate purine biosynthesis (19.Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (112) Google Scholar). In this study, we elucidated a mechanism for feedback metabolic regulation of MTHFS by 10-formylTHF and provide evidence that this regulatory mechanism supports a role for 5-formylTHF as storage form of folate in mammalian cells. Furthermore, we discovered a novel role for MTHFS-bound 10-formylTHF in the regulation of de novo purine biosynthesis. Materials—MES, HEPES, and Tris were purchased from Sigma. ATP was purchased from Roche Applied Science. [6S]-5-formylTHF (the natural isomer) and [6R,S]-5-FormylTHF were a generous gift from Eprova AG. Folic acid, 10-formylfolic acid, [6R,S]-5-formyltetrahydropteroate, 6-methylpterin, 5-formyltetrahydrofolate triglutamate, and folic acid polyglutamates were from Schircks Laboratories. Homofolate (NSC 79249) was a generous gift from Dr. Roy Kisliuk, Tufts University. All other materials were of high quality and obtained from various commercial vendors. Synthesis of Folate and Antifolate Derivatives—[6R]-10-formylTHF (the natural isomer) was synthesized from [6S]-5-formylTHF as described previously (23.Stover P. Schirch V. Anal. Biochem. 1992; 202: 82-88Crossref PubMed Scopus (35) Google Scholar). 5-Formyl, 6-methyltetrahydropterin and 5-formyltetrahydrohomofolic acid were synthesized from 6-methylpterin and homofolic acid, respectively, as described by others (11.Moran R.G. Semin. Oncol. 1999; 26: 24-32PubMed Google Scholar). Both homofolic acid and 6-methylpterin were reduced to tetrahydrohomofolic acid and 6-methyltetrahydropterin, respectively, by reduction over palladium(II) oxide hydrate in water or formic acid, respectively; formylation at N5 was achieved by the immediate addition of formic acid and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide at pH 4.0 as described previously (24.Moran R.G. Keyomarsi K. Colman P.D. Methods Enzymol. 1986; 122: 309-312Crossref PubMed Scopus (9) Google Scholar). All synthesized compounds were purified by anion exchange chromatography and desalted by gravity filtration on a 0.5 × 64-inch G-10 Sephadex column using water as the mobile phase as described elsewhere (23.Stover P. Schirch V. Anal. Biochem. 1992; 202: 82-88Crossref PubMed Scopus (35) Google Scholar). Samples were lyophilized and their identity verified by UV spectroscopy and 1H NMR. Homology Model Construction—A model of the mouse MTHFS protein was constructed using the crystal structure of Mycoplasma pneumoniae MTHFS with ADP, phosphate, and 5-formylTHF bound (PDB code 1U3G; Fig. 3) (25.Chen S. Shin D.H. Pufan R. Kim R. Kim S.H. Proteins. 2004; 56: 839-843Crossref PubMed Scopus (10) Google Scholar, 26.Chen S. Yakunin A.F. Proudfoot M. Kim R. Kim S.H. Proteins. 2005; 61: 433-443Crossref PubMed Scopus (13) Google Scholar). A BLAST search of the Swiss-Prot data base (27.Boeckmann B. Bairoch A. Apweiler R. Blatter M.C. Estreicher A. Gasteiger E. Martin M.J. Michoud K. O'Donovan C. Phan I. Pilbout S. Schneider M. Nucleic Acids Res. 2003; 31: 365-370Crossref PubMed Scopus (2822) Google Scholar), using the SIB BLAST Network Service (28.Altschul S.F. Madden T.L. Schaffer A.A. Zhang J. Zhang Z. Miller W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (60233) Google Scholar), revealed a number of sequences related to mouse MTHFS. A set of eight sequences comprising three mammalian and five bacterial proteins, including the murine and M. pneumoniae MTHFS enzymes, was aligned using ClustalW (29.Higgins D.G. Thompson J.D. Gibson T.J. Methods Enzymol. 1996; 266: 383-402Crossref PubMed Scopus (1288) Google Scholar). A small adjustment was made to the location of insertions in the murine relative to the M. pneumoniae sequence to minimize interruption of secondary structure elements by the insertions. The SWISS-MODEL homology modeling server (30.Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3381-3385Crossref PubMed Scopus (4537) Google Scholar) was used to generate a model of mouse MTHFS. The M. pneumoniae and mouse sequences are 28% identical and 70% similar, and the alignment of the portions of the sequence that are involved in ATP and THF binding is unambiguous. The conformations and locations of inserted loops are uncertain, but none of these is close enough to the active site to be directly involved in catalysis. Protein Expression and Purification—Recombinant murine MTHFS protein was expressed and purified as described elsewhere (31.Anguera M.C. Liu X. Stover P.J. Protein Expression Purif. 2004; 35: 276-283Crossref PubMed Scopus (13) Google Scholar). Steady-state Kinetics—MTHFS activity was monitored by measuring the increase in 5,10-methenylTHF production with time at 37 °C in 1-ml cuvettes using a spectrophotometer; 5,10-methenylTHF has a unique absorbance maximum at λ, 355 nm (ϵ, 25,100). Reaction mixtures contained 100 mm MES, pH 6.0, 20 mm MgATP. Reactions were initiated by the addition of MTHFS to 70 nm. Km and kcat values were determined from Lineweaver-Burke plots, and reported values are the average of three experiments. To determine the Km for [6R,S]-5-formylTHFGlu3, final substrate concentrations ranged from 0.1 to 1.5 μm. For all inhibition experiments, 70 nm MTHFS was preincubated with the inhibitors for 2–4 min in the reaction mixture; the reaction was then initiated by adding [6S]-5-formylTHF. Ki values were determined from Dixon plots, and reported values are the average of at least two independent determinations. Purine Biosynthesis Assay—SH-SY5Y neuroblastoma cells expressing the human MTHFS cDNA (SH-SY5YMTHFS) have been described elsewhere (19.Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 31.Anguera M.C. Liu X. Stover P.J. Protein Expression Purif. 2004; 35: 276-283Crossref PubMed Scopus (13) Google Scholar). These cells were maintained in Minimal Essential Medium, alpha modification (Hyclone) supplemented with 11% fetal bovine serum. For tracer experiments, fetal bovine serum was dialyzed against phosphate-buffered saline for over 24 h with four buffer changes to remove folate and other small molecules and then charcoal treated to remove any residual folate. The tracer medium was Defined Minimal Essential Medium (Hyclone) that lacked glycine, serine, methionine, hypoxanthine, and folate but was supplemented with 200 μm methionine, 20 nm leucovorin, 2 nm [3H]hypoxanthine, and 20 μm [14C]formate. Cells were split 1:3 and grown in 6-well plates at 37 °C, 5% CO2 in tracer medium until confluent and harvested. The cell pellets were stored at -40 °C. Nuclear DNA was isolated using a DNA blood kit from Quiagen and isotope levels quantified on a Beckman LS6500 scintillation counter in dual dpm mode. For HPLC separation of nucleosides, cells were grown to confluence and harvested. DNA was digested to nucleosides prior to separation by HPLC, using procedures described elsewhere (32.Friso S. Choi S.W. Dolnikowski G.G. Selhub J. Anal. Chem. 2002; 74: 4526-4531Crossref PubMed Scopus (220) Google Scholar). Peaks corresponding to nucleosides were identified from standards, collected, and isotope levels quantified. Determinants of MTHFS Substrate Specificity—The contribution of all folate chemical moieties to substrate binding and catalysis was elucidated for recombinant murine MTHFS (Table 1 and Fig. 1). [6R,S]-5-formylTHFGlu3, an endogenous form of cellular folate, is a substrate for murine MTHFS (Km, 0.4 μm; kcat/Km, 225). Previous studies have shown that the unnatural [6R]-5-formylTHF is not a substrate or inhibitor of mammalian MTHFS enzymes; therefore, the Km value for [6S]-5-formylTHFGlu3 is likely 0.2 μm (33.Hopkins S. Schirch V. J. Biol. Chem. 1984; 259: 5618-5622Abstract Full Text PDF PubMed Google Scholar, 34.Bertrand R. MacKenzie R.E. Jolivet J. Biochim. Biophys. Acta. 1987; 911: 154-161Crossref PubMed Scopus (28) Google Scholar). The Km for [6S]-5-formylTHF is 10 μm (kcat/Km, 6.0), in agreement with values determined for rabbit and human MTHFS (22.Bertrand R. Jolivet J. J. Biol. Chem. 1989; 264: 8843-8846Abstract Full Text PDF PubMed Google Scholar, 33.Hopkins S. Schirch V. J. Biol. Chem. 1984; 259: 5618-5622Abstract Full Text PDF PubMed Google Scholar). Therefore, the loss of the diglutamate chain increases the Km by 25-fold and decreases the substrate specificity (kcat/Km) by 97%. [6R,S]-5-formyltetrahydropteroate, an unnatural derivative that lacks a glutamate moiety, is also a substrate (Km, 33 μm; kcat/Km, 1.8), indicating that the Glu1 moiety of 5-formylTHF makes only minor contributions to the Km and substrate specificity (kcat/Km). [6R,S]-5-formyl, 6-methyltetrahydropterin, which lacks the p-aminobenzoic acid and glutamate moieties, at a concentration of 60 μm did not inhibit the MTHFS-catalyzed conversion of 10 μm [6S]-5-formylTHF to 5, 10-methenylTHF, demonstrating that the p-aminobenzoate moiety of folate is essential for substrate binding. Collectively, these data indicate that pterins lack affinity for MTHFS and that the glutamate polypeptide decreases Km and increases substrate specificity.TABLE 1Substrate specificity of MTHFS All reactions were carried out at 37 °C in 100 mm MES, pH 6.0.SubstrateKmkcatkcat/Kmμmmin–1[6R,S]-5-formylTHPteroate33 ± 1060 ± 201.8[6S]-5-formylTHF10 ± 364 ± 166.4[6R,S]-5-formylTHFGlu30.4 ± 0.190 ± 30225.0 Open table in a new tab Inhibition of MTHFS—Previous studies of human MTHFS protein have demonstrated that folic acid, THF, and 5-methylTHF are weak competitive inhibitors of human MTHFS (34.Bertrand R. MacKenzie R.E. Jolivet J. Biochim. Biophys. Acta. 1987; 911: 154-161Crossref PubMed Scopus (28) Google Scholar). Inhibition of MTHFS by 10-formylTHF has never been investigated. The inhibition of recombinant murine MTHFS by folic acid and 10-formyl-substituted folate derivatives was examined and compared with inhibition by the synthetic antifolate and known MTHFS competitive inhibitor, 5-formylTHHF (Table 2). 5-FormylTHHF inhibited MTHFS activity with a Ki of 700 nm. Folic acid, an oxidized and unnatural form of folate present in vitamin supplements, weakly inhibited MTHFS activity (Ki, 58 μm). The Ki for folic acid triglutamate and pentaglutamate was reduced by 70 and 98%, respectively, compared with folic acid; the Ki for folic acid pentaglutamate was similar to that determined for 5-formylTHHF. The tighter binding resulting from the addition of the polyglutamate chain illustrates the importance of the polyglutamate binding site as a critical determinant of inhibitor affinity. N10-Formyl substitution markedly decreased the Ki for folate derivatives; inhibition of all N10-substituted folates was competitive with the substrate, [6S]-5-formylTHF. N10-Formylation of folic acid decreased the Ki value by nearly 80%. Similarly, [6R,S]-10-formylTHHF was a more effective inhibitor than [6R,S]-5-formylTHHF (Table 2). The Ki for N10-formyl folates decreased with glutamate chain length; [6R]-10-formylTHF inhibited MTHFS with a Ki of 150 nm, whereas [6R,S]-10-formylTHF triglutamate inhibited MTHFS with a Ki of 30 nm. These results demonstrate that both N10-formyl substitution and the glutamate chain increase the affinity of folates for MTHFS. The effective inhibition of MTHFS by [6R]-10-formylTHF polyglutamate, a naturally occurring folate derivative, indicates that 10-formylTHF regulates MTHFS activity in vivo.TABLE 2Inhibition of MTHFS All reactions were carried out at 37 °C in 100 mm MES, pH 6.0 in the presence of 10 μm [6S]-5-formylTHF.InhibitorKiμmFolic acid58 ± 5Folic acid Glu317 ± 3Folic acid Glu51.0 ± 0.310-Formylfolic acid13 ± 6[6R,S]-10-formylTHPteroate5 ± 2[6R]-10-formylTHF0.15 ± 0.09[6R,S]-10-formylTHFGlu30.03 ± 0.01[6R,S]-5-formylTHHF0.7 ± 0.3[6R,S]-10-formylTHHF0.20 ± 0.02 Open table in a new tab Structural Analysis of MTHFS Active Site—The murine MTHFS model shows the calculated solvent-accessible surface colored according to electrostatic potential (Fig. 3, blue for positive and red for negative). The positioning of the substrate, 5-formylTHF (in green), is identical to that observed in the active site of the M. pneumoniae crystal structure of the MTHFS·ADP·Pi·5-formylTHF complex. In this model, the MTHFS N terminus is at the lower left, the entrance to the ATP binding site is on the left side, and the view is directly into the 5-formylTHF binding site. All the inserted loops (i.e. the poorly modeled portions of the structure) are in the top half of the molecule. The positively charged region at the bottom of the structure is the putative polyglutamate binding site. In this model, the conformation of 5-formylTHF exposes the γ-carboxyl of the 5-formylTHF glutamate moiety to the solvent, but this part of the molecule was not well defined in the crystal structure (zero occupancies are given in the Protein Data Bank for all atoms past C9). Adjustment of the 5-formylTHF model using O (35.Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13014) Google Scholar) reveals that it is easily possible for hydrogen-bonding interactions to be made between polar atoms in the substrate glutamate moiety and the side chains of residues Lys-17 and Arg-147. Glu-62 could also be part of a network of H-bonds involving 5-formylTHF and the protein surface. All three of these residues are conserved in known mammalian MTHFS sequences, and Lys-17 of rabbit liver MTHFS can be cross-linked to the α-carboxylate of 5-formylTHF (36.Maras B. Stover P. Valiante S. Barra D. Schirch V. J. Biol. Chem. 1994; 269: 18429-18433Abstract Full Text PDF PubMed Google Scholar). The lack of specific contacts for the 5-formylTHF Glu1 is consistent with its apparent weak contribution to substrate specificity (Table 1). Residues lining the 5-formylTHF binding site include amino acid sequence regions 58–64, 97–105, 133–136, and 144–152 (corresponding to 49–55, 76–84, 104–107, and 115–123, respectively, in the M. pneumoniae protein) (Fig. 4a). The pterin moiety of 5-formylTHF is coordinated by the side chain of Glu-64 and the main chain O of Asp-59 and probably by Gln-58 Oϵ1 and Ser-97 Oγ. The first two of these interactions are present in the M. pneumoniae structure and in the MTHFS of most species, whereas the latter two are not. Modeling an ATP into the MTHFS active site such that the γ-phosphate occupies the site of a phosphate in the M. pneumoniae structure illustrates that a simple rotation of the formyl group of 5-formylTHF places the formyl O in good approximation and orientation for nucleophilic attack on the γ-phosphorous (Fig. 4a). The importance of the highly conserved residues 144, 148, and 150–152 in defining the active site is apparent: Arg-144 occupies a critical position coordinating both the N5-formyl group of 5-formylTHF and the γ-phosphate of ATP. 10-FormylTHF can be built into the THF binding site without altering the position of side chain residues (Fig. 4b). Because the active site is not tightly constricted, several folate derivatives can be positioned to form good interactions between the pterin moiety and residues lining the binding site and between the glutamate moiety and surface hydrophilic residues. This is consistent with the kinetic data that demonstrate weak inhibition of MTHFS activity by most folate derivatives (Table 2). However, it is not possible to position the N10 formyl oxygen properly for attack on the γ-phosphorous of ATP. Approach of the formyl oxygen to the phosphorous is hindered by two of the phosphate oxygens. The angle (formyl O)-P-(opposite P-O bond) is nearly linear with the 5-formyl moiety but is bent by ∼40° with the 10-formyl moiety. In the case of N10 formyl species, it is possible that an H-bond could be formed between the formyl O and Lys-149 N; whether this is sufficient to explain the tighter binding of N10-formylated inhibitors is unclear (Table 2). Effect of MTHFS Expression on Purine Biosynthesis—The intracellular concentration of folate derivatives is less than the binding capacity of folate-utilizing enzymes, indicating that biosynthetic pathways within the one-carbon network compete for a limiting pool of folate cofactors (6.Suh J.R. Herbig A.K. Stover P.J. Annu. Rev. Nutr. 2001; 21: 255-282Crossref PubMed Scopus (224) Google Scholar, 34.Bertrand R. MacKenzie R.E. Jolivet J. Biochim. Biophys. Acta. 1987; 911: 154-161Crossref PubMed Scopus (28) Google Scholar). Folate-binding proteins can serve as "sinks" that sequester specific folates and thereby inhibit folate-dependent pathways or can interact with other enzymes to selectively "channel" cofactors and accelerate flux through individual biosynthetic pathways (12.Schirch V. Strong W.B. Arch. Biochem. Biophys. 1989; 269: 371-380Crossref PubMed Scopus (132) Google Scholar). 10-FormylTHF is required by GARFT and phosphoribosylaminoimidazole carboxamide formyltransferase, two folate-dependent enzymes involved in de novo purine biosynthesis (Fig. 2) (1.Shane B. Bailey L.B. Folate in Health and Disease. Marcel Dekker, Inc., New York1995: 1-22Google Scholar, 11.Moran R.G. Semin. Oncol. 1999; 26: 24-32PubMed Google Scholar). To determine the metabolic effects of 10-formylTHF sequestration by MTHFS on de novo purine biosynthesis, a "formate suppression" assay was developed. Mammalian cells expressing the MTHFS cDNA were cultured in the presence of [3H]hypoxanthine and [14C]formate. [3H]Hypoxanthine is converted to purine nucleotides via the folate-independent salvage pathway, whereas [14C]formate is incorporated into purine nucleotides via the de novo pathway after condensing with THF to form 10-formylTHF (Fig. 5). The ability of the de novo purine biosynthetic pathway to suppress contributions from the purine salvage pathway to DNA synthesis was investigated in human SHSY-5Y and SHSY-5YMTHFS neuroblastoma cells. SHSY-5YMTHFS cells display 100-fold increased MTHFS activity and protein levels (19.Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (112) Google Scholar, 34.Bertrand R. MacKenzie R.E. Jolivet J. Biochim. Biophys. Acta. 1987; 911: 154-161Crossref PubMed Scopus (28) Google Scholar). The ratio of 14C to 3H (dpm) in DNA and purine nucleotides serves as a measure of de novo purine synthesis efficiency. The 14C/3H ratio in nuclear DNA is 20% higher in SHSY-5YMTHFS cells compared with the parent cell line (Fig. 6). Because 14C could also be incorporated into deoxythymidine and methylcytosine (Fig. 2) via equilibration into the folate-activated one-carbon pool, the DNA was digested to nucleosides, which were fractionated by HPLC. The deoxyguanosine and deoxyadenosine 14C/3H ratio was increased by 43 and 69%, respectively, in SHSY-5YMTHFS cells compared with the parent cell line (Fig. 6). Comparison of 14C counts derived from purified deoxythymidine normalized to 3H counts from deoxyadenosine indicates that increased MTHFS expression does not affect the 14C deoxythymidine/3H deoxyadenosine ratio. The enhancement of de novo purine biosynthesis by MTHFS indicates that MTHFS-bound 10-formylTHF is available for de novo purine biosynthesis.FIGURE 6MTHFS enhances de novo purine biosynthesis. Mammalian cells expressing the MTHFS cDNA were cultured in the presence of [3H]hypoxanthine and [14C]formate. [3H]Hypoxanthine is converted to purines via the folate-independent salvage pathway, whereas [14C]formate is incorporated into purines via the de novo pathway. The 14C/3H dpm ratio was determined in SHSY-5Y and SHSY-5YMTHFS nuclear DNA. Nuclear DNA was isolated from SHSY-5Y (white bars) and SHSY-5YMTHFS (shaded bars) and digested to nucleosides, which were then separated by HPLC. The 14C and 3H content (dpm) in resulting fractions was quantified on a Beckman Coulter LS6500 scintillation counter. Variation is expressed as S.D. of the mean from three measurements for separated nucleosides and six experiments for nuclear DNA. dA, deoxyadenosine; dT, deoxythymidine; dG, deoxyguanosine.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The data presented in this study provide the first evidence that 5-formylTHF concentrations are regulated in mammalian cells. The product of the MTHFS reaction, 5, 10-methenylTHF, exists in chemical equilibrium with 10-formylTHF. Upon the hydrolysis of 5,10-methenylTHF to 10-formylTHF, the MTHFS reaction is subject to feedback inhibition by 10-formylTHF (Table 2) (Fig. 7). This discovery that MTHFS is regulated by 10-formylTHF informs the physiological function of 5-formylTHF. 5-FormylTHF can only accumulate when MTHFS is inhibited by 10-formylTHF. Therefore, 10-formylTHF accumulation both suppresses 5-formylTHF metabolism and, through its equilibrium conversion to 5,10-methenylTHF, serves as a substrate for 5-formylTHF synthesis by serine hydroxymethyltransferase. This regulatory mechanism does not support a role for physiological regulation of purine biosynthesis by 5-formylTHF. 5-FormylTHF can only accumulate when the substrate for de novo purine biosynthesis, 10-formylTHF, accumulates. Feedback regulation of MTHFS does indicate that 5-formylTHF represents a stable pool of excess formyl folates that can be mobilized by MTHFS only when 10-formylTHF levels are depleted. These studies have also identified MTHFS as a 10-formylTHF tight-binding protein. Folate cofactors are bound by enzymes that utilize one-carbon units in transfer reactions and also by folate tight-binding proteins that do not metabolize the cofactor but regulate its availability. For example, 5-methylTHF is a substrate for methionine synthase but is bound tightly to and inhibits glycine N-methyltransferase (37.Yeo E.J. Briggs W.T. Wagner C. J. Biol. Chem. 1999; 274: 37559-37564Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). THF is bound tightly by and inhibits 10-formyltetrahydrofolate dehydrogenase (38.Fu T.F. Maras B. Barra D. Schirch V. Arch. Biochem. Biophys. 1999; 367: 161-166Crossref PubMed Scopus (12) Google Scholar), and 5-formylTHF and 5-methylTHF are bound tightly by and inhibit serine hydroxymethyltransferase (39.Herbig K. Chiang E.P. Lee L.R. Hills J. Shane B. Stover P.J. J. Biol. Chem. 2002; 277: 38381-38389Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). MTHFS is the first 10-formylTHF tight-binding protein to be identified. Previously, we have shown that increased MTHFS expression in SHSY-5Y cells increased relative concentrations of 10-formylTHF at the expense of 5-methylTHF levels; 10-formylTHF levels constituted as much as 90% of total cellular folate in MTHFS-expressing cells (21.Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These results are consistent with MTHFS serving as a reservoir for 10-formylTHF that permits its accumulation. The results from this study indicate that MTHFS may determine the fate of cellular 10-formylTHF (Fig. 7). 10-FormylTHF can be used by three pathways. It is a cofactor for phosphoribosylaminoimidazole carboxamide formyltransferase and GARFT during de novo purine biosynthesis. Alternatively, the 10-formyl group can be reduced and the cofactor converted to other THF one-carbon forms through its conversion to 5,10-methenylTHF by the enzyme 5,10-methenylTHF cyclohydrolase. Finally, 10-formylTHF can be converted to THF and CO2 by the enzyme 10-formyltetrahydrofolate dehydrogenase, an enzyme that depletes the supply of 10-formylTHF (38.Fu T.F. Maras B. Barra D. Schirch V. Arch. Biochem. Biophys. 1999; 367: 161-166Crossref PubMed Scopus (12) Google Scholar). In this study, we have demonstrated that sequestration of 10-formylTHF by MTHFS enhances de novo purine biosynthesis, which supports a role for MTHFS in directing 10-formylTHF to de novo purine synthesis. 10-FormylTHF sequestration by MTHFS may function to protect purine biosynthesis from disruptions in the one-carbon network, including folate deficiency. Although there is no direct evidence that MTHFS associates with phosphoribosylaminoimidazole carboxamide formyltransferase or GARFT and channels 10-formylTHF, the electrostatic surface of MTHFS shows a large negatively charged area near the active site that is a potential protein-protein interaction site (Fig. 3). Previous studies have demonstrated that MTHFS and cytoplasmic serine hydroxymethyltransferase exhibit very different tissue expression patterns (40.Anguera M.C. Suh J.R. Ghandour H. Nasrallah I.M. Selhub J. Stover P.J. J. Biol. Chem. 2003; 278: 29856-29862Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), indicating that MTHFS may play other roles in one-carbon metabolism distinct from metabolizing 5-formylTHF, including serving as a 10-formylTHF-binding protein. These results inform the design of inhibitors that target MTHFS. The data in Table 2 indicate that high affinity MTHFS inhibitors should be N10 formyl substituted and be capable of conversion to polyglutamate derivatives in the cell. N5-substituted MTHFS inhibitors, including 5-formylTHHF, are not attractive in vivo inhibitors because they can be phosphorylated and slowly metabolized by some mammalian MTHFS enzymes and because they are not effective substrates for folylpolyglutamate synthetase (1.Shane B. Bailey L.B. Folate in Health and Disease. Marcel Dekker, Inc., New York1995: 1-22Google Scholar). A number of GARFT inhibitors have been synthesized (4.Clark S. Banfield K. Carmel R. Jacobsen D.W. Homocysteine in Health and Disease. Cambridge University Press, New York2001: 63-78Google Scholar, 8.Kisliuk R.L. Pharmacol. Ther. 2000; 85: 183-190Crossref PubMed Scopus (19) Google Scholar, 41.Marsilje T.H. Labroli M.A. Hedrick M.P. Jin Q. Desharnais J. Baker S.J. Gooljarsingh L.T. Ramcharan J. Tavassoli A. Zhang Y. Wilson I.A. Beardsley G.P. Benkovic S.J. Boger D.L. Bioorg. Med. Chem. 2002; 10: 2739-2749Crossref PubMed Scopus (18) Google Scholar, 42.McGuire J.J. Curr. Pharm. Des. 2003; 9: 2593-2613Crossref PubMed Scopus (276) Google Scholar), including 5,10-dideazatetrahydrofolate (DDATHF or Lometrexol). DDATHF inhibited mouse GARFT with a Ki of 6 nm and human GARFT with a Ki of 60 nm. It also proved to be an effective inhibitor of cell growth with an EC50 of 10–30 nm in several different cell lines (42.McGuire J.J. Curr. Pharm. Des. 2003; 9: 2593-2613Crossref PubMed Scopus (276) Google Scholar). A similar compound, 10-formyl-5,10-dideaza-acyclicTHF (10-formyl-DDACTHF), was shown to exhibit some selectivity for GARFT. 10-Formyl-DDACTHF is a substrate for folylpolyglutamate synthetase and accumulates in cell cultures over 100-fold (41.Marsilje T.H. Labroli M.A. Hedrick M.P. Jin Q. Desharnais J. Baker S.J. Gooljarsingh L.T. Ramcharan J. Tavassoli A. Zhang Y. Wilson I.A. Beardsley G.P. Benkovic S.J. Boger D.L. Bioorg. Med. Chem. 2002; 10: 2739-2749Crossref PubMed Scopus (18) Google Scholar). The pentaglutamate form effectively inhibits GARFT (Ki, 14 nm) and was an effective cytotoxic agent (IC50, 60 nm). Given the high affinity of MTHFS for the natural isomer of 10-formylTHFGlu3 (Ki, 15 nm) and its relaxed specificity for the pterin moiety, the results of the present work suggest that N10-substituted analogs of 10-formylTHF may be targeting MTHFS, which may contribute to their cytotoxicity.
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