2005 by The International Union of Biochemistry and Molecular Biology BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Printed in U.S.A. Vol. 33, No. 4, pp. 255 259, 2005 Articles Facilitating Understanding of the Purine Nucleotide Cycle and the One-carbon Pool PART II: METABOLISM OF THE ONE-CARBON POOL Received for publication, November 2, 2004, and in revised form, February 2, 2005 Ifeanyi J. Arinze From the Department of Biochemistry, Meharry Medical College, Nashville, Tennessee 37208 Some metabolic processes such as glycolysis, gluconeogenesis, and lipogenesis are readily understood because they are circumscribed in metabolic pathways that have clearly identifiable beginning points, end products, and other features. Other metabolic pathways that do not appear to be straightforward pose difficulties for students. In part I of this two-part series, one such metabolic process, the purine nucleotide cycle, was discussed (I. J. Arinze (2005) Biochem. Mol. Biol. Educ. 33, 165 168). In the present article, the focus is on the metabolism of one-carbon fragments, the so-called one-carbon pool. As with the first article in this series, the intent of this article is to facilitate teaching and learning of this rather complex area of metabolism. Keywords: S-adenosylmethionine, one-carbon metabolism, tetrahydrofolate, vitamin B 12. Understanding metabolism of amino acids could be a tedious undertaking because, unlike sugars and fatty acids, there is no unified metabolic pathway that is central to all amino acids. Hence, textbooks discuss amino acid metabolism by grouping them whenever possible under themes (such as their metabolic fate or the number of carbons in the molecule) that might make learning less cumbersome. Fundamentally important issues such as one-carbon metabolism often appear not only in chapters devoted to the metabolism of certain amino acids (e.g. serine or methionine) but also in chapters dealing with nucleotide metabolism. An integrated viewpoint regarding metabolic processes germane to the one-carbon pool would tremendously aid teaching and learning of this enormously important area of metabolism. BIOLOGICAL SIGNIFICANCE OF ONE-CARBON METABOLISM Deficiencies of vitamin B 12 and folate are the most common causes of megaloblastic anemia. Furthermore, a number of abnormalities constituting neural tube defects arise from defects in folate metabolism [1, 2]. Increasing folate in the diet early in pregnancy significantly reduces the incidence of spina bifida. The metabolic basis for these abnormalities is impaired DNA replication resulting from the inability to synthesize DNA precursors. This synthesis is dependent on one-carbon metabolism involving folate and vitamin B 12. Although biotin-dependent and biotinindependent CO 2 fixation reactions are also one-carbon metabolism, such reactions are not considered to be part To whom correspondence should be addressed: Dept. of Biochemistry, Meharry Medical College, 1005 Dr. David B. Todd, Jr., Blvd., Nashville, TN 37208-3599. Tel.: 615-327-6586; Fax: 615-327-6442, E-mail: iarinze@mmc.edu. This paper is available on line at http://www.bambed.org 255 of the so-called one-carbon pool, which involves the generation of one-carbon moieties from five different amino acids (serine, glycine, methionine, histidine, and tryptophan). Components from this pool are subsequently utilized in (a) the biosynthesis of purine nucleotides and thymidylic acid, (b) conversion of homocysteine to methionine, and (c) the methylation of DNA, RNA, and protein. In the absence of either folic acid or vitamin B 12, metabolism of the one-carbon pool (hereafter referred to as one-carbon metabolism) is impaired. Appling [3] has reviewed cellular compartmentalization of folate-mediated one-carbon metabolism in eukaryotes and discusses the regulatory and therapeutic implications of processes in one-carbon metabolism. THE ONE-CARBON FRAGMENTS For students of biochemistry, it is important to answer the following questions: What are the one-carbon fragments? How do they arise in metabolism? How are they used in the a, b, and c events referred to above, and what is meant by the term one-carbon pool? The individual one-carbon fragments (Table I) represent different oxidation states of the carbon atom, which when fully reduced exists as CH 4. They are considered to be in a pool because they are metabolically interconvertible (Fig. 1). Folic acid is involved in the reactions in which the one carbon is first transferred from a donor to an acceptor. This vitamin is able to bond to any of the one-carbon moieties only after it is fully reduced to tetrahydrofolate (THF), 1 a two-step reaction catalyzed by folate reductase and dihydrofolate reductase (Fig. 2). Structurally, the relevant por- 1 The abbreviations used are: THF, tetrahydrofolate; AdoMet, S-adenosylmethionine.
256 BAMBED, Vol. 33, No. 4, pp. 255 259, 2005 tion for linkage to one-carbon moiety is the N 5 or N 10 atom (see arrows on the structure of THF, Fig. 2) or both when a methylene (-CH 2 -) or methenyl ( CH-) group is involved. Whereas any of the members of the one-carbon pool can be linked to THF, only the methyl (-CH 3 ) group can be linked to cobalamin (to form methyl-cobalamin, also called methyl-b 12 ). In methyl-b 12, the methyl group is the sixth co-ordinate to the cobalt atom in the corrin ring of cobalamin. Methyl-B 12 is one of only two known coenzyme forms of vitamin B 12, the other being 5 -deoxyadenosyl- B 12, which functions in the isomerization of methylmalonyl- CoA to succinyl-coa; in that case, the 5 -deoxyadenosyl moiety replaces the methyl group as the sixth co-ordinate to the cobalt atom. METABOLIC ORIGIN OF ONE-CARBON FRAGMENTS AND THEIR EVENTUAL METABOLIC FATES The term pool denotes the five isoforms of the onecarbon moieties that can bond to THF (see circle in Fig. 1). How do one-carbon fragments enter and exit the pool? One of the major donors of one-carbon moieties into the pool is serine, which comes from dietary sources, from TABLE I Members of the one-carbon pool Common name Chemical form 1. Methyl -CH 3 2. Methylene -CH 2-3. Methenyl (also called methylidyne) CH- 4. Formyl -CHO 5. Formimino -CH NH breakdown of endogenous protein, or from glucose as a by-product of the glycolytic pathway (Fig. 3). Through conversion of serine to glycine, a reaction catalyzed by pyridoxal phosphate-requiring serine hydroxymethyltransferase, the third carbon of serine is transferred to THF to form N 5 N 10 -methylene-thf, which can also be generated by the glycine cleavage system (Fig. 1). Upon reduction, this member of the one-carbon pool is converted to the methyl form (i.e. CH 3 THF). Alternatively, it can be oxidized to the methenyl form; a cyclohydrolase converts the methenyl form to N 10 -formyl-thf, which can be isomerized to N 5 -formyl-thf. Not shown in Fig. 1 is the ATP-dependent conversion of N 5 -formyl-thf to N 5 N 10 -methenyl- THF; the reaction is reversible. Also not shown is the ATP-dependent activation of formate (derived from the catabolism of tryptophan) to N 10 -formyl-thf. The most stable of the members of the one-carbon pool is N 5 -CH 3 - THF, which is used primarily by homocysteine methyltransferase, a B 12 -dependent enzyme. The conversion of N 5 N 10 -CH 2 -THF to N 5 -CH 3 -THF is not reversible under physiological conditions. Because all other members of the pool can be converted to N 5 -CH 3 -THF, there is a tendency for it to accumulate during B 12 deficiency, essentially trapping THF. Given the fact that all dietary folate can virtually end up as N 5 -CH 3 -THF, the vital role of homocysteine methyltransferase (discussed below) for untrapping THF so that it can be used to pick up one-carbon units for purine and pyrimidine nucleotide synthesis becomes obvious. If the homocysteine methyltransferase reaction were to be impaired, for example by FIG. 1.Overview of the one-carbon pool. The figure shows the flow of substrates into and out of the one-carbon pool (in the circle colored yellow). The blue lines are used here to designate reactions or processes that drain the one-carbon pool, whereas the red lines are used to indicate reactions that replenish the pool. Enzymes catalyzing reactions 1 through 4, respectively, are reductase (1), reductase (2), cyclodeaminase (3), and cyclohydrolase (4). The inter-conversion of N 10 -formyl-thf to N 5 -formyl-thf is not shown in this figure. The glycine cleavage system (*) requires NAD, FAD, lipoic acid, and pyridoxal phosphate. -KG, -ketoglutarate; B 6, vitamin B 6 ; B 12, vitamin B 12 ; DHF, 7,8-dihydrofolate; THF, 5,6,7,8-tetrahydrofolate; dump, deoxy-uridiylic acid; TMP, thymidylic acid.
257 FIG. 2. Pathway for converting folic acid to fully reduced folate (tetrahydrofolate) that can bond to one-carbon moieties. Pteroylpolyglutamate (folate) is reduced by dihydrofolate reductase (reaction 1) followed by further reduction (reaction 2) by the same enzyme to 5,6,7,8-tetrahydrofolate (THF). The structure of THF is shown in the box; the arrows indicate the N 5 and N 10 atoms that are involved in bonding to one-carbon units. Either one of these atoms can bond to any of the members of the one-carbon units shown in Table I. In methylenetetrahydrofolate (N 5,N 10 -CH 2 -THF), the methylene carbon forms a bridge between these nitrogen atoms. In N 5,N 10 -methenyl-thf, the bridge is between the methenyl carbon and the two nitrogen atoms. B 12 deficiency, the methyl trap phenomenon creates an artificial folate deficiency resulting from insufficiency of other members of the one-carbon pool. As discussed in textbooks of biochemistry, the synthesis of thymidylic acid requires N 5 N 10 -CH 2 -THF, while the synthesis of purine nucleotides requires the formyl derivative. The only use of methyl-thf is for the methylation of homocysteine to methionine, a reaction catalyzed by homocysteine methyltransferase, in which the methyl group is initially transferred from methyl-thf to B 12 and subsequently to homocysteine. Thus, the one-carbon pool is drained directly by the synthesis of nucleotides used for nucleic acid synthesis, and by methylation of homocysteine to yield methionine. Because S-adenosylmethionine (AdoMet), formed from the methyl group that arises from the pool, is used in a variety of methylation reactions (see Table II), the pool is also drained indirectly by such methylation reactions. Besides the synthesis of purine nucleotides, prokaryotes also use N 10 -formyl-thf in a formylation reaction that yields formyl-methionine-trna, which they use to initiate protein synthesis. Also, another member of the one-carbon pool, namely N 5,N 10 -methylenetetrahydrofolate, functions in DNA repair that is catalyzed by FIG. 3. Glucose is the primary source of the one-carbon groups carried by the coenzyme forms of folic acid and B 12 in the one-carbon pool. Glucose is metabolized to the triose level via glycolysis. Following either the phosphorylated pathway (a) or the nonphosphorylated pathway (b), the triose is eventually converted to serine, a nonessential amino acid. The transfer of the third carbon of serine to THF, catalyzed by serine hydroxymethyltransferase, leads to the formation of methylenetetrahydrofolate (N 5,N 10 -CH 2 -THF), a member of the one-carbon pool from which the other members can be formed, as shown in Fig. 1. TABLE II Methylated products in which AdoMet provides the methyl group Methyl group acceptor Guanidoacetate Norepinephrine Acetylserotonin Nucleic acids Proteins Phosphatidylethanolamine Product Creatine Epinephrine Melatonin Methylated nucleic acids a Methylated proteins b Phosphatidylcholine a CAP structure in RNA; CpG dinucleotides in DNA. b At specific Arg and Lys residues. DNA photolyase in Escherichia coli. This enzyme contains bound N 5,N 10 -methylenetetrahydrofolate that absorbs a photon that is needed to form the excited state that cleaves the thymine dimer into its original bases [4]. How is the one-carbon pool replenished? Replenishment occurs by transfer of one carbon from serine and/or glycine to form N 5,N 10 -methylenetetrahydrofolate (Fig. 1). The pool can also be replenished from the catabolism of histidine through which the one carbon enters the pool in the form of formiminotetrahydrofolate and from formylkynurenine (a catabolite of tryptophan), although these are not considered as important as replenishment from serine and glycine. INTERCONNECTION BETWEEN THE METABOLISM OF S-ADENOSYLMETHIONINE AND THE ONE-CARBON POOL The first reaction in the catabolism of methionine (methionine ATP 3 AdoMet P i PP i ) is its activation to AdoMet, a universal donor of methyl group in a variety of biochemical reactions (Table II). The adenosylation of me-
258 BAMBED, Vol. 33, No. 4, pp. 255 259, 2005 FIG. 4. Input of one-carbon into the metabolism of homocysteine and methionine. The figure shows the pathway for the catabolism of methionine to generate the nonessential amino acid, cysteine, through transulfuration (the transfer of the sulfur atom of methionine through homocysteine to serine to form cystathionine, which upon hydrolysis yields cysteine and -ketobutyrate). About 50% of the homocysteine formed in this pathway is back converted to methionine by utilizing methyl group from the onecarbon pool. The reaction is catalyzed by homocysteine methyltransferase, which requires vitamin B 12 as an additional co-factor. After this methylation, the methyl group eventually becomes the methyl group of AdoMet, which is used to methylate several biologically important compounds (see Table II). thionine to synthesize AdoMet requires three high energy equivalents. AdoMet, which is a sulfonium ion, is a high energy compound. Its methyl group is a good leaving group, and in a subsequent reaction it is used to methylate various methyl acceptors. Upon donation of its methyl group to an acceptor, AdoMet becomes S-adenosylhomocysteine. Subsequent hydrolysis of the adenosyl moiety leads to the formation of homocysteine (see Fig. 4). About half of the homocysteine levels are catabolized by condensation with serine to cystathionine and eventually to cysteine and propionyl-coa. This is the transulfuration pathway by which the sulfur atom of methionine is transferred to serine to make the nonessential amino acid, cysteine. The other half of homocysteine levels is converted back to methionine (often called remethylation of homocysteine) in a reaction catalyzed by homocysteine methyltransferase. This reaction requires not only B 12 but also is allosterically activated by AdoMet. The take home message is that introduction of one carbon from the one-carbon pool into this reaction provides the methyl carbon that is eventually found in the methylated compounds listed in Table II. MULTIPLE USES FOR THE METHYL GROUP ARISING FROM THE ONE-CARBON POOL There are multiple uses for the methyl group in AdoMet. For example, the formation of epinephrine occurs via AdoMet-dependent methylation of norepinephrine by phenylethanolamine-n-methyltransferase. The pathway for the degradation of catecholamines involves the combined action of (i) catechol-o-methyltransferase (which requires AdoMet) and (ii) monoamine oxidase, which deaminates the catecholamine. Covalent modification of a protein by methylation may provide that protein with additional surfaces for hydrophobic interaction(s) with various components; this is probably the case with myelin basic protein in which the sole methylated arginine residue at position 108 of the polypeptide chain provides sites for cross-chain stabilization or for interaction with lipids and other proteins in the myelin sheath [5]. A variety of arginine N-methyltransferases that use AdoMet to modify arginyl residues are known in mammalian systems [6], and this modification has been implicated in transcription, signal transduction, RNA transport, and RNA splicing. Proteins can also be modified by methylation at lysine, histidine, or aspartic acid residues. Examples of the involvement of methyl group transfers involving AdoMet abound in nucleic acid metabolism. For transcription and gene expression to occur in eukaryotes, the DNA is made accessible to the transcriptional machinery by chromatin-modifying complexes, the activities of which include nucleosome positioning, and covalent modification of histone proteins in the nucleosome through phosphorylation, acetylation, and methylation [7]. In vertebrate DNA, about 80% of cytosine residues within the dinucleotide sequence CpG are modified by methylation in a pattern that is tissue-specific; this pattern is formed during embryonic development and maintained in somatic cells. Cytosine methylation is directed by DNA methyltransferases, primarily to these CpG dinucleotides, although cytosine methylation of CpNpG is also known [8]. Areas of the genome that are methylated are usually less expressed. The mechanism by which this occurs most likely involves recruitment of repressor complexes to methylated DNA via the binding of methyl-cpg-binding domain proteins, resulting in a more compact and transcriptionally inactive chromatin [9 14]. Another example of methylationrelated phenomenon in nucleic acid biology is the methyl-
259 directed repair of damaged DNA and the capping of the 5 ends of pre-mrna to provide stability that is vital during translocation out of the nucleus and during translation of the mrna in eukaryotes. In sum, one-carbon metabolism is vital to the basic biology of cell growth and differentiation. Acknowledgments I thank Richard W. Hanson (Case Western Reserve School of Medicine, Cleveland, OH) for expert suggestions during the preparation of this article and for critically reading several drafts of the document. I also thank Mulchand Patel and Alexander Brownie of State University of New York at Buffalo and Peter Dolce, Meharry Medical College, for providing critical comments during the preparation of the article. Nalo Hamilton, Jackie Akech, Marjelo Mines, and Latricia Fitzgerald provided excellent assistance with the electronic preparation of the figures. REFERENCES [1] A. Fleming, A. J. Copp (1998) Embryonic folate metabolism and mouse neural tube defects, Science 280, 2107 2109. [2] D. M. Juriloff, M. J. Harris (2000) Mouse models for neural tube defects, Human Mol. Gen. 9, 993 1000. [3] D. R. Appling (1991) Compartmentation of folate-mediated one-carbon metabolism in eukaryotes, FASEB J. 5, 2645 2651. [4] J. M. Berg, J. L. Tymoczko, L. Stryer (2001) Biochemistry, 5th Ed., W. H. Freeman and Company, New York. [5] G. L. Mendz (1992) in Myelin: Biology and Chemistry (R. E. Martenson, ed.) Structural and molecular interactions of myelin basic protein and its antigenic peptides, pp. 277 366, CRC Press, Boca Raton, FL. [6] T. B. Miranda, M. Miranda, A. Frankel, S. Clarke (2004) PRMT7 is a member of the protein arginine methyltransferase family with a distinct substrate specificity, J. Biol. Chem. 279, 22902 22907. [7] M. Gerber, A. Shilatifard (2003) Translocational elongation by RNA polymerase II and histone methylation, J. Biol. Chem. 278, 26303 26306. [8] M. C. Lorincz, D. Schubeler, S. C. Goeke, M. Walters, M. Groudine, D. I. K. Martin (2000) Dynamic analysis of proviral induction and de novo methylation: Implications for a histone deacetylase-independent, methylation density-dependent mechanism of transcriptional repression, Mol. Cell. Biol. 20, 842 850. [9] X. Nan, H.-H. Ng, C. A. Johnson, C. D. Laherty, B. M. Turner, R. N. Eisenman, A. Bird (1998) Transcriptional repression by the methyl CpG-binding protein MeCP2 involves a histone deacetylase complex, Nature 393, 386 389. [10] A. Razin (1998) CpG methylation, chromatin structure and gene silencing A three-way connection, EMBO J. 17, 4905 4908. [11] M. C. Lorincz, D. Schubeler, M. Groudine (2001) Methylation-mediated proviral silencing is associated with MeCP2 recruitment and localized histone H3 deacetylation, Mol. Cell. Biol. 21, 7913 7922. [12] K. M. Stimson, P. M. Vertino (2002) Methylation-mediated silencing of TMS1/ASC is accompanied by histone hypoacetylation and CpG island-localized changes in chromatin architecture, J. Biol. Chem. 277, 4951 4958. [13] N. Thakur, M. Kanduri, C. Holmgren, R. Mukhopadhyay, C. Kanduri (2003) Bidirectional silencing and DNA methylation-sensitive methylation-spreading properties of the Kcnq1 imprinting control region map to the same regions, J. Biol. Chem. 278, 9514 9519. [14] C. S. Swindle, H. G. Kim, C. A. Klug (2004) Mutation of CpGs in the murine stem cell virus retroviral vector long terminal repeat represses silencing in embryonic stem cells, J. Biol. Chem. 279, 34 41.