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Defining the steps of the folate one-carbon shuffle and homocysteine metabolism

来源:《美国临床营养学杂志》 作者:Robert J Cook 2008-12-28
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摘要: As a consequence of its role in glycine production, serine is also the major donor of folate-linked one-carbon units, which are used in the biosynthesis of purines and 2‘-deoxythymidine 5‘-monophosphate and the remethylation of homocysteine to methionine。 Note that for each glycine molecule de......


Robert J Cook

1 From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN.

2 Reprints not available. Address correspondence to RJ Cook, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232-0146. E-mail: cookrj{at}ctrvax.vanderbilt.edu.

See corresponding article on page1535.

In mammals, serine is derived from the diet and is biosynthesized from glycolysis via 3-phosphoglycerate. Serine plays a central role in intermediary metabolism as a contributor to phospholipid, sphingolipid, and cysteine biosynthesis and as a primary source of glycine (1, 2). There is a tremendous demand for glycine that probably exceeds dietary intake by 10–50-fold, not only for protein synthesis—in particular collagen—but also as a precursor for 5 major biosynthetic pathways: creatine, porphyrins, purines, bile acids, and glutathione (3). As a consequence of its role in glycine production, serine is also the major donor of folate-linked one-carbon units, which are used in the biosynthesis of purines and 2'-deoxythymidine 5'-monophosphate and the remethylation of homocysteine to methionine. Note that for each glycine molecule derived from serine there is also a one-carbon unit formed. Therefore, is glycine use equal to the use of one-carbon units? On the basis of the demand for glycine for its biosynthetic roles, it would be expected that there is a surplus of one-carbon units that are catabolized by oxidation to carbon dioxide.

The freely reversible interconversion of serine and glycine is catalyzed by serine hydroxymethyltransferase (SHMT), a reaction that is both folate- and pyridoxal 5-phosphate–dependent. Conversion of serine to glycine results in the removal of the C-3 serine and the formation of 5,10-methylenetetrahydrofolate, which can be used in folate-dependent one-carbon metabolism or oxidized to carbon dioxide via 10-formyltetrahydrofolate. There are 2 forms of SHMT, cytosolic (cSHMT) and mitochondrial (mSHMT), which are encoded by separate genes that are expressed in varying amounts in various tissues (4,5). Much of the work on serine and glycine interconversion has been done on cell extracts that contained both cSHMT and mSHMT; the precise roles of the SHMT isozymes are not clear. The current hypothesis holds that mSHMT is the primary supplier of glycine and one-carbon units to the cell (6). The supporting evidence comes from work in isolated mitochondria (6, 7); in serine-glycine flux studies in Chinese hamster ovary (CHO) Gly A mutants, which lack mSHMT and are glycine auxotrophs (8); and in transfection studies of CHO Gly A cells with human mSHMT that eliminated glycine auxotrophy (5). cSHMT is unable to provide sufficient glycine for normal growth in CHO Gly A cells (9). Another feature of cSHMT is its ability to catalyze the synthesis of 5-formyltetrahydrofolate, which is a slow, tight-binding inhibitor of cSHMT (10). Girgis et al (11) suggested that cSHMT may have more of a regulatory than of a catalytic role in directing the distribution of one-carbon units.

The article by Gregory et al (12) in this issue of the Journal offers a novel observation on serine, glycine, and one-carbon metabolism in a human subject. The inclusion of the analysis of VLDL apolipoprotein (apo) B-100 gives important insight into hepatic one-carbon metabolism. Given the free reversibility of SHMT, there is serious concern about the "scrambling" of the label at the C-3 of serine. Gregory et al should be congratulated for attempting the study and for interpreting the resulting labeling patterns.

The observed [2H1]methionine (M+1) labeling of apo B-100 is consistent with the mitochondrial processing of one-carbon units from serine via mSHMT (see Figure 5 in reference 12). During the oxidation of 5,10-[C-2H2]methylenetetrahydrofolate to 5,10-[C-2H1]methenyltetrahydrofolate, a deuterium atom is removed; thus, the one-carbon unit changes from M+2 to M+1. There was very rapid hepatic remethylation of homocysteine with an M+1 one-carbon unit, as evidenced by labeling of methionine in apo B-100 (see Figure 4 in reference 12), with the enrichment reaching 75% of the plateau value 30 min after infusion. These results are the first to provide in vivo proof that hepatic one-carbon units derived from serine by mSHMT can be assimilated in the cytosol and used for homocysteine remethylation and strongly support the model proposed by Appling (6). The presence of [2H2]methionine in both plasma and apo B-100 is proof that cSHMT is also able to supply one-carbon units for homocysteine remethylation. However, the results (see Figure 4 in reference 12) suggest that cSHMT was a minor contributor to the one-carbon pool during the initial 3 h of the experiment.

The absence of labeled glycine either in apo B-100 or in plasma probably indicates that glycine production from serine is tightly coupled to use in the biosynthetic pathways listed above. Alternatively, the glycine pool may be strictly regulated by the glycine cleavage system. The results also suggest that there is a separate glycine pool for hepatic protein synthesis.

The reversible nature of SHMT makes the results for serine impossible to interpret unless the specific labeling patterns of the C-2 and C-3 atoms can be determined. Theoretically, there are 3 possible routes each for the synthesis of [2H2]serine and [2H1]serine, starting with [2H1]glycine or unlabeled glycine, followed by the addition of an M+0, M+1, or M+2 folate-linked methyl group. If we assume that mSHMT converts serine in the direction of glycine (see Figure 5 in reference 12), then cSHMT must be operating in both directions to provide M+2 one-carbon units in the form of 5,10-methylenetetrahydrofolate for the remethylation of homocysteine, as seen in plasma and apo B-100. The use of [3-13C]serine, as outlined by Gregory et al (12), in future studies will considerably simplify the labeling patterns. In this instance, the remethylation of homocysteine will yield only M+1 methionine. Likewise, only M+1 cystathionine will be produced in the transsulfuration pathway. [3-13C]Serine will, however, allow for an estimation of the rate of oxidation of one-carbon units via 10-[13C]formyltetrahydrofolate to 13CO2.

Stable isotope–labeled serine infusion for investigating the remethylation and transsulfuration of homocysteine can now be used successfully to directly determine the effect of polymorphisms that may affect homocysteine metabolism. The most obvious application of this type of protocol would be to directly determine in vivo any differences in the rates of remethylation and transsulfuration of homocysteine in humans that carry the A222V mutation in methylenetetrahydrofolate reductase.

REFERENCES

  1. Schirch L. Folate in serine and glycine metabolism. In: Blakley RL, Benkovic Sj, eds. Folates and pterins. New York: John Wiley & Sons, 1984:399–431.
  2. MacKenzie RE. Biogenesis and interconversion of substituted tetrahydrofolates. In: Blakley RL, Bankovic SJ, eds. Folates and pterins. New York: John Wiley & Sons, 1984:255–306.
  3. Salway JG. Metabolism at a glance. London: Blackwell Scientific Publications, 1994.
  4. Snell K, Fell DA. Metabolic control analysis of mammalian serine metabolism. Adv Enzyme Regul 1990;30:13–32.
  5. Stover PJ, Chen LH, Suh JR, Stover DM, Keyomarsi K, Shane B. Molecular cloning, characterization, and regulation of the human mitochondrial serine hydroxymethyltransferase gene. J Biol Chem 1997;272:1842–8.
  6. Appling DR. Compartmentation of folate-mediated one-carbon metabolism in eukaryotes. FASEB J 1991;5:2645–51.
  7. Barlowe CK, Appling DR. In vitro evidence for the involvement of mitochondrial folate metabolism in the supply of cytoplasmic one-carbon units. Biofactors 1988;1:171–6.
  8. Narkewicz MR, Sauls SD, Tjoa SS, Teng C, Fennessey PV. Evidence for intracellular partitioning of serine and glycine metabolism in Chinese hamster ovary cells. Biochem J 1996;313:991–6.
  9. Chasin LA, Feldman A, Konstam M, Urlaub G. Reversion of a Chinese hamster cell auxotrophic mutant. Proc Natl Acad Sci U S A 1974;71:718–22.
  10. Stover P, Kruschwitz H, Schirch V. Evidence that 5-formyltetrahydropteroylglutamate has a metabolic role in one-carbon metabolism. Adv Exp Med Biol 1993;338:679–85.
  11. Girgis S, Suh JR, Jolivet J, Stover PJ. 5-Formyltetrahydrofolate regulates homocysteine remethylation in human neuroblastoma. J Biol Chem 1997;272:4729–34.
  12. Gregory JF III, Cuskelly GJ, Shane B, Toth JP, Baumgartner TG, Stacpoole PW. Primed, constant infusion with [2H3]serine allows in vivo kinetic measurement of serine turnover, homocysteine remethylation, and transsulfuration processes in human one-carbon metabolism. Am J Clin Nutr 2000;72:1535–41.


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