Abstract
Folate-dependent one-carbon metabolism is required for the synthesis of purines and thymidylate and for the remethylation of homocysteine to methionine. Methionine is subsequently adenylated to S-adenosylmethionine (SAM), a cofactor that methylates DNA, RNA, proteins, and many metabolites. Previous experimental and theoretical modeling studies have indicated that folate cofactors are limiting for cytoplasmic folate-dependent reactions and that the synthesis of DNA precursors competes with SAM synthesis. Each of these studies concluded that SAM synthesis has a higher metabolic priority than dTMP synthesis. The influence of cytoplasmic serine hydroxymethyltransferase (cSHMT) on this competition was examined in MCF-7 cells. Increases in cSHMT expression inhibit SAM concentrations by two proposed mechanisms: (1) cSHMT-catalyzed serine synthesis competes with the enzyme methylenetetrahydrofolate reductase for methylenetetrahydrofolate in a glycine-dependent manner, and (2) cSHMT, a high affinity 5-methyltetrahydrofolate-binding protein, sequesters this cofactor and inhibits methionine synthesis in a glycine-independent manner. Stable isotope tracer studies indicate that cSHMT plays an important role in mediating the flux of one-carbon units between dTMP and SAM syntheses. We conclude that cSHMT has three important functions in the cytoplasm: (1) it preferentially supplies one-carbon units for thymidylate biosynthesis, (2) it depletes methylenetetrahydrofolate pools for SAM synthesis by synthesizing serine, and (3) it sequesters 5-methyltetrahydrofolate and inhibits SAM synthesis. These results indicate that cSHMT is a metabolic switch that, when activated, gives dTMP synthesis higher metabolic priority than SAM synthesis.
Highlights
Folate is present in cells as a family of coenzymes that carry one-carbon units and function in both the mitochondrial and cytoplasmic compartments [1,2,3]
We conclude that cytoplasmic serine hydroxymethyltransferase (cSHMT) has three important functions in the cytoplasm: [1] it preferentially supplies one-carbon units for thymidylate biosynthesis, [2] it depletes methylenetetrahydrofolate pools for SAM synthesis by synthesizing serine, and [3] it sequesters 5-methyltetrahydrofolate and inhibits SAM synthesis
It is required for the conversion of dUMP to dTMP, catalyzed by thymidylate synthase (TS); for the conversion of glycine to serine, catalyzed by cSHMT; and for the synthesis of 5-methylTHF, catalyzed by methylenetetrahydrofolate reductase (MTHFR), a reaction that commits one-carbon units to the methionine cycle
Summary
Inhibition of MTHFR by SAM certainly contributes to the prevention of methyl trapping under normal conditions, there is evidence that competition among folate-dependent enzymes regulates the supply of one-carbon units to the methionine cycle. In fibroblasts where MS activity was deficient due to a variety of genetic mutations, serine formation was low compared with control cells, consistent with the formation of a folate methyl trap. Mutant fibroblasts with diminished MTHFR activity exhibited normal to high serine formation, indicating that MTHFR deficiency increased the availability of 5,10-methyleneTHF for cSHMT-catalyzed synthesis of serine from glycine [15]. The cSHMT enzyme is poised to regulate the metabolic competition between TS and MTHFR (see Fig. 1). We examined the effect of altered cSHMT expression and activity on the homocysteine remethylation pathway and the influence of glycine on cSHMT activity and folate metabolism
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