Molecular Cloning, Characterization, and Regulation of the Human Mitochondrial Serine Hydroxymethyltransferase Gene

Abstract

The human mitochondrial serine hydroxymethyltransferase (mSHMT) gene was isolated, sequenced, and characterized. The 4.5-kilobase gene contains 10 introns and 11 exons, with all splice junctions conforming to the GT/AG rule. The 5 promoter region contains consensus motifs for several regulatory proteins including PEA-3, Sp-1, AP-2, and a CCCTCCC motif common to many genes expressed in liver. Consensus TATA or CAAT sequence motifs are not present, and primer extension and 5-rapid amplification of cDNA ends studies suggest that transcription initiation occurs at multiple sites. The mitochondrial leader sequence region of the deduced mRNA contains two potential ATG start sites, which are encoded by separate exons. The intervening 891-base pair intron contains consensus promoter elements suggesting that mSHMT may be transcribed from alternate promoters. 5-Rapid amplification of cDNA ends analysis demonstrated that the first ATG is transcribed in human MCF-7 cells. However, transfection of Chinese hamster ovary cells deficient in mSHMT activity with the human mSHMT gene lacking exon 1 overcame the cell's glycine auxotrophy and restored intracellular glycine concentrations to that observed in wildtype cells, showing that exon 1 is not essential for mSHMT localization or activity and that translation initiation from the second ATG is sufficient for mSHMT import into the mitochondria. Mitochondrial SHMT mRNA levels in MCF-7 cells did not vary during the cell cycle and were not affected by the absence of glycine, serine, folate, thymidylate, or purines from the media.Folates function as a family of cofactors by carrying onecarbon units that are required for the synthesis of glycine, thymidylate, purines, methionine, and numerous methylation reactions in mammalian cells. Serine is the major source of one-carbon units that are generated in a reaction catalyzed by the enzyme serine hydroxymethyltransferase (SHMT).1 Alternatively, one-carbon units can also be generated from glycine in cells that contain a glycine cleavage activity. SHMT is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible interconversion of serine and H 4 PteGlu to glycine and 5,10-CH 2 -H 4 PteGlu. SHMT is present in both the mitochondria (mSHMT) and the cytoplasm (cSHMT) in mammalian cells. The human SHMT cDNAs encoding the two isozymes have been isolated and the genes localized to chromosomes 12q13 and 17p11.2, respectively (1). Currently, the metabolic role of the individual SHMT isozymes is not clearly understood. Chinese hamster ovary cells lacking mSHMT activity are auxotrophic for glycine, suggesting that the mitochondria are the primary site of glycine synthesis, whereas the enzymes responsible for thymidylate, purine, and methionine synthesis are present in the cytoplasm (2). The central role of SHMT isozymes in producing one-carbon-substituted folate cofactors has suggested that the regulation of these enzymes may influence cell growth and proliferation and that they may be targets for the development of antineoplastic agents. T...

Role of polymorphisms in MTHFR and MTHFD1 genes in the outcome of childhood acute lymphoblastic leukemia

“…For example, the enzymes affecting folate catabolism 37 or methylene-THF synthesis might be of importance. 38,39 Similarly, the mitochondrial counterpart of MTHFD1, the NAD-dependent methylenetetrahydrofolate dehydrogenase, can influence 10-formyl-THF generation that in turn contributes to the cytoplasmatic folate pools. 40 In order to analyze the whole range of polymorphisms underlying other enzymatic variants within the folate pathway, as well as their possible gene-gene interactions, it is necessary that much larger and more collaborative studies be undertaken, if we are to understand the role of gene polymorphisms in ALL outcome.…”

Cytoplasmic Serine Hydroxymethyltransferase Regulates the Metabolic Partitioning of Methylenetetrahydrofolate but Is Not Essential in Mice

“…SHMT isozymes are present in each of these three compartments. Whereas mSHMT is expressed ubiquitously among mammalian cell types, cSHMT exhibits tissue-specific expression patterns (5,7,8).…”

“…SHMT isozymes are present in each of these three compartments. Whereas mSHMT is expressed ubiquitously among mammalian cell types, cSHMT exhibits tissue-specific expression patterns (5,7,8).A definitive role for the essentiality of mitochondria in generating folate-activated single carbons for cytoplasmic metabolism has yet to be established. Although the genes that encode the mitochondrial pathway for the folate-dependent conversion of serine to glycine and formate are expressed in mammalian embryonic and tumor cells, the enzyme responsible for the * This work was supported, in whole or in part, by National Institutes of Health Grant DK58144 from the USPHS.…”

“…The hydroxymethyl group of serine enters the folate-activated onecarbon pool through its THF-dependent conversion to glycine and 5,10-methylene-THF in a reaction catalyzed by the enzyme serine hydroxymethyltransferase (SHMT). SHMT is present in both the cytoplasm (cSHMT) and mitochondria (mSHMT), and the SHMT isozymes are encoded on distinct genes, Shmt1 and Shmt2, respectively (7,8).Mitochondrial one-carbon metabolism generates formate, a single-carbon molecule, from the amino acids serine, glycine, sarcosine, and dimethylglycine ( Fig. 1) (2).…”

Mimosine Is a Cell-specific Antagonist of Folate Metabolism

“…Cells were lysed with 1 ml of 50 mM potassium phosphate, pH 7.2, containing 0.1% Triton X-100. SHMT activity was determined in the crude cell extracts by measuring the rate of exchange of the pro-2S proton of [2][3] H]glycine, as described previously (20). This assay measures both mitochondrial and cytoplasmic SHMT activity.…”