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...
Human cDNAs for methionine synthase (5-methyltetrahydrofolate:L-homocysteine S-transmethylase; EC 2.1.1.13) have been isolated from fetal and adult liver and HepG2 libraries. The cDNAs span 7.2 kilobases (kb) and consist of a 394-base pair upstream untranslated region, a 3795-base pair open reading frame encoding a 1265-residue 140.3-kDa protein, and about 3 kb of 3 region. The deduced protein sequence shares 53 and 63% identity with the Escherichia coli and the presumptive Caenorhabditis elegans proteins, respectively, and contains all residues implicated in B 12 binding to the E. coli protein. Several potential polymorphisms and a cryptic splice deletion were detected in the coding region of the cDNAs. A polymorphism that results in a D919G modification in the protein is fairly common in human DNA samples. Northern analyses of poly(A) mRNA indicated two major species of about 8 and 10 kb in human tissues and some minor, partially spliced species. mRNA levels were highest in the pancreas, skeletal muscle, and heart of the adult and in the kidney in the fetus and were low in adult liver. Genomic clones were isolated and the 5 region was analyzed. Exon 1 is preceded by a number of potential promoter sites, including an E box, CAAT boxes, and a GC box, but this region lacks a TATA element. The human methionine synthase gene was localized to chromosome region 1q42.3-43 by in situ hybridization.Methionine synthase, one of two B 12 -dependent mammalian enzymes, catalyzes the remethylation of homocysteine to methionine and the concurrent demethylation of 5-methyltetrahydrofolate to tetrahydrofolate (1). Under conditions of B 12 -depletion, such as pernicious anemia, loss of methionine synthase activity leads to a "methyl folate trap." The depletion of other folate coenzymes results in defective DNA synthesis and the development of megaloblastic anemia (1-3). Recently, homocysteine has received considerable attention as elevations in plasma homocysteine have been implicated as a risk factor for vascular disease (4, 5). Polymorphisms in methylenetetrahydrofolate reductase, the enzyme that catalyzes the synthesis of 5-methyltetrahydrofolate, and in cystathionine -synthase, which catalyzes the removal of homocysteine via the transsulfuration pathway, have been implicated in elevated homocysteine levels and in vascular disease risk (6, 7).Little is known about the regulation or properties of eukaryotic methionine synthases, partly because of the very limited distribution of B 12 -dependent enzymes in eukaryotes. The Escherichia coli methionine synthase gene has been cloned and the protein purified to homogeneity, and the structure of its B 12 -binding domain has been elucidated (8 -10). Other bacterial genes and the Caenorhabditis elegans methionine synthase gene have been tentatively identified by homology to the E. coli gene (Ref. 11; accession number Z46828). The pig liver enzyme has recently been purified to near homogeneity and some of its kinetic properties have been characterized (12). We are interested in the meta...
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