The recognition that mitochondria participate in folate-mediated one-carbon metabolism grew out of pioneering work beginning in the 1950s from the laboratories of D.M. Greenberg, C.G. Mackenzie, and G. Kikuchi. These studies revealed mitochondria as the site of oxidation of one-carbon donors such as serine, glycine, sarcosine, and dimethylglycine. Subsequent work from these laboratories and others demonstrated the participation of folate coenzymes and folate-dependent enzymes in these mitochondrial processes. Biochemical and molecular genetic approaches in the 1980s and 1990s identified many of the enzymes involved and revealed an interdependence of cytoplasmic and mitochondrial one-carbon metabolism. These studies led to the development of a model of eukaryotic one-carbon metabolism that comprises parallel cytosolic and mitochondrial pathways, connected by one-carbon donors such as serine, glycine, and formate. Sequencing of the human and other mammalian genomes has facilitated identification of the enzymes that participate in this intercompartmental one-carbon metabolism, and animal models are beginning to clarify the roles of the cytoplasmic and mitochondrial isozymes of these enzymes. Identifying the mitochondrial transporters for the one-carbon donors and elucidating how flux through these pathways is controlled are two areas ripe for exploration.

Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH 2 -THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH 2 -THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP ؉ -dependent CH 2 -THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH 2 -THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH 2 -THF to formate in adult mammalian mitochondria.

The Saccharomyces cerevisiae ADE16 and ADE17 genes encode 5-aminoimidazole-4-carboxamide ribonucleotide transformylase isozymes that catalyze the penultimate step of the de novo purine biosynthesis pathway. Disruption of these two chromosomal genes results in adenine auxotrophy, whereas expression of either gene alone is sufficient to support growth without adenine. In this work, we show that an ade16 ade17 double disruption also leads to histidine auxotrophy, similar to the adenine/histidine auxotrophy of ade3 mutant yeast strains. We also report the purification and characterization of the ADE16 and ADE17 gene products (Ade16p and Ade17p). Like their counterparts in other organisms, the yeast isozymes are bifunctional, containing both 5-aminoimidazole-4-carboxamide ribonucleotide transformylase and inosine monophosphate cyclohydrolase activities, and exist as homodimers based on cross-linking studies. Both isozymes are localized to the cytosol, as shown by subcellular fractionation experiments and immunofluorescent staining. Epitope-tagged constructs were used to study expression of the two isozymes. The expression of Ade17p is repressed by the addition of adenine to the media, whereas Ade16p expression is not affected by adenine. Ade16p was observed to be more abundant in cells grown on nonfermentable carbon sources than in glucose-grown cells, suggesting a role for this isozyme in respiration or sporulation. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR)1 transformylase catalyzes the ninth step of the de novo purine biosynthesis pathway. This reaction involves the formylation of AICAR using 10-formyltetrahydrofolate as the formyl donor ( Fig. 1). Saccharomyces cerevisiae possesses two genes, ADE16 and ADE17, that encode AICAR transformylase enzymes that share 85% identity (1). This reaction is the only purine biosynthesis step that is catalyzed by separate isozymes in yeast. Expression of either ADE16 or ADE17 alone is sufficient to support the growth of yeast in adenine-free media, but disruption of both genes results in adenine auxotrophy (1). AICAR transformylase assays of yeast crude extracts demonstrated that the ADE17 gene product is the more active of the two enzymes, but the low level of activity supplied by the ADE16 gene product is sufficient for wild-type growth rates. Given these results, we began to question why yeast possess two AICAR transformylase isozymes and whether these two enzymes have separate metabolic roles in the cell.Recent research has shown a difference in the regulation of the expression of ADE16 and ADE17 by adenine. Like other purine biosynthesis genes, optimal expression of ADE17 is dependent on the transcription factors Bas1p and Bas2p, and ADE17 expression is repressed by adenine (2). The ADE17 gene is one of the most strongly repressed purine biosynthesis genes, as shown using ADE17-lacZ fusion constructs. Unlike other purine biosynthesis genes, there are no consensus Bas1p and Bas2p binding sequences in the promoter region of the ADE16 gene, and Northern blot analys...

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