Human Mitochondrial C1-Tetrahydrofolate Synthase

Prasannan, Priya; Pike, Schuyler T.; Peng, Kun; Shane, Barry; Appling, Dean R.

doi:10.1074/jbc.m304319200

Cited by 51 publications

(54 citation statements)

References 55 publications

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“…We also examined the cellular localization of DHFRL1. The compartmentalization of folate metabolism has been previously established (26), with several mitochondrial-specific enzymes identified in recent years (30)(31)(32)(33). Moreover, a small fraction of DHFR has been reported to localize to the nucleus during the synthesis phase of the cell cycle (17).…”

Section: Discussionmentioning

confidence: 99%

The former annotated human pseudogene dihydrofolate reductase-like 1 ( DHFRL1 ) is expressed and functional

McEntee

Minguzzi

O’Brien

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Human dihydrofolate reductase (DHFR) was previously thought to be the only enzyme capable of the reduction of dihydrofolate to tetrahydrofolate; an essential reaction necessary to ensure a continuous supply of biologically active folate. DHFR has been studied extensively from a number of perspectives because of its role in health and disease. Although the presence of a number of intronless DHFR pseudogenes has been known since the 1980s, it was assumed that none of these were expressed or functional. We show that humans do have a second dihydrofolate reductase enzyme encoded by the former pseudogene DHFRP4, located on chromosome 3. We demonstrate that the DHFRP4, or dihydrofolate reductase-like 1 (DHFRL1), gene is expressed and shares some commonalities with DHFR. Recombinant DHFRL1 can complement a DHFR-negative phenotype in bacterial and mammalian cells but has a lower specific activity than DHFR. The K m for NADPH is similar for both enzymes but DHFRL1 has a higher K m for dihydrofolate when compared to DHFR. The need for a second reductase with lowered affinity for its substrate may fulfill a specific cellular requirement. The localization of DHFRL1 to the mitochondria, as demonstrated by confocal microscopy, indicates that mitochondrial dihydrofolate reductase activity may be optimal with a lowered affinity for dihydrofolate. We also found that DHFRL1 is capable of the same translational autoregulation as DHFR by binding to its own mRNA; with each enzyme also capable of replacing the other. The identification of DHFRL1 will have implications for previous research involving DHFR.

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Section: Discussionmentioning

confidence: 99%

The former annotated human pseudogene dihydrofolate reductase-like 1 ( DHFRL1 ) is expressed and functional

McEntee

Minguzzi

O’Brien

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…This enzyme is responsible for the synthesis of 10-formyl-THF in mitochondria (23). Mitochondrial 10-formyl-THF can be used in the generation of formate through FTHFS activity encoded by Mthfd1L (8,24) or used to formylate the initiator methionyl-tRNA. Changes in mitochondrial protein synthesis were not associated with in utero lethality in Mthfd2-deficient embryos (15).…”

Section: Discussionmentioning

confidence: 99%

Mthfd1 Is an Essential Gene in Mice and Alters Biomarkers of Impaired One-carbon Metabolism

MacFarlane

Perry

Girnary

et al. 2009

Journal of Biological Chemistry

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Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1 gt/؉ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1 gt/؉ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors. Folate-mediated one-carbon (1C)3 metabolism is compartmentalized in the cytoplasm, mitochondria, and nucleus of mammalian cells (1). In the cytoplasm, 1C metabolism functions to carry and chemically activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine (2) (see Fig. 1). Methionine can be adenosylated to form S-adenosylmethionine (AdoMet), the major cellular methyl group donor required for the methylation of DNA, RNA, histones, small molecules, and lipids. Nuclear 1C metabolism functions to synthesize thymidylate from dUMP and serine during S phase through the small ubiquitin-like modifier-dependent translocation of cytoplasmic serine hydroxymethyltransferase (cSHMT), dihydrofolate reductase, and thymidylate synthase into the nucleus (3).Serine, through its conversion to glycine by SHMT, is a primary source of 1Cs for nucleotide and methionine synthesis (4). SHMT generates 1Cs in the cytoplasm, mitochondria, and nucleus, although the generation of 1Cs through SHMT activity in the cytoplasm is not essential in mice, indicating the essentiality of mitochondria-derived 1Cs for cytoplasmic 1C metabolism (5). In mitochondria, the hydroxymethyl group of serine and the C2 carbon of glycine are transferred to tetrahydrofolate (THF) to generate 5,10-methylene-THF by the mitochondrial isozyme of SHMT and the glycine cleavage system, respectively (6). The 1C carried by methylene-THF is oxidized and hydrolyzed to generate formate by the NAD-dependent methylene-

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“…Cell culture and selection of stable transfectants were carried out as described previously (32,37). Cells were disrupted and processed to give cytosolic and mitochondrial fractions as described (32). Mitochondria were isolated essentially according to Rickwood et al (38) and subfractionated by a modified digitonin method (39, 40) using 0.12 mg of digitonin/mg of mitochondria.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Mammalian MTHFD2L Encodes a Mitochondrial Methylenetetrahydrofolate Dehydrogenase Isozyme Expressed in Adult Tissues

Swetha

Young²,

Cole

et al. 2011

Journal of Biological Chemistry

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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.

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Human Mitochondrial C1-Tetrahydrofolate Synthase

Cited by 51 publications

References 55 publications

The former annotated human pseudogene dihydrofolate reductase-like 1 ( DHFRL1 ) is expressed and functional

The former annotated human pseudogene dihydrofolate reductase-like 1 ( DHFRL1 ) is expressed and functional

Mthfd1 Is an Essential Gene in Mice and Alters Biomarkers of Impaired One-carbon Metabolism

Mammalian MTHFD2L Encodes a Mitochondrial Methylenetetrahydrofolate Dehydrogenase Isozyme Expressed in Adult Tissues

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