Cloning, expression, and purification of 5,10-methenyltetrahydrofolate synthetase from Mus musculus

Anguera, Montserrat C.; Liu, Xiaowen

doi:10.1016/j.pep.2004.02.010

Cited by 15 publications

(15 citation statements)

References 21 publications

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“…5A), it was PCR-amplified from M. smegmatis genomic DNA and cloned to pET15b vector, such that the recombinant protein would be expressed as a N-terminal 6xHis-tagged peptide from the built-in isopropyl 1-thio-␤-D-galactopyranoside-inducible T7 promoter. First attempts to express the protein in E. coli host BL21(DE3) using standard induction protocol (1 mM isopropyl 1-thio-␤-D-galactopyranoside, 37°C) resulted in majority of the recombinant protein insoluble (not shown), similar to previously reported studies with other MTHFS proteins (38). To optimize the solubility of the protein, the E. coli strain ArcticExpress (DE3)RP, that co-expresses the coldadapted chaperonin proteins from Oleispira Antarctica, was used to facilitate protein folding at reduced temperatures.…”

Section: Mthfs Activity Mediates Utilization Of 5-cho-h 4 Pteglu 1 Insupporting

confidence: 84%

Bacterial Conversion of Folinic Acid Is Required for Antifolate Resistance

Ogwang

Nguyen

Sherman

et al. 2011

Journal of Biological Chemistry

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Antifolates, which are among the first antimicrobial agents invented, inhibit cell growth by creating an intracellular state of folate deficiency. Clinical resistance to antifolates has been mainly attributed to mutations that alter structure or expression of enzymes involved in de novo folate synthesis. We identified a Mycobacterium smegmatis mutant, named FUEL (which stands for folate utilization enzyme for leucovorin), that is hypersusceptible to antifolates. Chemical complementation indicated that FUEL is unable to metabolize folinic acid (also known as leucovorin or 5-formyltetrahydrofolate), whose metabolic function remains unknown. Targeted mutagenesis, genetic complementation, and biochemical studies showed that FUEL lacks 5,10-methenyltetrahydrofolate synthase (MTHFS; also called 5-formyltetrahydrofolate cyclo-ligase; EC 6.3.3.2) activity responsible for the only ATP-dependent, irreversible conversion of folinic acid to 5,10-methenyltetrahydrofolate. In trans expression of active MTHFS proteins from bacteria or human restored both antifolate resistance and folinic acid utilization to FUEL. Absence of MTHFS resulted in marked cellular accumulation of polyglutamylated species of folinic acid. Importantly, MTHFS also affected M. smegmatis utilization of monoglutamylated 5-methyltetrahydrofolate exogenously added to the medium. Likewise, Escherichia coli mutants lacking MTHFS became susceptible to antifolates. These results indicate that folinic acid conversion by MTHFS is required for bacterial intrinsic antifolate resistance and folate homeostatic control. This novel mechanism of antimicrobial antifolate resistance might be targeted to sensitize bacterial pathogens to classical antifolates.

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Section: Mthfs Activity Mediates Utilization Of 5-cho-h 4 Pteglu 1 Insupporting

confidence: 84%

Bacterial Conversion of Folinic Acid Is Required for Antifolate Resistance

Ogwang

Nguyen

Sherman

et al. 2011

Journal of Biological Chemistry

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“…Protein Expression and Purification-Recombinant murine MTHFS protein was expressed and purified as described elsewhere (31).…”

Section: Methodsmentioning

confidence: 99%

“…Purine Biosynthesis Assay-SH-SY5Y neuroblastoma cells expressing the human MTHFS cDNA (SH-SY5YMTHFS) have been described elsewhere (19,31). These cells were maintained in Minimal Essential Medium, alpha modification (Hyclone) supplemented with 11% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

Regulation of de Novo Purine Biosynthesis by Methenyltetrahydrofolate Synthetase in Neuroblastoma

Field

Szebenyi

Stover

2006

Journal of Biological Chemistry

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“…Expression was induced overnight with isopropyl-beta-D-thiogalactopyranoside (750 μM). Proteins were purified according to the protocol designed for His-MTHFS (29). D activity was quantified using an enzyme-coupled assay with SHMT generating 5,10-methyleneTHF from serine and THF.…”

Section: Methodsmentioning

confidence: 99%

Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis

Field¹,

Kamynina²,

Watkins

et al. 2014

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

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An inborn error of metabolism associated with mutations in the human methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) gene has been identified. The proband presented with SCID, megaloblastic anemia, and neurologic abnormalities, but the causal metabolic impairment is unknown. SCID has been associated with impaired purine nucleotide metabolism, whereas megaloblastic anemia has been associated with impaired de novo thymidylate (dTMP) biosynthesis. MTHFD1 functions to condense formate with tetrahydrofolate and serves as the primary entry point of single carbons into folate-dependent one-carbon metabolism in the cytosol. In this study, we examined the impact of MTHFD1 loss of function on folate-dependent purine, dTMP, and methionine biosynthesis in fibroblasts from the proband with MTHFD1 deficiency. The flux of formate incorporation into methionine and dTMP was decreased by 90% and 50%, respectively, whereas formate flux through de novo purine biosynthesis was unaffected. Patient fibroblasts exhibited enriched MTHFD1 in the nucleus, elevated uracil in DNA, lower rates of de novo dTMP synthesis, and increased salvage pathway dTMP biosynthesis relative to control fibroblasts. These results provide evidence that impaired nuclear de novo dTMP biosynthesis can lead to both megaloblastic anemia and SCID in MTHFD1 deficiency.folate | MTHFD1 | SCID | megaloblastic anemia | thymidylate

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Cloning, expression, and purification of 5,10-methenyltetrahydrofolate synthetase from Mus musculus

Cited by 15 publications

References 21 publications

Bacterial Conversion of Folinic Acid Is Required for Antifolate Resistance

Bacterial Conversion of Folinic Acid Is Required for Antifolate Resistance

Regulation of de Novo Purine Biosynthesis by Methenyltetrahydrofolate Synthetase in Neuroblastoma

Human mutations in methylenetetrahydrofolate dehydrogenase 1 impair nuclear de novo thymidylate biosynthesis

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