Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase.
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ABSTRACT:
SUBMITTER: Varalakshmi K
PROVIDER: S-EPMC1146819 | biostudies-other | 1986 May
REPOSITORIES: biostudies-other
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Project description:The folate and methionine cycles, constituting one-carbon metabolism, are critical pathways for cell survival. Intersecting these two cycles, 5,10-methylenetetrahydrofolate reductase (MTHFR) directs one-carbon units from the folate to methionine cycle, to be exclusively used for methionine and S-adenosylmethionine (AdoMet) synthesis. MTHFR deficiency and upregulation result in diverse disease states, rendering it an attractive drug target. The activity of MTHFR is inhibited by the binding of AdoMet to an allosteric regulatory domain distal to the enzyme's active site, which we have previously identified to constitute a novel fold with a druggable pocket. Here, we screened 162 AdoMet mimetics using differential scanning fluorimetry, and identified 4 compounds that stabilized this regulatory domain. Three compounds were sinefungin analogues, closely related to AdoMet and S-adenosylhomocysteine (AdoHcy). The strongest thermal stabilisation was provided by (S)-SKI-72, a potent inhibitor originally developed for protein arginine methyltransferase 4 (PRMT4). Using surface plasmon resonance, we confirmed that (S)-SKI-72 binds MTHFR via its allosteric domain with nanomolar affinity. Assay of MTHFR activity in the presence of (S)-SKI-72 demonstrates inhibition of purified enzyme with sub-micromolar potency and endogenous MTHFR from HEK293 cell lysate in the low micromolar range, both of which are lower than AdoMet. Nevertheless, unlike AdoMet, (S)-SKI-72 is unable to completely abolish MTHFR activity, even at very high concentrations. Combining binding assays, kinetic characterization and compound docking, this work indicates the regulatory domain of MTHFR can be targeted by small molecules and presents (S)-SKI-72 as an excellent candidate for development of MTHFR inhibitors.
| S-EPMC8040968 | biostudies-literature
Project description:BACKGROUND: The polymorphism 5,10-methylenetetrahydrofolate reductase (MTHFR) c.1298A>C is associated with various diseases. 45 DNA samples homozygous for the A allele and 40 DNA probes homozygous for the C allele were taken from healthy German subjects of white Caucasian origin to analyze the haplotype of the two MTHFR c.1298A>C alleles. Samples were genotyped for the polymorphism MTHFR c.677C>T and for the silent polymorphisms MTHFR c.129C>T, IVS2 533 G>A, c.1068C>T and IVS10 262C>G. FINDINGS: Haplotype construction revealed that the C-allele of MTHFR c.1298A>C was more frequently observed in cis with c.129T, IVS2 533A, c.677C, c.1068T, and IVS10 262 G than expected from normal distribution. Estimation of the most recent common ancestor with the DMLE + 2.3 program resulted in an estimated age of approximately 36,660 years of the MTHFR c.1298C allele. CONCLUSION: Given that the era from 30,000 to 40,000 years ago is characterised by the spread of modern humans in Europe and that the prevalence of the MTHFR c.1298C allele is significantly higher in Central Europe in comparison to African populations, a selective advantage of MTHFR c.1298C could be assumed, e. g. by adaption to changes in the nutritional environment. The known founder ancestry of the T allele of MTHFR c.677C>T allele, together with the present data suggests that the MTHFR mutant alleles c.677T and 1298C arose from two independent ancestral alleles, that both confer a selective advantage.
| S-EPMC3212962 | biostudies-literature
Project description:The objective of this study was to examine the relation between the 5, 10-methylenetetrahydrofolate reductase (MTHFR) gene and behaviors related to attention- deficit/hyperactivity disorder (ADHD) in individuals with myelomeningocele. The rationale for the study was twofold: folate metabolizing genes, (e.g. MTHFR), are important not only in the etiology of neural tube defects but are also critical to cognitive function; and individuals with myelomeningocele have an elevated incidence of ADHD. Here, we tested 478 individuals with myelomeningocele for attention-deficit hyperactivity disorder behavior using the Swanson Nolan Achenbach Pelham-IV ADHD rating scale. Myelomeningocele participants in this group for whom DNAs were available were genotyped for seven single nucleotide polymorphisms (SNPs) in the MTHFR gene. The SNPs were evaluated for an association with manifestation of the ADHD phenotype in children with myelomeningocele. The data show that 28.7% of myelomeningocele participants exhibit rating scale elevations consistent with ADHD; of these 70.1% had scores consistent with the predominantly inattentive subtype. In addition, we also show a positive association between the SNP rs4846049 in the 3'-untranslated region of the MTHFR gene and the attention-deficit hyperactivity disorder phenotype in myelomeningocele participants. These results lend further support to the finding that behavior related to ADHD is more prevalent in patients with myelomeningocele than in the general population. These data also indicate the potential importance of the MTHFR gene in the etiology of the ADHD phenotype.
| S-EPMC3515551 | biostudies-literature
Project description:Cleft lip with or without cleft palate (CL/P) is considered a multifactorial genetic disorder. Folic acid metabolism has been suggested to underlie the development of CL/P. The gene for the enzyme 5,10-methylentetrahydrofolate reductase (MTHFR) contributes to folic acid metabolism, and polymorphisms of this gene at C677T (rs1801133) and A1298C (rs1801131) are reported to alter its enzyme activity and are suggested to be involved in CL/P development. We investigated C677T and A1298C polymorphisms of the MTHFR gene in Japanese patients with nonsyndromic CL/P and cleft palate only (CPO). We examined 240 patients with CL/P, 103 fathers and 153 mothers of the patients, and 68 healthy controls. Restriction fragment length polymorphisms (RFLPs) of C677T and A1298C of MTHFR were analyzed. We determined the frequencies of the polymorphisms in the patients and controls and performed a transmission equilibrium test and haplotype analysis of both MTHFR C677T and A1298C. There were no significant differences in the frequencies of MTHFR C677T and A1298C polymorphisms between the patients and controls. We did not observe transmission equilibrium or linkage equilibrium among the cases. In this experimental condition, we did not detect an association of MTHFR C677T and/or A1298C polymorphisms with the development of CL/P in this Japanese cohort.
| S-EPMC7583695 | biostudies-literature
Project description:The folate and methionine cycles are crucial for biosynthesis of lipids, nucleotides and proteins, and production of the methyl donor S-adenosylmethionine (SAM). 5,10-methylenetetrahydrofolate reductase (MTHFR) represents a key regulatory connection between these cycles, generating 5-methyltetrahydrofolate for initiation of the methionine cycle, and undergoing allosteric inhibition by its end product SAM. Our 2.5 Å resolution crystal structure of human MTHFR reveals a unique architecture, appending the well-conserved catalytic TIM-barrel to a eukaryote-only SAM-binding domain. The latter domain of novel fold provides the predominant interface for MTHFR homo-dimerization, positioning the N-terminal serine-rich phosphorylation region near the C-terminal SAM-binding domain. This explains how MTHFR phosphorylation, identified on 11 N-terminal residues (16 in total), increases sensitivity to SAM binding and inhibition. Finally, we demonstrate that the 25-amino-acid inter-domain linker enables conformational plasticity and propose it to be a key mediator of SAM regulation. Together, these results provide insight into the molecular regulation of MTHFR.
| S-EPMC5995969 | biostudies-literature
Project description:The high-affinity folate-binding protein (FBP) is primarily involved in the uptake of the 5-methyltetrahydrofolate, and its expression may be physiologically regulated by the intracellular folate content. The overexpression of FBP on the cell surface of ovarian carcinoma cells may be responsible for an increased folate uptake. We tested the hypothesis of the existence of a defect in the 5, 10-methylenetetrahydrofolate reductase (MTHFR) in ovarian tumours that could cause reduced intracellular regeneration of the 5-methyltetrahydrofolate and induce increased FBP expression. No sequence mutations were found in the MTHFR gene, but allelic deletions of this gene were frequently detected in ovarian tumours (59%). Chromosomal losses appeared to be confined to the 1p36.3 region to which the MTHFR gene maps. Although it cannot be stated that MTHFR is the target gene of the chromosomal loss involving the 1p36.3 region, a correlation between loss of heterozygosity at this locus and decrease in MTHFR activity was shown, suggesting a role of these allelic deletions in generating a biochemical defect in folate metabolism. Further studies are needed to assess further the relationship between MTHFR and FBP overexpression, but the demonstration of the alteration of a key metabolic enzyme of the folate cycle in a subset of human ovarian tumours is in accordance with the hypothesis of an altered folate metabolism in these neoplasias and might be exploited for therapeutic purposes.
| S-EPMC2222800 | biostudies-other
Project description:Serine hydroxymethyltransferase (SHMT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyses the transfer of the hydroxymethyl group from serine to tetrahydrofolate to yield glycine and N (5), N (10)-methylenetetrahydrofolate. An analysis of the known SHMT sequences indicated that several amino acid residues were conserved. In this paper, we report the identification of the amino acid residues essential for maintaining the oligomeric structure of sheep liver cytosolic recombinant SHMT (scSHMT) through intra- and inter-subunit interactions and by stabilizing the binding of PLP at the active site. The mutation of Lys-71, Arg-80 and Asp-89, the residues involved in intra-subunit ionic interactions, disturbed the oligomeric structure and caused a loss of catalytic activity. Mutation of Trp-110 to Phe was without effect, while its mutation to Ala resulted in the enzyme being present in the insoluble fraction. These results suggested that Trp-110 located in a cluster of hydrophobic residues was essential for proper folding of the enzyme. Arg-98 and His-304, residues involved in the inter-subunit interactions, were essential for maintaining the tetrameric structure. Mutation of Tyr-72, Asp-227 and His-356 at the active site which interact with PLP resulted in the loss of PLP, and hence loss of tetrameric structure. Mutation of Cys-203, located away from the active site, weakened PLP binding indirectly. The results demonstrate that in addition to residues involved in inter-subunit interactions, those involved in PLP binding and intra-subunit interactions also affect the oligomeric structure of scSHMT.
| S-EPMC1223116 | biostudies-other
Project description:The common 5,10-methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism causes decreased activity of this enzyme and can be associated with mild-to-moderate hyperhomocysteinemia in homozygotes, particularly when there is folic acid deficiency, as well as with vascular dementia, arterial thrombosis, venous thrombosis, neural-tube defects, and fetal loss. When folic acid intake is sufficient, homozygotes for MTHFR 677T appear to be protected against colon cancer and acute lymphatic leukemia, and fetuses bearing this genotype have an augmented survival. The distribution of MTHFR 677T is worldwide, but its frequency in different populations varies extensively. In the present study, we addressed the question of whether the MTHFR 677T alteration has an ancestral origin or has occurred repeatedly. We analyzed the frequency distribution of the previously described polymorphism A1298C in exon 7 and of three intronic dimorphisms, in white Israelis (Jews and Arabs), Japanese, and Ghanaian Africans. The 677T allele was, remarkably, associated with one haplotype, G-T-A-C, in white and Japanese homozygotes. Among the Africans, analysis of maximum likelihood also disclosed an association with the G-T-A-C haplotype, although none of the 174 subjects examined was homozygous for MTHFR 677T. These results suggest that the MTHFR 677T alteration occurred on a founder haplotype that may have had a selective advantage.
| S-EPMC384952 | biostudies-literature
Project description:Background: Severe 5,10-methylenetetrahydrofolate reductase (MTHFR) deficiency is a heterogeneous metabolic disorder inherited in an autosomal recessive manner. Pathogenic mutations in MTHFR gene have been associated with severe MTHFR deficiency. The clinical presentation of MTHFR deficiency is highly variable and associated with several neurological anomalies. Methods: Direct whole-exome sequencing (WES) was performed in all the five available individuals from the family, including the affected individual (III-7) using standard procedures. Results: We observed a proband (III-7) with an abnormality in the cerebral white matter, apnoea, and microcephaly. WES analysis identified a novel homozygous non-sense mutation (c.154C>T; p.Arg52*) in MTHFR gene that segregated with the disease phenotype within the family. Conclusion: We identified a novel non-sense mutation in MTHFR gene in a single Egyptian family with severe MTHFR deficiency. The present investigation is clinically important, as it adds to the growing list of MTHFR mutations, which might help in genetic counseling of families of affected children and proper genotype-phenotype correlation.
| S-EPMC6491806 | biostudies-literature
Project description:Hyperhomocysteinemia is a common medical condition observed in patients with aminoaciduria. Deficiency in cystathionine beta-synthase, metabolism of cobalamin associated C, peroxiredoxin 1, 5-methyltetrahydrofolate-homocysteine methyltransferase reductase, LMBR1 domain containing 1, 5-methyltetrahydrofolate-homocysteine methyltransferase or 5,10-methylenetetrahydrofolate reductase (MTHFR) all can result in an elevation in plasma homocysteine, which has been reported to be a risk factor of vascular events, such as atherosis, acute myocardial infarction and cerebral stroke. Hyperhomocysteinemia due to the deficiency of 5,10-methylenetetrahydrofolate reductase (MTHFR; also known as 5,10-methyl THR reductase) is an autosomal recessive rare disease caused by defects in the MTHFR gene. The clinical manifestations of this disorder are heterogeneous, ranging from asymptomatic to severe neurological disorders. However, hydrocephalus has seldomly been reported in patients with MTHFR deficiency. The present study thus describes a case of severe MTHFR deficiency in an infant, whose main manifestation was hydrocephalus. The clinical course and genotype of the patient were also examined. Specifically, a 4-month-old boy with hydrocephalus was admitted to hospital. Clinical examinations and genetic sequencing of the patient were performed to determine the probable causative factors. A physical examination revealed that the patient had developmental delay and progressive hydrocephalus. Amino acid analysis of the blood revealed an enhancement in serum homocysteine levels and a decrease in blood methionine and free carnitine levels. The organic acid levels in urine were normal. Therefore, he was diagnosed with hyperhomocysteinemia. Targeted next-generation sequencing was performed to determine the pathogenetic gene in this case. A paternal mutation c.1530G>A (p.K510K) and a maternal mutation c.233C>A (p.S78X) were identified. Previous experimental evidence indicated that these two mutations were all pathogenic; therefore, this patient was ultimately diagnosed with MTHFR deficiency. The patient in described herein study presented with severe progressive hydrocephalus in association with a delayed developmental milestone. According to the clinical and genetic tests, the patient was diagnosed with severe MTHFR deficiency. It thus is recommended that screening for metabolites and performing gene sequencing in infants presenting with undisclosed hydrocephalus.
| S-EPMC9829199 | biostudies-literature