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Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism.

ABSTRACT: Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by SAM for decades, but the importance of this regulatory control to one-carbon metabolism has never been adequately understood. To shed light on this issue, we exchanged selected amino acid residues in a highly conserved stretch within the regulatory region of yeast MTHFR to create a series of feedback-insensitive, deregulated mutants. These were exploited to investigate the impact of defective allosteric regulation on one-carbon metabolism. We observed a strong growth defect in the presence of methionine. Biochemical and metabolite analysis revealed that both the folate and methionine cycles were affected in these mutants, as was the transsulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. ¹³C isotope labeling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CH₃THF) to form methionine. This reaction also drives SAM formation and further depletes ATP reserves. SAM was then cycled back to methionine, leading to futile cycles of SAM synthesis and recycling and explaining the necessity for MTHFR to be regulated by SAM. The study has yielded valuable new insights into the regulation of one-carbon metabolism, and the mutants appear as powerful new tools to further dissect out the intersection of one-carbon metabolism with various pathways both in yeasts and in humans.

SUBMITTER: Bhatia M

PROVIDER: S-EPMC7681022 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Diabetes was induced by treating rats with alloxan, and was confirmed by blood glucose values greater than 250 mg/dl. In perfused livers from both normal and diabetic rats, basal rates of O2 uptake were similar (120-130 mumol/h per g). In livers from diabetic rats, basal rates of glucose output of 60 mumol/h per g declined to around 20 mumol/h per g during 1 h of perfusion. Basal glucose production was abolished by pretreatment with an inhibitor of glycogen synthesis, galactosamine (1.5 g/kg), injected 3 h before perfusion. The subsequent infusion of lactate (2 mM) increased O2 uptake and glucose production about 40-50 mumol/h per g in both groups; however, the average maximal increase in glucose output was nearly twice as high in livers from normal (33 mumol/h per g) as from diabetic (18 mumol/h per g) rats. Rates of lactate uptake were also about 50% lower in livers from diabetic than from normal rats, yet rates of ketone-body formation were similar. Miniature O2 electrodes placed on periportal and pericentral regions of the liver lobule were employed to measure local rates of O2 uptake before, during and after infusion of lactate by stopping the flow of perfusate through the liver and measuring the decrease in local [O2]. Local rates of glucose production were calculated from the extra O2 consumed and the known stoichiometry between O2 uptake and glucose production from lactate. In livers from normal rats, glucose was synthesized predominantly in periportal regions of the liver lobule; however, glucose was produced exclusively in periportal regions in livers from diabetic rats. In pericentral regions, O2 uptake increased slightly in livers from normal rats, but declined significantly by 10 mumol/h per g in livers from diabetic rats. These data are consistent with the hypothesis that gluconeogenesis from lactate occurs exclusively in periportal regions of the liver lobule in livers from diabetic rats. A portion of this glucose is metabolized back to lactate in pericentral areas, leading to increased rates of glycolytic ATP production, thereby decreasing the demands for O2. This production of glucose from lactate in periportal regions, followed by conversion of glucose back into lactate in pericentral areas, raises the possibility of intercellular futile cycling, stimulated by diabetes.

Project description:UnlabelledMetabolism plays an important role in the persister phenotype, as evidenced by the number of strategies that perturb metabolism to sabotage this troublesome subpopulation. However, the absence of techniques to isolate high-purity populations of native persisters has precluded direct measurement of persister metabolism. To address this technical challenge, we studied Escherichia coli populations whose growth had been inhibited by the accumulation of the MazF toxin, which catalyzes RNA cleavage, as a model system for persistence. Using chromosomally integrated, orthogonally inducible promoters to express MazF and its antitoxin MazE, bacterial populations that were almost entirely tolerant to fluoroquinolone and ?-lactam antibiotics were obtained upon MazF accumulation, and these were subjected to direct metabolic measurements. While MazF model persisters were nonreplicative, they maintained substantial oxygen and glucose consumption. Metabolomic analysis revealed accumulation of all four ribonucleotide monophosphates (NMPs). These results are consistent with a MazF-catalyzed RNA futile cycle, where the energy derived from catabolism is dissipated through continuous transcription and MazF-mediated RNA degradation. When transcription was inhibited, oxygen consumption and glucose uptake decreased, and nucleotide triphosphates (NTPs) and NTP/NMP ratios increased. Interestingly, the MazF-inhibited cells were sensitive to aminoglycosides, and this sensitivity was blocked by inhibition of transcription. Thus, in MazF model persisters, futile cycles of RNA synthesis and degradation result in both significant metabolic demands and aminoglycoside sensitivity.ImportanceMetabolism plays a critical role in controlling each stage of bacterial persistence (shutdown, stasis, and reawakening). In this work, we generated an E. coli strain in which the MazE antitoxin and MazF toxin were artificially and independently inducible, and we used this strain to generate model persisters and study their metabolism. We found that even though growth of the model persisters was inhibited, they remained highly metabolically active. We further uncovered a futile cycle driven by continued transcription and MazF-mediated transcript degradation that dissipated the energy derived from carbon catabolism. Interestingly, the existence of this futile cycle acted as an Achilles' heel for MazF model persisters, rendering them vulnerable to killing by aminoglycosides.

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Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism.

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