Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism. The enzyme is known to be allosterically inhibited by S-adenosyl methionine (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 transulfuration pathway, leading also to a disruption in redox homeostasis. The major consequences, however, appeared to be in the depletion of nucleotides. 13C isotope labelling 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.

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In vitro studies involving cell lines or primary cells, provide critical insights into their physiology under normal and perturbed conditions like cancer and infection. Given that there are multiple sources of carbon, nitrogen, and other nutrients available in routinely used standard media (such as DMEM, RPMI), it is vital to quantify their contribution to cellular metabolism. 13C based Isotopic tracers of the media components can be used to kinetically track their oxidation by the cell systems such as Human Lung Carcinoma (A549) cells. In this study, a universally labelled glucose tracer ([13C6]glucose) was used to quantify its metabolic contribution that provided further insights into the central carbon metabolism of A549 cells. Gas chromatography and mass spectrometry (GC-MS) based mass isotopomer analysis (average 13C) of methanolic extracts (glycerol: 5.46±3.53 % and lactate: 74.4±2.65 %), amino acids derived from acid hydrolysates of protein (Serine: 4.51±0.21 %, Glycine: 2.44±0.31 %, Alanine: 24.56±0.59 %, Glutamate: 8.81±0.85 %, Proline: 6.96±0.53 % and Aspartate: 10.72±0.95 %) and the metabolites of the culture filtrate (glycerol: 43.14±1.45 % and lactate: 81.67±0.91 %), allowed to capture the relative contribution of glucose. We observed the Warburg effect and a significant amount of lactate contributed from glucose, was released to the media. 13C glycerol of glucogenic origin was kinetically released to the culture filtrate and might be playing a critical role in metabolic reprogramming of A549 cells. Part of the protein biomass contributed from amino acids were of glucogenic origin. Besides, the workflow adopted for 13C analysis and derived average 13C of each metabolite provided a standard methodology that could be useful in defining the metabolic phenotypes of cells in normal and perturbed conditions. Understanding precisely the altered cellular metabolism to meet the biomass demand under a range of physiological conditions, kinetically, may identify pathways for targeted and effective therapeutic interventions.

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