@article {2248, title = {Allosteric inhibition of MTHFR prevents futile SAM cycling and maintains nucleotide pools in one-carbon metabolism.}, journal = {J Biol Chem}, volume = {295}, year = {2020}, month = {2020 11 20}, pages = {16037-16057}, 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. C isotope labeling and metabolic studies revealed that the deregulated MTHFR cells undergo continuous transmethylation of homocysteine by methyltetrahydrofolate (CHTHF) 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.

}, keywords = {Adenosine Triphosphate, Allosteric Regulation, Humans, Methylation, Methylenetetrahydrofolate Reductase (NADPH2), S-Adenosylmethionine, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins}, issn = {1083-351X}, doi = {10.1074/jbc.RA120.015129}, author = {Bhatia, Muskan and Thakur, Jyotika and Suyal, Shradha and Oniel, Ruchika and Chakraborty, Rahul and Pradhan, Shalini and Sharma, Monika and Sengupta, Shantanu and Laxman, Sunil and Masakapalli, Shyam Kumar and Bachhawat, Anand Kumar} } @article {2203, title = {Genome-scale reconstruction of Gcn4/ATF4 networks driving a growth program.}, journal = {PLoS Genet}, volume = {16}, year = {2020}, month = {2020 12}, pages = {e1009252}, abstract = {

Growth and starvation are considered opposite ends of a spectrum. To sustain growth, cells use coordinated gene expression programs and manage biomolecule supply in order to match the demands of metabolism and translation. Global growth programs complement increased ribosomal biogenesis with sufficient carbon metabolism, amino acid and nucleotide biosynthesis. How these resources are collectively managed is a fundamental question. The role of the Gcn4/ATF4 transcription factor has been best studied in contexts where cells encounter amino acid starvation. However, high Gcn4 activity has been observed in contexts of rapid cell proliferation, and the roles of Gcn4 in such growth contexts are unclear. Here, using a methionine-induced growth program in yeast, we show that Gcn4/ATF4 is the fulcrum that maintains metabolic supply in order to sustain translation outputs. By integrating matched transcriptome and ChIP-Seq analysis, we decipher genome-wide direct and indirect roles for Gcn4 in this growth program. Genes that enable metabolic precursor biosynthesis indispensably require Gcn4; contrastingly ribosomal genes are partly repressed by Gcn4. Gcn4 directly binds promoter-regions and transcribes a subset of metabolic genes, particularly driving lysine and arginine biosynthesis. Gcn4 also globally represses lysine and arginine enriched transcripts, which include genes encoding the translation machinery. The Gcn4 dependent lysine and arginine supply thereby maintains the synthesis of the translation machinery. This is required to maintain translation capacity. Gcn4 consequently enables metabolic-precursor supply to bolster protein synthesis, and drive a growth program. Thus, we illustrate how growth and starvation outcomes are both controlled using the same Gcn4 transcriptional outputs that function in distinct contexts.

}, keywords = {Basic-Leucine Zipper Transcription Factors, Cell Proliferation, Gene Expression Regulation, Fungal, Gene Regulatory Networks, Genome, Fungal, Ribosomes, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcriptional Activation}, issn = {1553-7404}, doi = {10.1371/journal.pgen.1009252}, author = {Srinivasan, Rajalakshmi and Walvekar, Adhish S and Rashida, Zeenat and Seshasayee, Aswin and Laxman, Sunil} } @article {2150, title = {The Rad53-Spt21 and Tel1 axes couple glucose tolerance to histone dosage and subtelomeric silencing.}, journal = {Nat Commun}, volume = {11}, year = {2020}, month = {2020 08 19}, pages = {4154}, abstract = {

The DNA damage response (DDR) coordinates DNA metabolism with nuclear and non-nuclear processes. The DDR kinase Rad53 controls histone degradation to assist DNA repair. However, Rad53 deficiency causes histone-dependent growth defects in the absence of DNA damage, pointing out unknown physiological functions of the Rad53-histone axis. Here we show that histone dosage control by Rad53 ensures metabolic homeostasis. Under physiological conditions, Rad53 regulates histone levels through inhibitory phosphorylation of the transcription factor Spt21 on Ser276. Rad53-Spt21 mutants display severe glucose dependence, caused by excess histones through two separable mechanisms: dampening of acetyl-coenzyme A-dependent carbon metabolism through histone hyper-acetylation, and Sirtuin-mediated silencing of starvation-induced subtelomeric domains. We further demonstrate that repression of subtelomere silencing by physiological Tel1 and Rpd3 activities coveys tolerance to glucose restriction. Our findings identify DDR mutations, histone imbalances and aberrant subtelomeric chromatin as interconnected causes of glucose dependence, implying that DDR kinases coordinate metabolism and epigenetic changes.

}, keywords = {Acetylation, Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins, Checkpoint Kinase 2, DNA Damage, DNA Repair, Gene Silencing, Glucose, Histone Deacetylases, Histones, Intracellular Signaling Peptides and Proteins, Mutation, Phosphorylation, Protein-Serine-Threonine Kinases, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Serine, Telomere, Transcription Factors}, issn = {2041-1723}, doi = {10.1038/s41467-020-17961-4}, author = {Bruhn, Christopher and Ajazi, Arta and Ferrari, Elisa and Lanz, Michael Charles and Batrin, Renaud and Choudhary, Ramveer and Walvekar, Adhish and Laxman, Sunil and Longhese, Maria Pia and Fabre, Emmanuelle and Smolka, Marcus Bustamente and Foiani, Marco} } @article {616, title = {Mechanistic implications from structures of yeast alcohol dehydrogenase complexed with coenzyme and an alcohol.}, journal = {Arch Biochem Biophys}, volume = {591}, year = {2016}, month = {2016 Feb 1}, pages = {35-42}, abstract = {

Yeast alcohol dehydrogenase I is a homotetramer of subunits with 347 amino acid residues, catalyzing the oxidation of alcohols using NAD(+) as coenzyme. A new X-ray structure was determined at 3.0 {\r A} where both subunits of an asymmetric dimer bind coenzyme and trifluoroethanol. The tetramer is a pair of back-to-back dimers. Subunit A has a closed conformation and can represent a Michaelis complex with an appropriate geometry for hydride transfer between coenzyme and alcohol, with the oxygen of 2,2,2-trifluoroethanol ligated at 2.1 {\r A} to the catalytic zinc in the classical tetrahedral coordination with Cys-43, Cys-153, and His-66. Subunit B has an open conformation, and the coenzyme interacts with amino acid residues from the coenzyme binding domain, but not with residues from the catalytic domain. Coenzyme appears to bind to and dissociate from the open conformation. The catalytic zinc in subunit B has an alternative, inverted coordination with Cys-43, Cys-153, His-66 and the carboxylate of Glu-67, while the oxygen of trifluoroethanol is 3.5 {\r A} from the zinc. Subunit B may represent an intermediate in the mechanism after coenzyme and alcohol bind and before the conformation changes to the closed form and the alcohol oxygen binds to the zinc and displaces Glu-67.

}, keywords = {Alcohol Dehydrogenase, Binding Sites, Catalysis, Coenzymes, Computer Simulation, Enzyme Activation, Models, Chemical, Models, Molecular, NAD, Protein Binding, Protein Conformation, Saccharomyces cerevisiae Proteins, Substrate Specificity, Trifluoroethanol}, issn = {1096-0384}, doi = {10.1016/j.abb.2015.12.009}, author = {Plapp, Bryce V and Charlier, Henry A and Ramaswamy, S} }