Methylenetetrahydrofolate reductase (MTHFR) is a pivotal flavoprotein connecting the folate and methionine methyl cycles, catalyzing the conversion of methylenetetrahydrofolate to methyltetrahydrofolate. Human MTHFR (
Cobalamin-dependent methionine synthase (MS) is a key enzyme in methionine and folate one-carbon metabolism. MS is a large multi-domain protein capable of binding and activating three substrates: homocysteine, folate, and
- Award ID(s):
- 1945174
- PAR ID:
- 10468637
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract h MTHFR) undergoes elaborate allosteric regulation involving protein phosphorylation andS -adenosylmethionine (AdoMet)-dependent inhibition, though other factors such as subunit orientation and FAD status remain understudied due to the lack of a functional structural model. Here, we report crystal structures ofChaetomium thermophilum MTHFR (c MTHFR) in both active (R) and inhibited (T) states. We reveal FAD occlusion by Tyr361 in the T-state, which prevents substrate interaction. Remarkably, the inhibited form ofc MTHFR accommodates two AdoMet molecules per subunit. In addition, we conducted a detailed investigation of the phosphorylation sites inh MTHFR, three of which were previously unidentified. Based on the structural framework provided by ourc MTHFR model, we propose a possible mechanism to explain the allosteric structural transition of MTHFR, including the impact of phosphorylation on AdoMet-dependent inhibition. -
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Tetrahydrofolate and its derivatives participate in one-carbon transfer reactions in all organisms. The cellular form of tetrahydrofolate (THF) is modified by multiple glutamate residues and polyglutamylation plays a key role in organellar and cellular folate homeostasis. In addition, polyglutamylation of THF is known to increase the binding affinity to enzymes in the folate cycle, many of which can utilize polyglutamylated THF as a substrate. Here, we use X-ray crystallography to provide a high-resolution view of interactions between the enzyme serine hydroxymethyltransferase (SHMT), which provides one carbon precursors for the folate cycle, and a polyglutamylated form of THF. Our 1.7 Å crystal structure of soybean SHMT8 in complex with diglutamylated 5-formyl-THF reveals, for the first time, a structural rearrangement of a loop at the entrance to the folate binding site accompanied by the formation of novel specific interactions between the enzyme and the diglutamyl tail of the ligand. Biochemical assays show that additional glutamate moieties on the folate ligand increase both enzyme stability and binding affinity. Together these studies provide new information on SHMT structure and function and inform the design of anti-folate agents.
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