Inhibition of Folate Metabolism Drives Autophagy-Dependent Differentiation and Reduces Survival of Therapy-Resistant Leukaemic Stem Cells
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Serine hydroxymethyltransferase
Purine metabolism
Metabolic pathway
Serine hydroxymethyltransferase (SHMT) catalyzes the reversible conversion of l-serine and tetrahydrofolate into glycine and 5,10-methylenetetrahydrofolate. This enzyme, which plays a pivotal role in one-carbon metabolism, is involved in cancer metabolic reprogramming and is a recognized target of chemotherapy intervention. In humans, two isoforms of the enzyme exist, which are commonly termed cytosolic SHMT1 and mitochondrial SHMT2. Considerable attention has been paid to the structural, mechanistic, and metabolic features of these isozymes. On the other hand, a detailed comparison of their catalytic and regulatory properties is missing, although this aspect seems to be considerably important, considering that SHMT1 and SHMT2 reside in different cellular compartments, where they play distinct roles in folate metabolism. Here we performed a full kinetic characterization of the serine hydroxymethyltransferase reaction catalyzed by SHMT1 and SHMT2, with a focus on pH dependence and substrate inhibition. Our investigation, which allowed the determination of all kinetic parameters of serine hydroxymethyltransferase forward and backward reactions, uncovered a previously unobserved substrate inhibition by l-serine and highlighted several interesting differences between SHMT1 and SHMT2. In particular, SHMT2 maintains a pronounced tetrahydrofolate substrate inhibition even at the alkaline pH characteristic of the mitochondrial matrix, whereas with SHMT1 this is almost abolished. At this pH, SHMT2 also shows a catalytic efficiency that is much higher than that of SHMT1. These observations suggest that such different properties represent an adaptation of the isoforms to the respective cellular environments and that substrate inhibition may be a form of regulation.
Serine hydroxymethyltransferase
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Serine hydroxymethyltransferase
Purine metabolism
Pyridoxal phosphate
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Serine hydroxymethyltransferase
Grey matter
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Serine hydroxymethyltransferase
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Serine hydroxymethyltransferase
Pyridoxal phosphate
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Plasmodium parasites, the causative agent of malaria, rely heavily on de novo folate biosynthesis, and the enzymes in this pathway have therefore been explored extensively for antimalarial development. Serine hydroxymethyltransferase (SHMT) from Plasmodium spp., an enzyme involved in folate recycling and dTMP synthesis, has been shown to catalyze the conversion of L- and D-serine to glycine (Gly) in a THF-dependent reaction, the mechanism of which is not yet fully understood. Here, the crystal structures of P. vivax SHMT (PvSHMT) in a binary complex with L-serine and in a ternary complex with D-serine (D-Ser) and (6R)-5-formyltetrahydrofolate (5FTHF) provide clues to the mechanism underlying the control of enzyme activity. 5FTHF in the ternary-complex structure was found in the 6R form, thus differing from the previously reported structures of SHMT-Gly-(6S)-5FTHF from other organisms. This suggested that the presence of D-Ser in the active site can alter the folate-binding specificity. Investigation of binding in the presence of D-Ser and the (6R)- or (6S)-5FTHF enantiomers indicated that both forms of 5FTHF can bind to the enzyme but that only (6S)-5FTHF gives rise to a quinonoid intermediate. Likewise, a large surface area with a highly positively charged electrostatic potential surrounding the PvSHMT folate pocket suggested a preference for a polyglutamated folate substrate similar to the mammalian SHMTs. Furthermore, as in P. falciparum SHMT, a redox switch created from a cysteine pair (Cys125-Cys364) was observed. Overall, these results assert the importance of features such as stereoselectivity and redox status for control of the activity and specificity of PvSHMT.
Serine hydroxymethyltransferase
Ternary complex
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The hydroxymethyl group of serine is a primary source of tetrahydrofolate (THF)-activated one-carbon units that are required for the synthesis of purines and thymidylate and for S-adenosylmethionine (AdoMet)-dependent methylation reactions. Serine hydroxymethyltransferase (SHMT) catalyzes the reversible and THF-dependent conversion of serine to glycine and 5,10-methylene-THF. SHMT is present in eukaryotic cells as mitochondrial SHMT and cytoplasmic (cSHMT) isozymes that are encoded by distinct genes. In this study, the essentiality of cSHMT-derived THF-activated one-carbons was investigated by gene disruption in the mouse germ line. Mice lacking cSHMT are viable and fertile, demonstrating that cSHMT is not an essential source of THF-activated one-carbon units. cSHMT-deficient mice exhibit altered hepatic AdoMet levels and uracil content in DNA, validating previous in vitro studies that indicated this enzyme regulates the partitioning of methylenetetrahydrofolate between the thymidylate and homocysteine remethylation pathways. This study suggests that mitochondrial SHMT-derived one-carbon units are essential for folate-mediated one-carbon metabolism in the cytoplasm.
Serine hydroxymethyltransferase
Purine metabolism
Uracil
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fromspinach leaves wasabsolutely dependent ontetrahydrofolate; pyridoxal phosphate hasnoeffect ontheactivity. Thestability ofthis activity intheisolated mitochondria wasdependent onthepresence ofsulfhydryl compounds. Itwasapparently morestable atpH7.0to7.5thanathigher pHeventhough thepHoptimum ofserine hydroxymethyltransferase was 8.5forboththemitochondrial andcytoplasmic fractions. Distribution studies haveindicated thatserine hydroxymethyltransferase waspredominantly located inthemitochondria. Theactivity ofserine hydroxymethyltransferase wasobserved tobeco-compartmented withglycine decarboxylation andmalate dehydrogenase behind themitochondrial inner membrane. Thisactivity could besolubilized byKCIfromosmotically ruptured mitochondrial membrane fractions butsubstantial activity (35to40%)was still retained withthemembrane fractions at0.3MKCI.Thissuggests that theglycine decarboxylation-serine hydroxymethyltransferase complex may beclosely bound totheinternal surface ofthemitochondrial inner membrane. Therelationship ofthis integrated enzymecomplex toCO2evolution andserine synthesis during photorespiration andthephysiological role of thedicarboxylate shuttle werediscussed.
Serine hydroxymethyltransferase
Photorespiration
Decarboxylation
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The activity of serine hydroxymethyltransferase in mitochondria isolated from spinach leaves was absolutely dependent on tetrahydrofolate; pyridoxal phosphate has no effect on the activity. The stability of this activity in the isolated mitochondria was dependent on the presence of sulfhydryl compounds. It was apparently more stable at pH 7.0 to 7.5 than at higher pH even though the pH optimum of serine hydroxymethyltransferase was 8.5 for both the mitochondrial and cytoplasmic fractions. Distribution studies have indicated that serine hydroxymethyltransferase was predominantly located in the mitochondria. The activity of serine hydroxymethyltransferase was observed to be co-compartmented with glycine decarboxylation and malate dehydrogenase behind the mitochondrial inner membrane. This activity could be solubilized by KCl from osmotically ruptured mitochondrial membrane fractions but substantial activity (35 to 40%) was still retained with the membrane fractions at 0.3 m KCl. This suggests that the glycine decarboxylation-serine hydroxymethyltransferase complex may be closely bound to the internal surface of the mitochondrial inner membrane. The relationship of this integrated enzyme complex to CO2 evolution and serine synthesis during photorespiration and the physiological role of the dicarboxylate shuttle were discussed.
Serine hydroxymethyltransferase
Photorespiration
Glycine cleavage system
Decarboxylation
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Serine hydroxymethyltransferase
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