Inhibition of 5,10-methenyltetrahydrofolate synthetase

doi:10.1016/j.abb.2006.12.023

Archives of Biochemistry and Biophysics

Volume 458, Issue 2, 15 February 2007, Pages 194-201

https://doi.org/10.1016/j.abb.2006.12.023 Get rights and content

Abstract

The interaction of 5-formyltetrahydrofolate analogs with murine methenyltetrahydrofolate synthetase (MTHFS) was investigated using steady-state kinetics, molecular modeling, and site-directed mutagenesis. MTHFS catalyzes the irreversible cyclization of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. Folate analogs that cannot undergo the rate-limiting step in catalysis were inhibitors of murine MTHFS. 5-Formyltetrahydrohomofolate was an effective inhibitor of murine MTHFS (K_i = 0.7 μM), whereas 5-formyl,10-methyltetrahydrofolate was a weak inhibitor (K_i = 10 μM). The former, but not the latter, was slowly phosphorylated by MTHFS. 5-Formyltetrahydrohomofolate was not a substrate for murine MTHFS, but was metabolized when the MTHFS active site Y151 was mutated to Ala. MTHFS active site residues do not directly facilitate N10 attack on the on the N5-iminium phosphate intermediate, but rather restrict N10 motion around N5. Inhibitors specifically designed to block N10 attack appear to be less effective than the natural 10-formyltetrahydrofolate polyglutamate inhibitors.

Section snippets

Materials

MES, HEPES, and Tris were purchased from Sigma. ATP was purchased from Roche. [6S]-5-formylTHF (the natural isomer) and [6R,S]-5-formylTHF were a generous gift from Eprova AG. 5-Formyltetrahydrofolate triglutamate was from Schircks Laboratories (Switzerland). Homofolate (NSC 79249) was the generous gift of Dr. Roy Kisliuk, Tufts University. All other materials were of high quality and obtained from various commercial vendors.

Synthesis of folate derivatives

5-Formyl,10-methylTHF was synthesized by incubating 5-formylTHF,

Mechanism-based inhibition of MTHFS

The mechanism of MTHFS inhibition by 5-formylTHF analogs (Fig. 2) was investigated using recombinant murine MTHFS protein. The ability of folate analogs that cannot undergo the rate-limiting nucleophilic attack by N10 on the N5-iminium phosphate intermediate to inhibit MTHFS was investigated. Nucleophilic attack by N10 can be impaired by N10 substitution (i.e. methylation of N10) and/or by increasing the distance between the N5-iminium phosphate and N10 (i.e. 5-formylTHHF) (Fig. 2). As

Acknowledgments

This work was supported by PHS HD35687 to P.J.S. The authors thank Bhumit Patel for protein purification.

References (21)

P. Stover et al.
Trends Biochem. Sci.
(1993)
T. Huang et al.
J. Biol. Chem.
(1995)
S. Girgis et al.
J. Biol. Chem.
(1997)
M.S. Field et al.
J. Biol. Chem.
(2006)
R. Bertrand et al.
J. Biol. Chem.
(1989)
R.G. Moran et al.
Methods Enzymol.
(1986)
P. Stover et al.
Anal. Biochem.
(1992)
M.C. Anguera et al.
Protein Expr. Purif.
(2004)
R.A. Sayle et al.
Trends Biochem. Sci.
(1995)
E. Merritt et al.
Methods Enzymol.
(1997)

There are more references available in the full text version of this article.

Cited by (18)

A new mathematical model of folate homeostasis in E. coli highlights the potential importance of the folinic acid futile cycle in cell growth
2024, BioSystems
Folate (vitamin B9) plays a central role in one-carbon metabolism in prokaryotes and eukaryotes. This pathway mediates the transfer of one-carbon units, playing a crucial role in nucleotide synthesis, methylation, and amino acid homeostasis. The folinic acid futile cycle adds a layer of intrigue to this pathway, due to its associations with metabolism, cell growth, and dormancy. It also introduces additional complexity to folate metabolism. A logical way to deal with such complexity is to examine it by using mathematical modelling. This work describes the construction and analysis of a model of folate metabolism, which includes the folinic acid futile cycle. This model was tested under three in silico growth conditions. Model simulations revealed: 1) the folate cycle behaved as a stable biochemical system in three growth states (slow, standard, and rapid); 2) the initial concentration of serine had the greatest impact on metabolite concentrations; 3) 5-formyltetrahydrofolate cyclo-ligase (5-FCL) activity had a significant impact on the levels of the 7 products that carry the one-carbon donated from folates, and the redox couple NADP/NADPH; this was particularly evident in the rapid growth state; 4) 5-FCL may be vital to the survival of the cells by maintaining low levels of homocysteine, as high levels can induce toxicity; and 5) the antifolate therapeutic trimethoprim had a greater impact on folate metabolism with higher nutrient availability. These results highlight the important role of 5-FCL in intracellular folate homeostasis and mass generation under different metabolic scenarios.
5-Formyltetrahydrofolate promotes conformational remodeling in a methylenetetrahydrofolate reductase active site and inhibits its activity
2023, Journal of Biological Chemistry
The flavoprotein methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of N5, N10-methylenetetrahydrofolate (CH₂-H₄folate) to N5-methyltetrahydrofolate (CH₃-H₄folate), committing a methyl group from the folate cycle to the methionine one. This committed step is the sum of multiple ping-pong electron transfers involving multiple substrates, intermediates, and products all sharing the same active site. Insight into folate substrate binding is needed to better understand this multifunctional active site. Here, we performed activity assays with Thermus thermophilus MTHFR (tMTHFR), which showed pH-dependent inhibition by the substrate analog, N5-formyltetrahydrofolate (CHO-H₄folate). Our crystal structure of a tMTHFR•CHO-H₄folate complex revealed a unique folate-binding mode; tMTHFR subtly rearranges its active site to form a distinct folate-binding environment. Formation of a novel binding pocket for the CHO-H₄folate p-aminobenzoic acid moiety directly affects how bent the folate ligand is and its accommodation in the active site. Comparative analysis of the available active (FAD- and folate-bound) MTHFR complex structures reveals that CHO-H₄folate is accommodated in the active site in a conformation that would not support hydride transfer, but rather in a conformation that potentially reports on a different step in the reaction mechanism after this committed step, such as CH₂-H₄folate ring-opening. This active site remodeling provides insights into the functional relevance of the differential folate-binding modes and their potential roles in the catalytic cycle. The conformational flexibility displayed by tMTHFR demonstrates how a shared active site can use a few amino acid residues in lieu of extra domains to accommodate chemically distinct moieties and functionalities.
Moonlighting glutamate formiminotransferases can functionally replace 5-Formyltetrahydrofolate cycloligase
2010, Journal of Biological Chemistry
Citation Excerpt :
The only known removal mechanism for 5-CHO-THF is a salvage reaction in which it is reconverted to 5,10-CH=THF by the enzyme 5-CHO-THF cycloligase (5-FCL, EC 6.3.3.2) (6–9). This reaction requires MgATP and is essentially irreversible (Fig. 1B) (10). The 5-FCL reaction is medically significant because, by recycling 5-CHO-THF back to the active C1-folate pool, it allows use of 5-CHO-THF as an antifolate rescue agent in cancer chemotherapy (6).
5-Formyltetrahydrofolate (5-CHO-THF) is formed by a side reaction of serine hydroxymethyltransferase. Unlike other folates, it is not a one-carbon donor but a potent inhibitor of folate enzymes and must therefore be metabolized. Only 5-CHO-THF cycloligase (5-FCL) is generally considered to do this. However, comparative genomic analysis indicated (i) that certain prokaryotes lack 5-FCL, implying that they have an alternative 5-CHO-THF-metabolizing enzyme, and (ii) that the histidine breakdown enzyme glutamate formiminotransferase (FT) might moonlight in this role. A functional complementation assay for 5-CHO-THF metabolism was developed in Escherichia coli, based on deleting the gene encoding 5-FCL (ygfA). The deletion mutant accumulated 5-CHO-THF and, with glycine as sole nitrogen source, showed a growth defect; both phenotypes were complemented by bacterial or archaeal genes encoding FT. Furthermore, utilization of supplied 5-CHO-THF by Streptococcus pyogenes was shown to require expression of the native FT. Recombinant bacterial and archaeal FTs catalyzed formyl transfer from 5-CHO-THF to glutamate, with k_cat values of 0.1–1.2 min⁻¹ and K_m values for 5-CHO-THF and glutamate of 0.4–5 μm and 0.03–1 mm, respectively. Although the formyltransferase activities of these proteins were far lower than their formiminotransferase activities, the K_m values for both substrates relative to their intracellular levels in prokaryotes are consistent with significant in vivo flux through the formyltransferase reaction. Collectively, these data indicate that FTs functionally replace 5-FCL in certain prokaryotes.
Chapter 1 Folate-Mediated One-Carbon Metabolism
2008, Vitamins and Hormones
Citation Excerpt :
MTHFS enzymatic activity is regulated primarily by folate coenzymes. 5‐MethylTHF and 10‐formylTHF, the latter of which is in chemical equilibrium with the product of the MTHFS reaction, act as tight‐binding inhibitors of MTHFS (Field et al., 2006). To date, two metabolic roles have been ascribed to MTHFS.
Tetrahydrofolate (THF) polyglutamates are a family of cofactors that carry and chemically activate one‐carbon units for biosynthesis. THF‐mediated one‐carbon metabolism is a metabolic network of interdependent biosynthetic pathways that is compartmentalized in the cytoplasm, mitochondria, and nucleus. One‐carbon metabolism in the cytoplasm is required for the synthesis of purines and thymidylate and the remethylation of homocysteine to methionine. One‐carbon metabolism in the mitochondria is required for the synthesis of formylated methionyl‐tRNA; the catabolism of choline, purines, and histidine; and the interconversion of serine and glycine. Mitochondria are also the primary source of one‐carbon units for cytoplasmic metabolism. Increasing evidence indicates that folate‐dependent de novo thymidylate biosynthesis occurs in the nucleus of certain cell types. Disruption of folate‐mediated one‐carbon metabolism is associated with many pathologies and developmental anomalies, yet the biochemical mechanisms and causal metabolic pathways responsible for the initiation and/or progression of folate‐associated pathologies have yet to be established. This chapter focuses on our current understanding of mammalian folate‐mediated one‐carbon metabolism, its cellular compartmentation, and knowledge gaps that limit our understanding of one‐carbon metabolism and its regulation.
Increasing Dosage of Leucovorin Results in Pharmacokinetic and Gene Expression Differences When Administered as Two-Hour Infusion or Bolus Injection to Patients with Colon Cancer
2023, Cancers
A Novel Immunomodulatory Mechanism by Which Vitamin D Influences Folate Receptor 3 Expression to Reduce COVID-19 Severity
2022, Anticancer Research

View all citing articles on Scopus

View full text

Inhibition of 5,10-methenyltetrahydrofolate synthetase

Abstract

Section snippets

Materials

Synthesis of folate derivatives

Mechanism-based inhibition of MTHFS

Acknowledgments

Trends Biochem. Sci.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Methods Enzymol.

Anal. Biochem.

Protein Expr. Purif.

Trends Biochem. Sci.

Methods Enzymol.