Structure of the synthetase domain of human CTP synthetase, a target for anticancer therapy

Kursula, Petri; Flodin, S.; Ehn, M.; Hammarstrom, M.; Schüler, H.; Nordlund, P.; Stenmark, Pål

doi:10.1107/s1744309106018136

Cited by 36 publications

(34 citation statements)

References 32 publications

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“…At concentrations less than 0.15 mM, GTP activates Gln-dependent CTP formation by CTPS; however, at concentrations greater than 0.15 mM, Gln-dependent CTP formation is inhibited. In the absence of crystal structures of CTPS with bound GTP, it is difficult to identify potential interactions between CTPS and GTP that might contribute to this behavior (3,8,9,23). Therefore, we undertook a structure-activity study using GTP/ guanosine analogues ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax

Lunn

MacDonnell

Bearne

2008

Journal of Biological Chemistry

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Cytidine 5-triphosphate synthase catalyzes the ATPdependent formation of CTP from UTP using either NH 3 or L-glutamine (Gln) as the source of nitrogen. GTP acts as an allosteric effector promoting Gln hydrolysis but inhibiting Glndependent CTP formation at concentrations of >0.15 mM and NH 3 -dependent CTP formation at all concentrations. A structure-activity study using a variety of GTP and guanosine analogues revealed that only a few GTP analogues were capable of activating Gln-dependent CTP formation to varying degrees: GTP ≈ 6-thio-GTP > ITP ≈ guanosine 5-tetraphosphate > O 6 -methyl-GTP > 2-deoxy-GTP. No activation was observed with guanosine, GMP, GDP, 2,3-dideoxy-GTP, acycloguanosine, and acycloguanosine monophosphate, indicating that the 5-triphosphate, 2-OH, and 3-OH are required for full activation. The 2-NH 2 group plays an important role in binding recognition, whereas substituents at the 6-position play an important role in activation. The presence of a 6-NH 2 group obviates activation, consistent with the inability of ATP to substitute for GTP. Nucleotide and nucleoside analogues of GTP and guanosine, respectively, all inhibited NH 3 -and Gln-dependent CTP formation (often in a cooperative manner) to a similar extent (IC 50 ≈ 0.2-0.5 mM). This inhibition appeared to be due solely to the purine base and was relatively insensitive to the identity of the purine with the exception of inosine, ITP, and adenosine (IC 50 ≈ 4 -12 mM). 8-Oxoguanosine was the best inhibitor identified (IC 50 ‫؍‬ 80 M). Our findings suggest that modifying 2-aminopurine or 2-aminopurine riboside may serve as an effective strategy for developing cytidine 5-triphosphate synthase inhibitors.CTP synthase (CTPS 2 ; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)) catalyzes the ATP-dependent formation of CTP from UTP using either L-glutamine (Gln) or NH 3 as the nitrogen source (Scheme 1) (1, 2). This glutamine amidotransferase is a single polypeptide chain consisting of two domains. The C-terminal Gln amide transfer domain catalyzes the hydrolysis of Gln. The nascent NH 3 derived from this glutaminase activity is transferred via an NH 3 tunnel (3) to the N-terminal synthase domain, where it reacts with UTP that has been activated by ATP-dependent phosphorylation at the 4-position (4 -7). CTPS from Escherichia coli is the most thoroughly characterized CTPS with respect to its physical, kinetic, and structural properties. Indeed, E. coli CTPS serves as a good model for understanding catalysis by other CTPSs, including human CTPS (8), since CTPSs exhibit high conservation of functionally and structurally important residues and have few insertion/ deletion differences (3, 9).The de novo biosynthesis of pyrimidine nucleotides is highly regulated in E. coli, and consequently, CTPS is regulated in a complex fashion (1). GTP is required as a positive allosteric effector to increase the efficiency (k cat /K m ) of Gln-dependent CTP synthesis (10) by stabilizing the enzyme conformation that binds the tetrahedral intermediates formed during Gln ...

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Section: Resultsmentioning

confidence: 99%

Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax

Lunn

MacDonnell

Bearne

2008

Journal of Biological Chemistry

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“…For both yeast and E. coli, CTPS exists as a dimer in the absence of ATP and UTP, but in the presence of saturating concentrations of ATP and UTP, CTPS exists as a tetramer (8,25,26). The crystal structure of the hCTPS1 synthase domain shows that it is also a dimer of dimers with a synthase domain very similar to that of E. coli (27). However, the influence of ATP and UTP on tetramerization has not been determined for either human isoform.…”

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confidence: 99%

Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation*

Kassel

Higgins

et al. 2010

Journal of Biological Chemistry

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Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K m and IC 50 values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. 568 is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V max of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser 568 as a major site of CTPS2 regulation by phosphorylation.Cytidine nucleotides are precursors for the synthesis of nucleic acids and phospholipids (1-4) as well as the post-translational modification of proteins by sialylation (1-3) and the regulation of enzymes such as CTP-phosphocholine cytidylyltransferase (2), carbamoyl phosphate synthetase, and phosphatidylserine synthase (5). CTP is the nucleotide with the lowest cellular concentration (1, 6), making it rate-limiting for RNA synthesis and other CTP-dependent events. Hence, understanding the regulation of CTP synthesis is important for many growth-related processes.CTP synthetase (CTPS, EC 6.3.4.2) 4 is the rate-limiting enzyme for the synthesis of cytosine nucleotides from both the de novo and uridine salvage pathways (7). CTPS consists of two enzymatic domains. In the synthase domain, UTP is phosphorylated by the ␥-phosphoryl group of ATP to form 4-phospho-UTP via an ATPase reaction (8 -11). In the glutaminase domain, the substrate glutamine is hydrolyzed to glutamate and NH 3 (8), and the resultant NH 3 is then transported to the synthase domain active site through an NH 3 tunnel (11, 12). NH 3 is then transferred to the phosphorylated UTP to form CTP (8). GTP activates the reaction by accelerating the formation of a covalent glutamyl-enzyme catalytic intermediate (8). CTPS has three functionally distinct sites: the glutaminase site for glutamine hydrolysis, the active site where CTP is formed, and the allosteric site where GTP binds (13). CTPS is also regulated by feedback inhibition, allowing the product CTP to inhibit the activity of the enzyme (14 -17).There are two isoforms of CTPS in humans, CTPS1 and CTPS2, and they share 74% amino acid identity (...

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“…Moreover, structures for the bacterial [28,29] and human [30] enzymes have been solved. The yeast CTP synthetases have high homology to bacterial and mammalian CTP synthetase enzymes [28][29][30][31][32]. All CTP synthetases identified from bacteria, parasites, yeast and mammals contain conserved CTP synthetase and glutamine amide transfer domains that are involved in catalysis (Fig.…”

Section: Ctp Synthetases Of S Cerevisiaementioning

confidence: 99%

“…All CTP synthetases identified from bacteria, parasites, yeast and mammals contain conserved CTP synthetase and glutamine amide transfer domains that are involved in catalysis (Fig. 3) [28][29][30][33][34][35][36][37][38][39]. The analysis of the Escherichia coli structure has shown that CTP synthetase has a novel product inhibition mechanism in which shared substrate and product moieties bind to a single subsite while specificity is conferred by separate sites [34].…”

Section: Ctp Synthetases Of S Cerevisiaementioning

confidence: 99%

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

Chang

Carman

2008

Progress in Lipid Research

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CTP synthetase is a cytosolic-associated glutamine amidotransferase enzyme that catalyzes the ATPdependent transfer of the amide nitrogen from glutamine to the C-4 position of UTP to form CTP. In the yeast Saccharomyces cerevisiae, the reaction product CTP is an essential precursor of all membrane phospholipids that are synthesized via the Kennedy (CDP-choline and CDP-ethanolamine branches) and CDP-diacylglycerol pathways. The URA7 and URA8 genes encode CTP synthetase in S. cerevisiae, and the URA7 gene is responsible for the majority of CTP synthesized in vivo. The CTP synthetase enzymes are allosterically regulated by CTP product inhibition. Mutations that alleviate this regulation result in an elevated cellular level of CTP and an increase in phospholipid synthesis via the Kennedy pathway. The URA7-encoded enzyme is phosphorylated by protein kinases A and C, and these phosphorylations stimulate CTP synthetase activity and increase cellular CTP levels and the utilization of the Kennedy pathway. The CTPS1 and CTPS2 genes that encode human CTP synthetase enzymes are functionally expressed in S. cerevisiae, and rescue the lethal phenotype of the ura7Δ ura8Δ double mutant that lacks CTP synthetase activity. The expression in yeast has revealed that the human CTPS1-encoded enzyme is also phosphorylated and regulated by protein kinases A and C.

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Structure of the synthetase domain of human CTP synthetase, a target for anticancer therapy

Cited by 36 publications

References 32 publications

Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax

Structural Requirements for the Activation of Escherichia coli CTP Synthase by the Allosteric Effector GTP Are Stringent, but Requirements for Inhibition Are Lax

Regulation of Human Cytidine Triphosphate Synthetase 2 by Phosphorylation*

CTP synthetase and its role in phospholipid synthesis in the yeast Saccharomyces cerevisiae

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