Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells

Whelan, Jeremy; Phear, Geraldine; Yamauchi, Masatake; Meuth, Mark

doi:10.1038/ng0493-317

Cited by 44 publications

(63 citation statements)

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“…The black oval indicates the location of the 2-fold crystallographic symmetry axis, which relates subunits of the tightly bound dimer. The previously characterized CTP resistance substitutions are D147E [Chlamydia trachomatis (25)], V*116F, G146E, I148T, M*151I, R158H, and H*229K [hamster (35)], and E155K [hamster (35) and yeast (18)] (an asterisk denotes residues different from those of EcCTPS). Note the potential for substitutions at residues 148 and 151 to disrupt binding at both sites, and the potential for binding at one CTP site to influence binding at the two-fold-related site.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex^,

et al. 2005

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Cytidine triphosphate synthetases (CTPSs) synthesize CTP and regulate its intracellular concentration through direct interactions with the four ribonucleotide triphosphates. In particular, CTP product is a feedback inhibitor that competes with UTP substrate. Selected CTPS mutations that impart resistance to pyrimidine antimetabolite inhibitors also relieve CTP inhibition and cause a dramatic increase in intracellular CTP concentration, indicating that the drugs act by binding to the CTP inhibitory site. Resistance mutations map to a pocket that, although adjacent, does not coincide with the expected UTP binding site in apo Escherichia coli CTPS [EcCTPS; Endrizzi, J. A., et al. (2004) Biochemistry 43, 6447-6463], suggesting allosteric rather than competitive inhibition. Here, bound CTP and ADP were visualized in catalytically active EcCTPS crystals soaked in either ATP and UTP substrates or ADP and CTP products. The CTP cytosine ring resides in the pocket predicted by the resistance mutations, while the triphosphate moiety overlaps the putative UTP triphosphate binding site, explaining how CTP competes with UTP while CTP resistance mutations are acquired without loss of catalytic efficiency. Extensive complementarity and interaction networks at the interfacial binding sites provide the high specificity for pyrimidine triphosphates and mediate nucleotide-dependent tetramer formation. Overall, these results depict a novel product inhibition strategy in which shared substrate and product moieties bind to a single subsite while specificity is conferred by separate subsites. This arrangement allows for independent adaptation of UTP and CTP binding affinities while efficiently utilizing the enzyme surface.Cytidine triphosphate (CTP) is synthesized de novo from uridine triphosphate (UTP), adenosine triphosphate (ATP), and glutamine by cytidine triphosphate synthetases (CTPSs, 1 EC 6.4. 1 Abbreviations: CTPS, cytidine triphosphate synthetase; CPEC, cyclopentenylcytosine; 3deazaU, 3-deazauridine; EcCTPS, Escherichia coli cytidine triphosphate synthetase; ALase, ammonia ligase reaction, i.e., the formation of a carbon-nitrogen bond by displacement of a phosphorylated oxygen; GATase, glutamine amidotransferase, which refers to a conserved glutamine hydrolysis domain that provides ammonia for a number of diverse enzyme-catalyzed reactions; DTBS, E. coli dethiobiotin synthetase (BioD) enzyme; rmsd, root-meansquare difference in position between atom sets, in angstroms. (Figure 1b) (3,4,(11)(12)(13). Further, the product CTP provides negative feedback by acting as a competitive inhibitor of substrate UTP (3,8,9,14), with a comparable K i for CTP (110 μM) and K m for UTP (150 μM) (3) (Figure 1a). This particular interaction determines the upper limit for intracellular CTP, and Saccharomyces cerevisiae carrying CTPS mutants defective in product inhibition exhibit 16-20-fold increased levels (18). NIH Public AccessAntimetabolite pyrimidine analogues cyclopentenylcytosine (CPEC) and 3-deazauridine (3-deazaU) are effectiv...

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Section: Discussionmentioning

confidence: 99%

Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex^,

et al. 2005

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“…Similar CTPS mutations also mediate cancer resistance to cytosine arabinoside and 5-fluorouracil, drugs that do not target this enzyme, by dramatically increasing 1 Abbreviations: CTPS, cytidine triphosphate synthetase; EcCTPS, Escherichia coli cytidine triphosphate synthetase; CPEC, cyclopentenyl cytosine; MAD, multiwavelength anomalous dispersion; TCEP, tricarboxyethyl-phosphine; MPD, 2,4-2-methylpentanediol; DTBS, E. coli dethiobiotin synthetase (BioD) enzyme; CPS, E. coli carbamoyl phosphate synthetase enzyme, CarA subunit; 10 GSA, glutamyl γ-semialdehyde; DAPA, diaminoperlargonic acid; rmsd, root-mean-squared difference in position between atom sets, in angstrom units; ALase, amidoligase reaction, formation of a carbon-nitrogen bond by displacement of a phosphorylated oxygen; GATase, glutamine amidotransferase, refers to the conserved glutamine hydrolysis domain that provides ammonia for a number of diverse reactions. (57,58). These observations suggest that CTPSs are master regulators of CTP levels and that resistance-evading anti-CTPS drugs could be effective therapeutics.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 95%

Crystal Structure of Escherichia coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets^,

et al. 2004

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Cytidine triphosphate synthetases (CTPSs) produce CTP from UTP and glutamine, and regulate intracellular CTP levels through interactions with the four ribonucleotide triphosphates. We solved the 2.3-Å resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifunctional monomer contains a dethiobiotin synthetase-like amidoligase N-terminal domain and a Type 1 glutamine amidotransferase C-terminal domain. For each amidoligase active site, essential ATP-and UTPbinding surfaces are contributed by three monomers, suggesting that activity requires tetramer formation, and that a nucleotide-dependent dimer-tetramer equilibrium contributes to the observed positive cooperativity. A gated channel that spans 25 Å between the glutamine hydrolysis and amidoligase active sites provides a path for ammonia diffusion. The channel is accessible to solvent at the base of a cleft adjoining the glutamine hydrolysis active site, providing an entry point for exogenous ammonia. Guanine nucleotide binding sites of structurally related GTPases superimpose on this cleft, providing insights into allosteric regulation by GTP. Mutations that confer nucleoside drug resistance and release CTP inhibition map to a pocket that neighbors the UTP-binding site and can accommodate a pyrimidine ring. Its location suggests that competitive feedback inhibition is affected via a distinct product/drug binding site that overlaps the substrate triphosphate binding site. Overall, the E. coli structure provides a framework for homology modeling of other CTPSs and structure-based design of anti-CTPS therapeutics. † This work was funded by the UC Systemwide Biotechnology Training Program Grant #2002-07, and the National Institutes of Health, General Medical Sciences, #GM63109. ‡ Protein Data Bank accession number 1S1M. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 June 24. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptCytidine triphosphate synthetases (CTPSs, 1 E. C. 6.4.3.2, uridine triphosphate ammonia lyases, 525-630 amino acid residues) are essential ubiquitous enzymes that catalyze the ratelimiting step of de novo CTP biosynthesis (1,2). Genes pyrG (bacteria), ctrA (some gram positive bacteria), URA7 and URA8 (Saccharomyces cerevisiae), and CTPS1 and CTPS2 (human) encode CTP synthetase enzymes, with most eukaryotes expressing two isoforms.CTPSs not only provide a key precursor for synthesis of DNA, RNA, and phospholipid (3,4), they also control the intracellular CTP concentrations that limit these processes (5-9).CTP is derived from UTP in three reaction steps catalyzed by CTPSs (Figure 1a). In one active site, the UTP O4 oxygen is activated by Mg-ATP-dependent phosphorylation, followed by displacement of the resulting 4-phosphate moiety by ammonia (10,11). In a separate site, ammonia is generated via rate-limiting glutamine hydrolysis (glutaminase) activity (12). CTPS activity was first re...

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“…Sequence analysis of the CTP synthetase gene from these mutants has revealed that most of the mutations are clustered within a stretch of 14 amino acids [41]. The most frequent mutations in the clustered region of the enzyme are glutamate-to-lysine and histidine-to-lysine changes [41]. The clustered sites are in a highly conserved region of the CTP synthetases from human cells [42], E. coli [31], Chlamydia trachomatis [43], Bacillus subtilis [44], and S. cerevisiae [4,5].…”

Section: Ctp Regulates Ctp Synthetase Activity and The Synthesis Of Mmentioning

confidence: 99%

“…The mutants were selected for their resistance to the growth inhibitory effects of the chemotherapeutic drugs arabinosyl cytosine and 5-fluorouracil [41]. Sequence analysis of the CTP synthetase gene from these mutants has revealed that most of the mutations are clustered within a stretch of 14 amino acids [41].…”

Section: Ctp Regulates Ctp Synthetase Activity and The Synthesis Of Mmentioning

confidence: 99%

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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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Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells

Cited by 44 publications

References 31 publications

Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex^,

Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex^,

Crystal Structure of Escherichia coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets^,

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

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Clustered base substitutions in CTP synthetase conferring drug resistance in Chinese hamster ovary cells

Cited by 44 publications

References 31 publications

Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex,

Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex,

Crystal Structure of Escherichia coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets,

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

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Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex^,

Mechanisms of Product Feedback Regulation and Drug Resistance in Cytidine Triphosphate Synthetases from the Structure of a CTP-Inhibited Complex^,

Crystal Structure of Escherichia coli Cytidine Triphosphate Synthetase, a Nucleotide-Regulated Glutamine Amidotransferase/ATP-Dependent Amidoligase Fusion Protein and Homologue of Anticancer and Antiparasitic Drug Targets^,