Crystal Structures of CTP Synthetase Reveal ATP, UTP, and Glutamine Binding Sites

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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“…This idea finds support in the structural similarity of EcCTPS and CTPS from Thermus thermophilus (37).…”

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confidence: 67%

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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“…Both adenosine-5 0 -triphosphate (ATP) and uridine-5 0 -triphosphate (UTP), as substrates, bind CTP synthase with positive co-operativity [10][11][12][13][14][15][16].…”

Section: How Is the Cytoophidium Assembled?mentioning

confidence: 99%

“…Guanosine-5 0 -triphosphate (GTP) binding allosterically activates glutamine hydrolysis and generates ammonia [15][16][17][18][19]. The CTP product provides a negative feedback by competitively inhibiting the UTP substrate [10,15,[20][21][22][23].…”

Section: How Is the Cytoophidium Assembled?mentioning

confidence: 99%

“…Biochemical studies have shown that CTP synthase can form inactive dimers and active tetramers [13]. Polymerisation of CTP synthase could result from conformational changes induced by binding to various ligands [15,16,23]. Polymerisation of CTP synthase into filamentary structures might stabilise the enzyme in a particular state, whether active, inactive or a mixture of both.…”

Section: How Is the Cytoophidium Assembled?mentioning

confidence: 99%

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The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

Liu

2011

BioEssays

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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%

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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Crystal Structures of CTP Synthetase Reveal ATP, UTP, and Glutamine Binding Sites

Cited by 65 publications

References 53 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^,

The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

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

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Crystal Structures of CTP Synthetase Reveal ATP, UTP, and Glutamine Binding Sites

Cited by 65 publications

References 53 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,

The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

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

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Product

Resources

About

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^,