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^,

Abstract

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...

The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

“…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].…”

“…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].…”

“…The CTP product provides a negative feedback by competitively inhibiting the UTP substrate [10,15,[20][21][22][23]. Despite bacterial and mammalian lineages having diverged approximately three billion years ago [24], their CTP synthase molecules Filament-formation appears to be a conserved, and thus an essential, property of CTP synthases.…”

“…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.…”

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

“…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).…”