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

Abstract

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...

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

“…For example, the enzyme has two overlapping binding sites for UTP and CTP (9). This arrangement permits the spontaneous development of resistance to drugs such as cyclopentenylcytosine and release from feedback inhibition by CTP (45-48) through clustered CTPS gene mutations altering residues in the CTP-binding site while retaining the ability of CTPS to bind UTP and catalyze CTP formation.…”

“…The detailed mechanism by which GTP induces the conformational changes to activate the glutaminase activity and inhibit NH 3 -dependent CTP formation remains unknown despite the recent solution of the structures of apo-E. coli CTPS (no nucleotides bound) (3), E. coli CTPS with bound CTP and ADP (9), and Thermus thermophilus CTPS with bound glutamine (23). Although Baldwin and co-workers (3) crystallized E. coli CTPS in the presence of GTP, no GTP was bound in the crystalline enzyme.…”

The enigmatic cytoophidium: Compartmentation of CTP synthase via filament formation

“…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.…”

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

“…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].…”