Limited proteolysis of Escherichia coli cytidine 5′‐triphosphate synthase. Identification of residues required for CTP formation and GTP‐dependent activation of glutamine hydrolysis

Abstract

Cytidine 5¢-triphosphate synthase catalyses the ATPdependent formation of CTP from UTP using either ammonia or L-glutamine as the source of nitrogen. When glutamine is the substrate, GTP is required as an allosteric effector to promote catalysis. Limited trypsin-catalysed proteolysis, Edman degradation, and site-directed mutagenesis were used to identify peptide bonds C-terminal to three basic residues (Lys187, Arg429, and Lys432) of Escherichia coli CTP synthase that were highly susceptible to proteolysis. Lys187 is located at the CTP/UTP-binding site within the synthase domain, and cleavage at this site destroyed all synthase activity. Nucleotides protected the enzyme against proteolysis at Lys187 (CTP > ATP > UTP > GTP). The K187A mutant was resistant to proteolysis at this site, could not catalyse CTP formation, and exhibited low glutaminase activity that was enhanced slightly by GTP. K187A was able to form tetramers in the presence of UTP and ATP. Arg429 and Lys432 appear to reside in an exposed loop in the glutamine amide transfer (GAT) domain. Trypsin-catalyzed proteolysis occurred at Arg429 and Lys432 with a ratio of 2.6 : 1, and nucleotides did not protect these sites from cleavage. The R429A and R429A/ K432A mutants exhibited reduced rates of trypsin-catalyzed proteolysis in the GAT domain and wild-type ability to catalyse NH 3 -dependent CTP formation. For these mutants, the values of k cat /K m and k cat for glutamine-dependent CTP formation were reduced % 20-fold and % 10-fold, respectively, relative to wild-type enzyme; however, the value of K m for glutamine was not significantly altered. Activation of the glutaminase activity of R429A by GTP was reduced 6-fold at saturating concentrations of GTP and the GTP binding affinity was reduced 10-fold. This suggests that Arg429 plays a role in both GTP-dependent activation and GTP binding.Keywords: activation; amidotransferase; CTP synthase; glutaminase; proteolysis; site-directed mutagenesis.CTP synthase [CTPS; EC 6.3.4.2; UTP:ammonia ligase (ADP-forming)] catalyses the ATP-dependent formation of CTP from UTP using either L-glutamine or NH 3 as the nitrogen source (Scheme 1) [1,2]. This glutamine amidotransferase is a single polypeptide chain containing 545 amino acids and consisting of two domains. The C-terminal glutamine amide transfer (GAT) domain catalyses the hydrolysis of glutamine, and the nascent NH 3 derived from glutamine hydrolysis is transferred to the N-terminal synthase domain where the amination of UTP is catalysed [3,4]. CTPS belongs to the Triad family of glutamine amidotransferases [5,6] which utilizes a Cys-His-Glu triad to catalyse glutamine hydrolysis and also includes anthranilate synthase, carbamoyl phosphate synthase, formylglycinamidine synthase, GMP synthase, imidazole glycerol phosphate synthase, and aminodeoxychorismate synthase.CTPS catalyses the final step in the de novo synthesis of cytosine nucleotides. Because CTP has a central role in the biosynthesis of nucleic acids [7] and membrane phospholipids [8], CTPS is a recog...

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,

“…A P-loop-like structure, composed of conserved residues Lys187, Thr188, and Lys189, forms the iodide/sulfate-binding site and is reasonably placed to interact with the UTP γ-phosphate (Figure 8c). Consistent with this idea, Lys187 is protected from proteolysis in the presence of UTP and the Lys187-Ala substituted enzyme does not synthesize CTP (83). The importance of the γ-phosphate-binding site is supported by the lack of reactivity of UDP and γ-phosphoryl methyl diester UTP (14,19).…”

“…Insertion 2 (residues 418-437, Figures 3b and 4) contains the only undefined region of the EcCTPS structure, residues 428-437, and is followed by conserved residue block 436-442. CTPS is proteolyzed by trypsin at Arg429 and Lys432, indicating that this region is solvent-accessible and unconstrained (83).…”

“…Bound magnesium would activate the ATP γ-phosphate for transfer to UTP O4, while the Gly143 amide and Lys18 and Lys40 side chains would stabilize the anionic transition state. A key role for Lys18 is supported by loss of CTP synthesis activity from Ala-substitution (83).…”

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

“…Within the nucleoside-binding reference subunit, the α-phosphate is recognized directly by the Ser14 side chain and via water by the Thr144 side chain. The critical role of the Lys187 side chain-γ-phosphate interaction in UTP bindng is underscored by the inability of Lys187Ala EcCTPS to perform Gln-or NH 3 -dependent CTP synthesis (51).…”

Cloning, expression, and characterization of a novel CTP synthase gene from Anoxybacillus gonensis G2

“…In all compared species, and many other species not mentioned here, the localization of pyrG is different and manifests differences among the compared bacteria. In the glutaminase domain of CTP synthase proteins, the GxxxRLG sequence is highly conserved between many prokaryotic and eukaryotic species (Simard et al, 2003) (Endrizzi et al, 2004). Lys 187 residue in the synthase domain is extremely conserved among CTP synthases from different organisms.…”