FIGURE 6 - uploaded by James Anthony Endrizzi
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Stereo diagrams of the glutaminase active site and the putative ammonia diffusion channel in the A subunit GATase domain. (a) Active site comparison of EcCTPS (cyan backbone coils and colored ball-and-sticks) to the CPS-GSA 1CS0 transition state analogue complex (beige backbone coils and sticks). The CPS was superimposed on EcCTPS using Cys379, Leu380, Gly351 and Gly352 main chain atoms. Covalently bound GSA from 1CS0 is indicated by the pink carbon atoms ( " Gln " ). Conserved residues are indicated by ball-andsticks , with black carbon atoms for those suggested to be directly involved with substrate binding. Hydrogen bonds between CPS and GSA are indicated by purple dotted lines, and those between EcCTPS and bound solvent molecules (red balls) are indicated by yellow dotted lines. Two solvent molecules, present in both subunits, occupy the expected positions of the amide nitrogen and carbonyl oxygen of the Gln acylation transition state. The structures differ significantly in the conformation of the L11 " lid " , and the positioning of catalytic His and Glu residues. (b) A solvent-containing vestibule and adjacent tubular channel connects the glutaminase and ALase active sites. Backbone positions from the A subunit (yellow) or A′ subunit (green) are indicated by ribbons or coils. The dot molecular surface was generated by GRASP using a 1.2-Å probe (77). The hypothetical positions of bound GTP, UTP, and glutamine are indicated by translucent sticks. Seven buried solvent molecules that are present in both subunits define a possible ammonia trajectory, and are shown as cyan balls, with their associated hydrogen bonds indicated by yellow dotted lines. Conserved residues that are suggested to be catalytically important (black), that line the proposed UTP (purple) or GTP (red-purple) binding sites, or that line the proposed ammonia channel (gray bonds) are indicated by sticks. Potential hydrogen-bond acceptors are provided by the carbonyl oxygens of Met52, His57, Gly58, Glu59, Val 60, Asp70, Ala304, Arg468, and the carboxylate oxygens of Glu59, Asp70, and Glu517 (red balls). Hydrogen-bond donors are furnished by the amides of Val60, Leu71, and Glu351, and the Arg468 NH1 atom (blue balls), with Tyr298 and His57 side chains contributing additional hydrogenbonding potential. Both side chain conformations for His57 are shown. The arrow indicates the suggested location of the ultimate or penultimate position of the conducted ammonia based on hydrogen bonding environment. A 3-Å opening at the base of the proposed GTP-binding site may provide entry for exogenous ammonia.
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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-A resolution crystal structure of Escherichia coli CTPS using Hg-MAD phasing. The structure reveals a nearly symmetric 222 tetramer, in which each bifu...
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... were generated with MOLSCRIPT (75) and rendered with RASTER3D (76). The surface points in Figure 6b were calculated using GRASP (77), with a 1.2-Å probe. ...
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... of the GATase Domain. The CTPS GATase domain sequences contain two conserved residue blocks, residues 374-383 and 513-529 (Figure 4), that are shared with other Type 1 GATase domains and participate in catalysis and substrate binding (Figure 6a). Other blocks, unique to CTPSs, may be involved in GTP activation and coupling GATase and ALase activities (18). ...
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... superimposed the EcCTPS GATase domain on CPS, using residues 191-380 from the 1CS0 structure (Figure 3b), and inferred residue functions from comparisons with liganded CPS structures determined by Holden and co- workers (66) (Figure 6a ...
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... the empty EcCTPS GATase site, important glutamine- binding and catalytic residues are positioned differently from those in the liganded and apo-CPS structures (66) ( Figure 6a). Relative to the nucleophile and oxyanion hole in the CPS-GSA complex, the main chain atoms of residues 513- 517 are shifted 0.8 Å rmsd. ...
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... recognition of the glutamine substrate R-amino acid moiety also differs from that of other GATases. In CPS, imidazole glycerol phosphate synthetase, and GMP synthetase, hydrogen bonds from the Arg470 and Tyr471 main chain and the Gln383 side chain amides are positioned to recognize the glutamine carboxylate (Figure 6a). Residues equivalent to EcCTPS 351-356 further sequester the substrate by forming a "lid" over the glutami- nase site (the "L11 lid"), with main chain carbonyl atoms of residues 352 and 354 providing hydrogen-bond acceptors for the substrate R-amino group. ...
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... equivalent to EcCTPS 351-356 further sequester the substrate by forming a "lid" over the glutami- nase site (the "L11 lid"), with main chain carbonyl atoms of residues 352 and 354 providing hydrogen-bond acceptors for the substrate R-amino group. In EcCTPS, residues 353- 357 are shifted away, primarily via backbone rotations at Phe353 and Val358, opening one end of the active site and preventing substrate R-amino group recognition by Gly354 (Figure 6a). However, the conserved Glu403 carboxylate occupies the analogous position, perhaps providing an alternative hydrogen bond. ...
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... solvent-filled vestibule (∼230 Å 3 ) connects the GATase active site and the GATase/ALase interface (Figure 6b). This volume is occupied by polypeptide in CPS but a conserved insertion, EcCTPS residues 297-301, diverts the chain and forms part of the vestibule wall (Figure 3b). ...
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... propose the vestibule and the tubular passage provide a path for ammonia diffusion between GATase and ALase active sites (see Discussion). In addition, bulk solvent can access the vestibule through a 3-Å wide opening to the surface (Figure 6b) into the proposed GTP-binding site (Figure 7b, see below). We suggest that this opening provides entry for exogenous ammonia. ...
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... channels are readily apparent in some structures, such as the prototypical 45 Å tunnel in CPS (95), while in others, their formation requires conformational changes induced by substrate, effector, or protein docking (92,94). In CTPS, the most apparent ammonia diffusion path is defined by two voids that span the 25 Å distance between GATase and ALase active sites (Figure 6b). The channel is distinct from that in CPS, although they share a common entrance from the glutaminase site. ...
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... enously added ammonia, which can be utilized for CTP synthesis (13,23), may also enter through this opening. This idea is supported by inhibition of ammonia-dependent CTP synthesis when GTP is present in combination with glutamine, the glutamine analogue affinity label DON (18, 96) or the covalent transition-state analogue GSA (23). Together, these ligands would block ammonia entry through both the glutaminase site and the vestibule opening. ...
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CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme in the yeast Saccharomyces cerevisiae. In this work we examined the regulation of CTP synthetase activity by S. cerevisiae protein kinase C (Pkc1p) phosphorylation. The results of labeling experiments with S. cerevisiae mutants expressing different l...
CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activitie...
CTP synthase is encoded by the pyrG gene and catalyzes the conversion of UTP to CTP. A Lactococcus lactis pyrG mutant with a cytidine requirement was constructed, in which β-galactosidase activity in a pyrG-lacLM transcriptional fusion was used to monitor gene expression of pyrG. A 10-fold decrease in the CTP pool induced by cytidine limitation was...
Citations
... Cytidine triphosphate synthase (CTPS) plays a critical role in catalyzing the final and rate-limiting step of de novo cytidine triphosphate (CTP) synthesis. CTPS is composed of a glutamine amidotransferase (GAT) domain and a kinase-like ammonia ligase (AL) domain 1,2 . It utilizes ammonia generated from glutamine and adenine triphosphate (ATP) as an energy source to convert the substrate uridine triphosphate (UTP) into CTP 3 . ...
... Upon the addition of the substrates UTP and ATP, it transitions into an active tetrameric form, which is also observed in eukaryotic CTPS 14 . The 1 School of Life Science and Technology, ShanghaiTech University, Shanghai, China. 2 Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK. 3 Shanghai Clinical Research and Trial Center, Shanghai, China. presence of product CTP prompts CTPS to shift into an inactive tetrameric state for both prokaryotic and eukaryotic enzymes 15 . ...
Cytidine triphosphate synthase (CTPS) plays a pivotal role in the de novo synthesis of cytidine triphosphate (CTP), a fundamental building block for RNA and DNA that is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: adenine triphosphate, uridine triphosphate, CTP, and guanidine triphosphate. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by viral or prokaryotic pathogens. Utilizing cryo‐electron microscopy, we determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, nicotinamide adenine dinucleotide (NADH), and the covalent inhibitor 6‐diazo‐5‐oxo‐ l‐norleucine (DON), achieving a resolution of 2.9 Å. We constructed a phylogenetic tree based on differences in filament‐forming interfaces and designed a variant to validate our hypothesis, providing an evolutionary perspective on CTPS filament formation. Our computational analysis revealed a solvent‐accessible ammonia tunnel upon DON binding. Through comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS that differs from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determined and validated the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of the diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.
... 24 Baprs m mutation from Bacillus amyloliquefaciens was reported to eliminate feedback inhibition. 25 UMP kinases (pyrH) and CTP synthetase (pyrG) suffered from feedback inhibition by UTP and CTP, respectively, 26,27 and the mutants pyrH m and CgpyrG m could desensitize the feedback inhibition. 26,28 pyrE and nudG were the rate-limiting step genes in cytidine biosynthesis. ...
Cytidine 5′-monophosphate (CMP) was widely applied in the food and pharmaceutical industries. Currently, CMP is mainly produced by enzyme catalysis. However, the starting materials for enzyme catalysis were relatively expensive. Therefore, seeking a low-cost production process for CMP was attractive. In this study, Escherichia coli (E. coli) was systematically modified to produce CMP. First, a the cytidine-producing strain was constructed by deleting cdd, rihA, rihB, and rihC. Second, the genes involved in the pyrimidine precursor competing pathway and negative regulation were deleted to increase cyti dine biosynthesis. Third, the deletion of the genes that caused the loss of CMP phosphatase activity led to the accumulation of CMP, and the overexpression of the rate-limiting step genes and feedback inhibition resistance genes greatly increased the yield of CMP. The yield of CMP was further increased to 1013.6 mg/L by blocking CMP phosphorylation. Ultimately, the yield of CMP reached 15.3 g/L in a 50 L bioreactor. Overall, the engineered E. coli with a high yield of CMP was successfully constructed and showed the potential for industrial production.
... CTP synthase (CTPS) plays a critical role in catalyzing the final and ratelimiting step of de novo CTP synthesis. CTPS is composed of a glutamine amidotransferase (GAT) domain and a kinase-like ammonia ligase (AL) domain [1,2]. It converts the substrate UTP into CTP, utilizing ammonia generated from glutamine and ATP as an energy source [3]. ...
CTP synthase (CTPS) plays a pivotal role in the de novo synthesis of CTP, a fundamental building block for RNA and DNA, which is essential for life. CTPS is capable of directly binding to all four nucleotide triphosphates: ATP, UTP, CTP, and GTP. Furthermore, CTPS can form cytoophidia in vivo and metabolic filaments in vitro, undergoing regulation at multiple levels. CTPS is considered a potential therapeutic target for combating invasions or infections by virus or prokaryotic pathogens. Utilizing cryo-electron microscopy, we have determined the structure of Escherichia coli CTPS (ecCTPS) filament in complex with CTP, NADH, and the covalent inhibitor DON, achieving a resolution of 2.8Å. We construct a phylogenetic tree based on differences in filament-forming interfaces and design a variant to validate our hypothesis, providing an evolutionary perspective on the CTPS filament formation. Our computational analysis reveals a solvent-accessible ammonia tunnel upon DON binding. By comparative structural analysis, we discern a distinct mode of CTP binding of ecCTPS, differing from eukaryotic counterparts. Combining biochemical assays and structural analysis, we determine and validate the synergistic inhibitory effects of CTP with NADH or adenine on CTPS. Our results expand our comprehension of diverse regulatory aspects of CTPS and lay a foundation for the design of specific inhibitors targeting prokaryotic CTPS.
... The crystal structures of ecCTPS in its unliganded form and with both CTP and ADP bound (Endrizzi et al., 2004;Endrizzi et al., 2005), in conjunction with the body of in vitro biochemical data, suggested that the CTPand UTP-binding sites partially overlap via a common triphosphate-binding pocket, while the pyrimidine rings of the respective nucleotides are bound at unique sites. Both binding sites were wedged between the A-B-A 0 interface of the tetramer (Figure 1b) (Endrizzi et al., 2005). ...
... In general, all structures were well-modeled to the corresponding electron density; however, insufficient density was available to model the surface loop comprising residues 429-436 in all structures. While the final maps in the inter-domain tunnel region containing H57 and Y44 suggest this region is conformationally dynamic, the modeled conformation presented in the structures here is consistent with the ammonia tunnel predominantly populating the hypothesized "constricted" state (Endrizzi et al., 2004;Endrizzi et al., 2005). The density assigned to the cysteine residues was consistent with their reduced state, as was previously observed in the ecCTPS holo crystal structure (PDB ID: 1S1M) (Endrizzi et al., 2004), but contrasts with the ecCTPS•CTP + ADP complex (PDB ID: 2 AD5) (Endrizzi et al., 2005), which possessed a disulfide bridge between C261 and C268. ...
... While the final maps in the inter-domain tunnel region containing H57 and Y44 suggest this region is conformationally dynamic, the modeled conformation presented in the structures here is consistent with the ammonia tunnel predominantly populating the hypothesized "constricted" state (Endrizzi et al., 2004;Endrizzi et al., 2005). The density assigned to the cysteine residues was consistent with their reduced state, as was previously observed in the ecCTPS holo crystal structure (PDB ID: 1S1M) (Endrizzi et al., 2004), but contrasts with the ecCTPS•CTP + ADP complex (PDB ID: 2 AD5) (Endrizzi et al., 2005), which possessed a disulfide bridge between C261 and C268. When modeling the nucleotideassociated metal (or water), the CheckMyMetal server (Zheng et al., 2014), interaction distances (Table S1), crystal conditions, and anomalous Fourier difference maps were all taken into consideration. ...
CTP synthases (CTPS) catalyze the de novo production of CTP using UTP, ATP, and l‐glutamine with the anticancer drug metabolite gemcitabine‐5′‐triphosphate (dF‐dCTP) being one of its most potent nucleotide inhibitors. To delineate the structural origins of this inhibition, we solved the structures of Escherichia coli CTPS (ecCTPS) in complex with CTP (2.0 Å), 2′‐ribo‐F‐dCTP (2.0 Å), 2′‐arabino‐F‐CTP (2.4 Å), dF‐dCTP (2.3 Å), dF‐dCTP and ADP (2.1 Å), and dF‐dCTP and ATP (2.1 Å). These structures revealed that the increased binding affinities observed for inhibitors bearing the 2′‐F‐arabino group (dF‐dCTP and F‐araCTP), relative to CTP and F‐dCTP, arise from interactions between the inhibitor's fluorine atom exploiting a conserved hydrophobic pocket formed by F227 and an interdigitating loop from an adjacent subunit (Q114‐V115‐I116). Intriguingly, crystal structures of ecCTPS•dF‐dCTP complexes in the presence of select monovalent and divalent cations demonstrated that the in crystallo tetrameric assembly of wild‐type ecCTPS was induced into a conformation similar to inhibitory ecCTPS filaments solely through the binding of Na⁺‐, Mg²⁺‐, or Mn²⁺•dF‐dCTP. However, in the presence of potassium, the dF‐dCTP‐bound structure is demetalated and in the low‐affinity, non‐filamentous conformation, like the conformation seen when bound to CTP and the other nucleotide analogues. Additionally, CTP can also induce the filament‐competent conformation linked to high‐affinity dF‐dCTP binding in the presence of high concentrations of Mg²⁺. This metal‐dependent, compacted CTP pocket conformation therefore furnishes the binding environment responsible for the tight binding of dF‐dCTP and provides insights for further inhibitor design.
... One of the most strongly induced proteins in these samples was the CTP synthase. This enzyme is responsible for the production of CTP from UTP and glutamine, and for the regulation of intracellular levels of CTP (Endrizzi et al., 2004), which is an essential precursor of membrane phospholipids (Chang and Carman, 2008). It also plays a key role in the metabolism of pyrimidines and is very important for the growth rate and the concentration of ribonucleotides and deoxyribonucleotides (Jørgensen et al., 2004). ...
... Cytidine triphosphate synthetase (PyrG) The highenergy compound cytidine triphosphate (CTP) is involved in various metabolic processes and impacts cell growth as well as ATP [293]. PyrG, ATP-dependent CTP synthetase, is responsible for catalyzing the amination of uridine triphosphate (UTP) to form CTP in the last step of the pyrimidine nucleotide biosynthesis pathway [294]. ...
... The structure of apo-PyrG consists of an N-terminal amidoligase (ALase) domain (referred to as the synthetase domain; residues 1 to 278), and a C-terminal glutamine amido-transferase (GATase) domain (residues 299 to 552) ( Fig. 7a) [295]. The two domains are composed of nearly identical Rossmann-like folds, which are connected by an interdomain linker (residues [279][280][281][282][283][284][285][286][287][288][289][290][291][292][293][294][295][296][297][298]. However, the presence of bound molecules (UTP, or UTP/ATP analog AMP-PCP/glutamine analog 5-oxo-L-norleucine) change the oligomeric state of PyrG from monomer to tetramer. ...
... The ATP-and UTP-binding pockets located on the concave surface of PyrG are defined by residues from two and three adjacent subunits, respectively (Fig. 7b-c). The active site of PyrG glutaminase is indicated by the characteristic GATase catalytic triad (Cys393-His524-Glu526) [294,295,297]. In addition, a putative ammonia diffusion channel, which is located between the active site of glutaminase and the amidoligase domain, provides an entrance point for exogenous ammonia. in which the phenyl ring forms π-π stacking with Arg223, and its nitro group forms hydrogen bonds with Ala253 and Asp252 [295]. ...
Mycobacterium tuberculosis ( Mtb ), the causative agent of tuberculosis (TB), is a tenacious pathogen that has latently infected one third of the world’s population. However, conventional TB treatment regimens are no longer sufficient to tackle the growing threat of drug resistance, stimulating the development of innovative anti-tuberculosis agents, with special emphasis on new protein targets. The Mtb genome encodes ~4000 predicted proteins, among which many enzymes participate in various cellular metabolisms. For example, more than 200 proteins are involved in fatty acid biosynthesis, which assists in the construction of the cell envelope, and is closely related to the pathogenesis and resistance of mycobacteria. Here we review several essential enzymes responsible for fatty acid and nucleotide biosynthesis, cellular metabolism of lipids or amino acids, energy utilization, and metal uptake. These include InhA, MmpL3, MmaA4, PcaA, CmaA1, CmaA2, isocitrate lyases (ICLs), pantothenate synthase (PS), Lysine-ε amino transferase (LAT), LeuD, IdeR, KatG, Rv1098c, and PyrG. In addition, we summarize the role of the transcriptional regulator PhoP which may regulate the expression of more than 110 genes, and the essential biosynthesis enzyme glutamine synthetase (GlnA1). All these enzymes are either validated drug targets or promising target candidates, with drugs targeting ICLs and LAT expected to solve the problem of persistent TB infection. To better understand how anti-tuberculosis drugs act on these proteins, their structures and the structure-based drug/inhibitor designs are discussed. Overall, this investigation should provide guidance and support for current and future pharmaceutical development efforts against mycobacterial pathogenesis.
... Cytidine triphosphate synthase/synthetase (CTPsyn) catalyzes the rate-limiting step of the de novo CTP biosynthesis pathway by converting the uridine triphosphate (UTP) to CTP. The conversion proceeds through three reactions: a kinase reaction to phosphorylate uracil O4 atom in an Mg 2+ -ATP-dependent manner, a glutaminase reaction to generate ammonia from glutamine hydrolysis, and a ligase reaction to displace the uracil O4 phosphate with ammonia ( Fig. 1) (Bhagavan and Ha 2011;Endrizzi et al. 2004;Kent and Carman 1999). ...
CTP biosynthesis is carried out by two pathways: salvage and de novo. CTPsyn catalyzes the latter. The study of CTPsyn activity in mammalian cells began in the 1970s, and various fascinating discoveries were made regarding the role of CTPsyn in cancer and development. However, its ability to fit into a cellular serpent-like structure, termed ‘cytoophidia,’ was only discovered a decade ago by three independent groups of scientists. Although the self-assembly of CTPsyn into a filamentous structure is evolutionarily conserved, the enzyme activity upon this self-assembly varies in different species. CTPsyn is required for cellular development and homeostasis. Changes in the expression of CTPsyn cause developmental changes in Drosophila melanogaster. A high level of CTPsyn activity and formation of cytoophidia are often observed in rapidly proliferating cells such as in stem and cancer cells. Meanwhile, the deficiency of CTPsyn causes severe immunodeficiency leading to immunocompromised diseases caused by bacteria, viruses, and parasites, making CTPsyn an attractive therapeutic target. Here, we provide an overview of the role of CTPsyn in cellular and disease perspectives along with its potential as a drug target.
... One of the most strongly induced proteins in these samples was the CTP synthase. This enzyme is responsible for the production of CTP from UTP and glutamine, and for the regulation of intracellular levels of CTP (Endrizzi et al., 2004), which is an essential precursor of membrane phospholipids (Chang and Carman, 2008). It also plays a key role in the metabolism of pyrimidines and is very important for the growth rate and the concentration of ribonucleotides and deoxyribonucleotides (Jørgensen et al., 2004). ...
The old debate of nature (genes) vs. nurture (environmental variables) is once again topical concerning the effect of climate change on environmental microorganisms. Specifically, the Polar Regions are experiencing a drastic increase in temperature caused by the rise in greenhouse gas emissions. This study, in an attempt to mimic the molecular adaptation of polar microorganisms, combines proteomic approaches with a classical microbiological analysis in three bacterial species Shewanella oneidensis , Shewanella frigidimarina , and Psychrobacter frigidicola . Both shewanellas are members of the same genus but they live in different environments. On the other hand, Shewanella frigidimarina and Psychrobacter frigidicola share the same natural environment but belong to a different genus. The comparison of the strategies employed by each bacterial species estimates the contribution of genome vs. environmental variables in the adaptation to temperature. The results show a greater versatility of acclimatization for the genus Shewanella with respect to Psychrobacter . Besides, S. frigidimarina was the best-adapted species to thermal variations in the temperature range 4–30°C and displayed several adaptation mechanisms common with the other two species. Regarding the molecular machinery used by these bacteria to face the consequences of temperature changes, chaperones have a pivoting role. They form complexes with other proteins in the response to the environment, establishing cooperation with transmembrane proteins, elongation factors, and proteins for protection against oxidative damage.
... Consequently, because of the central role of CTP in metabolism, the enzyme is recognized as a potential drug target for viral [7], protozoal [8][9][10][11][12][13][14], and Mycobacterium tuberculosis infections [15][16][17], as well as for cancer [4,[18][19][20][21][22][23][24][25][26][27] and immunosuppression [28,29]. As a member of the class I (or triad) subfamily of glutamine-dependent amidotransferases [30], CTPS utilizes a Cys-Glu-His triad to catalyze the hydrolysis of L-glutamine (Gln) to generate nascent NH 3 in its C-terminal Gln amide transfer (or GAT/glutaminase) domain (Escherichia coli CTPS (EcCTPS) residues 287-544) [31,32]), which is then transferred through an intramolecular tunnel to the N-terminal synthase (or amidoligase) domain (EcCTPS residues 1-266 connected to the GAT domain via an interdomain linker, residues 267-286) [32]. The lack of equilibration of the nascent NH 3 with the solvent supported the notion of such a tunnel [33]. ...
... Consequently, because of the central role of CTP in metabolism, the enzyme is recognized as a potential drug target for viral [7], protozoal [8][9][10][11][12][13][14], and Mycobacterium tuberculosis infections [15][16][17], as well as for cancer [4,[18][19][20][21][22][23][24][25][26][27] and immunosuppression [28,29]. As a member of the class I (or triad) subfamily of glutamine-dependent amidotransferases [30], CTPS utilizes a Cys-Glu-His triad to catalyze the hydrolysis of L-glutamine (Gln) to generate nascent NH 3 in its C-terminal Gln amide transfer (or GAT/glutaminase) domain (Escherichia coli CTPS (EcCTPS) residues 287-544) [31,32]), which is then transferred through an intramolecular tunnel to the N-terminal synthase (or amidoligase) domain (EcCTPS residues 1-266 connected to the GAT domain via an interdomain linker, residues 267-286) [32]. The lack of equilibration of the nascent NH 3 with the solvent supported the notion of such a tunnel [33]. ...
... Considering the central role of CTP in metabolism, it is not surprising that CTPS is highly regulated. While the substrates ATP and UTP promote oligomerization of the enzyme from inactive monomers and dimers to active tetramers [37,[39][40][41][42][43] by binding at an interfacial active site formed by the synthase domains [32,44,45], the product CTP can also induce tetramerization [43] as well as act as a feedback inhibitor [37,44]. Eukaryotic homologues of CTPS are also regulated by phosphorylation [46][47][48][49][50][51]. ...
Cytidine-5′-triphosphate (CTP) synthase (CTPS) is the class I glutamine-dependent amidotransferase (GAT) that catalyzes the last step in the de novo biosynthesis of CTP. Glutamine hydrolysis is catalyzed in the GAT domain and the liberated ammonia is transferred via an intramolecular tunnel to the synthase domain where the ATP-dependent amination of UTP occurs to form CTP. CTPS is unique among the glutamine-dependent amidotransferases, requiring an allosteric effector (GTP) to activate the GAT domain for efficient glutamine hydrolysis. Recently, the first cryo-electron microscopy structure of Drosophila CTPS was solved with bound ATP, UTP, and, notably, GTP, as well as the covalent adduct with 6-diazo-5-oxo-l-norleucine. This structural information, along with the numerous site-directed mutagenesis, kinetics, and structural studies conducted over the past 50 years, provide more detailed insights into the elaborate conformational changes that accompany GTP binding at the GAT domain and their contribution to catalysis. Interactions between GTP and the L2 loop, the L4 loop from an adjacent protomer, the L11 lid, and the L13 loop (or unique flexible “wing” region), induce conformational changes that promote the hydrolysis of glutamine at the GAT domain; however, direct experimental evidence on the specific mechanism by which these conformational changes facilitate catalysis at the GAT domain is still lacking. Significantly, the conformational changes induced by GTP binding also affect the assembly and maintenance of the NH3 tunnel. Hence, in addition to promoting glutamine hydrolysis, the allosteric effector plays an important role in coordinating the reactions catalyzed by the GAT and synthase domains of CTPS.
... The binding of a maximum of two DON molecules per tetramer suggested that only half of the sites were able to react with glutamine at a given time 30 , a finding that was confirmed much later 31 . The same study also suggested that the nascent NH3 generated during glutamine hydrolysis was delivered to the active site to directly react with UTP -using what would years later be shown to be an "ammonia tunnel" 32,33 . This ligand-induced tetramer formation was observed again in a study showing the sequential and cooperative ATP and UTP binding 34 . ...
... Tetramer existence in absence of substrates has also been observed in the cases of L. lactis CTPS 59,68 , E. coli CTPS 69,70 and human CTPS2 using a mutant (V352C) that is unable to disassemble once aggregated as filaments 64 . While the glutaminase domain has been shown to be active in dimeric form, at least in bacteria 34,59 , the synthetase domain seems to require tetramer formation 32,71 . ...
... Recent data generated using the D. melanogaster enzyme suggest that the binding of GTP depends on the prior binding of the substrates (ATP and/or UTP) that induces a rotation between the glutaminase (GAT) and synthetase (AL) domain 72 . The global shift induced by the substrates binding to the CTPS tetramer "creates" the GTP binding site and aligns two cavities ("Ammonia tunnel", 32,33,64,65,69 ) that channel the NH3 released from the glutamine hydrolysis towards the phospho-UTP intermediate. Then, the binding of glutamine stabilizes the F373 residue that can then interact and potentially stabilize the binding of GTP. ...
The cytidine triphosphate synthases (CTPS) are a highly conserved family of enzymes responsible for the de novo synthesis of CTP using UTP, ATP and glutamine as their substrate. The CTP synthases form inactive dimers, and active tetramers in presence of their substrates ATP and UTP as well as of their product CTP. These tetramers can further assemble as massive intracellular structures called filaments whose role in their regulation is still debated. There are two CTP synthases in human: CTPS1 and CTPS2. Previous works published in this lab demonstrated the crucial role of CTPS1 in the immune system, especially during lymphocyte expansion after TCR (T-cell receptor) stimulation in response to pathogen infections. Moreover, numerous anterior studies link increased CTP synthase activity to cancer development and progression. The available data suggest that specifically targeting the activity of CTPS1 could be a relevant therapeutic approach for the treatment of pathologies that include cancers and some immune diseases. We have generated and characterized CTPS1- and CTPS2-deficient cellular models using the HEK 293T and Jurkat cell lines. Jurkat cells do not express CTPS2, and the inactivation of CTPS1 abrogates their CTPS activity and ability to proliferate, leading to apoptosis. CTPS1 and CTPS2 were found to be partially redundant in HEK cells. In CTPS2 knock-out (KO) HEK, no detectable changes in CTPS activity and proliferation were observed. CTPS1-KO HEK were found to have a strongly decreased CTPS activity associated with a moderate decrease in proliferation. CTPS1 and CTPS2-KO HEK were unable to proliferate, yet able to survive a prolonged CTP deprivation despite a null CTPS activity. Exogenous complementation approaches confirmed that both CTPS1 and CTPS2 were able to restore CTPS-deficient cell viability and proliferation. The enzymatic activity of CTPS2 seems lower than that of CTPS1, and CTPS2 appears to have a reduced capacity to complement cellular models deficient in CTP synthases. Finally, our data suggest that CTPS1 and CTPS2 might interact at the tetramer level. The CTPS1-KO Jurkat model was used to study two CTPS1 mutants expressed in patients with immunodeficiency. CTPS1Δ18 results from the mutation of a splice site, leading to exon skipping and to the expression of an alternative C-terminal domain. Our data confirms that CTPS1Δ18 is an hypomorphic, yet active mutant. The second mutant, CTPS1Δ19, for which we had neither functional data nor patient samples, lacks the last 19 residues of the wild-type protein. Although our preliminary data suggests that his mutant is active and more stable than CTPS1Δ18, further studies are required to fully characterize the impact of this mutation. Our cellular models have allowed us to study the impact of mutations previously used in the literature but tested on other species as well as on non-CTPS-deficient models. In particular, we have been interested in their impact on the capacity of CTPS1 to sustain cell proliferation, form filaments and interact with CTPS2. We have also been interested in testing the impact of targeted mutations of CTPS1 expressed in CTP synthase-deficient cellular models on its capacity to sustain cell proliferation, form filaments and interact with CTPS2. This project has been established in collaboration with a company, Step Pharma, who develops CTPS1-specific inhibitors. We have observed that small molecule inhibitors developed by Step Pharma that were shown to be highly selective for human CTPS1 in cellular and enzyme models unexpectedly lost their selectivity on murine enzymes. Through sequence analysis, mutagenesis and generation of cellular models, we have validated the key role of a residue the selectivity for CTPS1 of Step Pharma compounds.
