CTP_Synthetase_from_M_

   CTP synthetase PyrG (PDB: 4ZDI) from Mycobacterium tuberculosis is required for the ATP-assisted amination of UTP to form CTP (Figure 1) in the biosynthesis of pyrimidine nucleotides, which are necessary for DNA and RNA biosynthesis. Inhibiting this essential protein, CTP synthetase PyrG, is therefore a potential method of preventing the M. tuberculosis infection from replicating (1, 2). Second only to HIV/AIDS in number of deaths caused by an infection, tuberculosis caused 1.5 million deaths worldwide out of 9 million reported cases in 2013, though the number of cases is slightly decreasing each year (3). In many cases, a six-month course of various antimicrobial drugs will completely cure the infection, but a growing number of multidrug-resistant strains have arisen, causing about 480,000 cases in 2013 alone (3). One way to combat this resistance is to develop new prodrugs with different targets, and since CTP synthetase PyrG inhibition would prevent M. tuberculosis replication, investigation of this protein's structure,active sites, functions, and mutations is potentially important to curing this deadly infection.
       Both wild and drug-resistant mutation strains of CTP synthetase PyrG were crystallized (1). After isolation by standard methods of the M. tuberculosis proteins of interest grown in E. coli, the proteins were each purified by multiple methods including the use of nickel affinity columns. Crystallization was induced at 18 ºC by combining the isolated apo form of the protein with various ligands such as 200 mM calcium (II) acetate, a UTP complex, and 100 mM MgCl₂, or a complex of the substrates L-DON (the glutamine analogue of UTP), UTP, and AMP-PCP with 100 mM NaCl (1). The ligands occupied the binding sites and allowed the protein structure to crystallize. Since substrates were used as ligands, their orientation could be determined, allowing drugs to be designed to suit these orientations. No ligands aside from Ca²⁺ were found naturally that associated with the protein, and calcium (II) was added to the crystallization mixture, making it difficult to identify where in the protein it would be located naturally. The added Mg²⁺ ion was present at the synthetase active site, indicating that a ligand could be involved in the mechanism of CTP synthesis (1). The UTP added was present at both active sites, supporting the proposed mechanism of CTP synthesis (Figure 1).
       With a molecular weight of 509.4 kDa and an isoelectic point of 5.41 as calculated by Expasy, CTP synthetase PyrG is a homotetramer containing eight identical subunits (1, 4, 5). Each subunit contains two active sites, glutaminase and synthetase, allowing it to bind multiple UTP substrates at once to efficiently synthesize CTP (1). The two domains each organize into an "X" shape and slightly coil around each other. The N-terminal synthetase binding pocket consists of nonpolar residues and points toward the center of the protein, whereas the C-terminal glutaminase binding pocket containing Cys-393 points outward, which is consistent with the CTP synthetase crystal structures solved for other organisms (1). The synthetase active site contains the ATP and UTP binding pockets that contribute toward the overall synthesis reaction of CTP, and the glutaminase binding pocket is also called the CTP binding pocket, as the catalytic triad of Cys-393, His-524, and Glu-526 hydrolyzes the glutamine of L-DON to produce CTP (1). These two active sites are linked by a partially-constricted NH4+ channel that allows the substrate to move from the synthetase to the glutaminase active site once the correct UTP intermediate forms (1, 6).
       These two active sites and the potential channel contribute toward the overall reaction shown by Figure 1. The mechanism for the synthesis of CTP by CTP synthetase PyrG is likely similar to that for CTP synthetases in other organisms. CTP synthetase occurs in many organisms, including Homo sapiens and Escherichia coli, and its primary structure in E. coli is identical to that of M. tuberculosis as given by its E value of 0.0 (7, 8). The BLAST server uses a sequence to find proteins of similar sequences and ranks their similarities by E values, where an E value of 0.0 indicates identical primary structure, and an E value of less than 0.05 is considered significant between sequences. The CTP synthetase in E. coli (PDB: 1N1M), however, has only two subunits as compared to the eight in CTP synthetase from M. tuberculosis, giving the protein significantly different tertiary and quaternary structures (8). The Dali search provided a tertiary structure agreement of Z = 49.7 between the two CTP synthetases, with a Z-score above 2 showing significant agreement between tertiary structures, indicating the E. coli and M. tuberculosis proteins had similar tertiary structures even with different numbers of subunits. These proteins serve the same function, to synthesize CTP so that DNA and RNA have this required nucleotide to replicate (1, 2). Though the proteins have identical primary structure, they do not function identically. Since the E. coli CTP synthetase has fewer subunits, it has a GTP active site not present in the M. tuberculosis analogue. The GTPase superimposes on a cleft of the CTP synthetase's ammonia channel not present in that of the M. tuberculosis, whose subunits arrange such that there is no space for the GTPase to superimpose (1, 2). Interestingly, the M. tuberculosis CTP synthetase PyrG has identical primary structure to that of the host in which the M. tuberculosis infection was grown in order to obtain enough protein for crystallization.
       Assuming the mechanism for CTP biosynthesis is the same for M. tuberculosis as for E. coli, since they have the same sequence, the equation in Figure 1 occurs in three steps. First, UTP is phosphorylated by ATP in the synthetase active site. This intermediate is then transferred via the ammonium channel to the glutaminase active site, where the UTP attacks the NH₃. Finally the release of the phosphate group on UTP yields CTP as well as ADP and Pi (6).
       The protein's secondary structure consists of 25 (35%) alpha helices and 27 (21%) beta sheets, with turns, 3/10 helices, the occasional random coil, and a segment of unknown secondary structure from Pro-430 to Gly-444 (2). Unknown secondary structure indicates a high degree of flexibility in that region and therefore many potential but unknown functions (5). At each of the active sites, a mixture of secondary structures is observed. Neither active site contained α-helices, which are extensively hydrogen-bonded, indicating the increased flexibility or more more distinct orientation given by other secondary structures, like that of the P-loop in the synthetase active site, is necessary to bind the intended substrates. At the synthetase active site, the Val-186, Val-14, Ile-221, and Leu-188 residues are each part of a Β-sheet, and these Β-sheets run parallel to each other (1, 2). These sheets create the right amount of space and the right hydropathy of residues to bind the substrates. At the glutaminase active site, Cys-393 and His-524 each have unspecified secondary structure between a Β-sheet and a helix, whereas Glu-526 is part of a 3/10 helix (2). The Cys-393 and His-524 residues are therefore bound to a degree by their surrounding secondary structures, but they are not held tightly in one place since they have unspecified secondary structure as opposed to a turn or a random coil.
       The hydrophobic pocket that makes up the synthetase active site contains a P-loop and nonpolar residues including Val-14, Ile-221, Val-186, and Leu-188 (1). These residues do not exhibit notable hydrogen bonding, but rather exert van der Waals forces. These weak forces are suited to pocket formation since there must be space for a substrate so that the protein can act on the substrate and carry out its intended function. These hydrophobic residues will orient the substrate by attracting the nonpolar region of a substrate and pushing away hydrophilic regions.
       In the glutaminase pocket, the catalytic triad of Cys-393, His-524, and Glu-526 displayed different forces, as these amino acid residues are significantly more polar than those of the synthetase active site. The composition of these residues indicates possible hydrogen bonding sites, as the Cys-393 could engage in hydrogen bonds through its sulfur, the His-524 through its nitrogens, the Glu-526 through its oxygens, and all of them through the hydrogens on their amino nitrogens. According to the crystal structure, the glutamate oxygen atoms possess a slightly negative charge, and these oxygen atoms form hydrogen bonds with the hydrogen atoms attached to the slightly positive amino nitrogen of the cysteine residue, as shown in Figure3. The dipole moments of all three residues in the triad makes their catalysis efficient for orienting the UTP and L-DON substrates.
       The drug-resistant strain of M. tuberculosis shows a Val-186-Gly mutation in the synthetase active site of CTP synthetase PyrG (1). The mutation inhibits potential pro-drugs since the glycine residue is more polar than the valine that is present in the wild strain. This increased polarity of the glycine alters the secondary structure at the active site, requiring even more potent pro-drugs than the compounds investigated thus far. These potential prodrugs, the S-dioxides of 5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide and 3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide (Figure 2), were effective at inhibiting activity only on the wild strain protein, and the variations of these structures investigated did not lead to more potent pro-drug discovery, as the variations were found to be less potent than the original compounds (1). Substitutions were made at the thiophene ring and at the 4-nitroaniline, which reduced rather than augmented the effect of the potential pro-drugs, so more investigation is required to find a suitable compound that can overcome the Val-186-Gly mutation.
   Another conformation of CTP synthetase PyrG exists (PDB: 4ZDJ), consisting of one subunit, but is linked to the same article (1) that this protein's crystal structure (PDB: 4ZDI) comes from. The article was only published in June of 2015, so more crystal structures of CTP synthetase from M. tuberculosis or different conformations of this protein could be determined in the future to show how these proteins are distinct. The crystal structure could also be solved again by another group to ensure the credibility of the data such that new pro-drugs can be developed accordingly.