Human Thymidylate Synthase (PDB ID 1I00)
Created by Hillary Goldstein
Thymidylate Synthase catalyzes the first step in the de novo biosynthesis of thymine, and such is an essential enzyme for almost all known organisms (8). Thymidylate synthase is a transferase, meaning that it catalyzes the transfer of a chemical group from one molecule to another. Specifically, thymidylate synthase catalyzes the conversion of 2-deoxyuridine-5-monophosphate (dUMP) in conjunction with its cofactor 5,10-methylenetetrahydrofolate (represented here by analogue tomudex) to thymidine monophosphate (dTMP) and dihydrofolate through reductive methylation (1,2). This reaction represents the only means by which dTMP is synthesized de novo within a cell. Figure 4 illustrates the larger context in which thymidylate synthase acts in the general pathway to dTMP synthesis (7). Thymidylate synthase has been identified in numerous species including E.coli, yeast, viruses, and mice (2, 3, 4). Searching the FASTA sequence using BLAST confirms that the protein is highly conserved, as all of the sequences compared had a greater than 95% query coverage and greater than 65% maximum identity. This is a logical result, as DNA is the basic unit of genetic information for all prokaryotes and eukaryotes as well as some viruses, and high conservation of primary structure of DNA between all organisms is consistent with high conservation of an enzyme the affects a DNA precursor.
Thymidylate synthase is a homodimeric protein consisting of two identical monomeric subunits, each of which has a molecular weight of approximately 35 kD and contains 290-313 amino acids. The structure of thymidylate synthase is estimated to have a unit evolutionary period of 22.9 million years, indicating that its structure directly leads to its essential function in organisms (8). Although several species display unique inserted sequences, none of these interfere with the core structure of the protein. A comparison between the primary structures thymidylate synthase of eight different species demonstrated 55 invariant residues, the majority of which were found in the protein core. Sixteen of the invariant residues are found in the active site of the protein, and ten are structurally determinant. Of the residues that were not invariant, 38 displayed changes that did not alter the protein's shape or the properties of the residue. (8) Although surface residues 37-40, 203-208, and 271-306-terminus that interact with the solvent displayed the least conservation (57%), the invariance of Thr-46, Lys-169, and Tyr-301 suggest that these residues may have functions not yet understood. (8)
The secondary structure of the protein is, like the primary structure, highly conserved between species. Each subunit contains two domains. The larger domain of consists of residues at the N- and C-termini, that display 5 α-helices and a five-stranded β-sheet. The smaller domain consisting of residues 70-139 is more variable, and includes 4 alpha-helices. The two monomeric units of thymidylate synthase are connected by a five-stranded β-sheet containing 29 residues within each monomer. Whereas β-sheets associated in this manner almost always display a twist of -30 degrees relative to each other, these display a unique orientation as one sheet has a right-handed twist of +28 degrees relative to the other (8). The helices and beta strands arrange in three layers to form the tertiary structure, the central layer of which is composed of alpha helices (8).
Both monomeric units contain active sites that bind to the substrate dUMP formed by 12 residues, Arg-50, Arg-175 located on the other monomer, Arg-215, Asp-218, Asn-226, His-256, and Tyr-259 interact with the substrate through hydrogen bonding. In humans, Cys-195 and Ser-216 interact through electrostatic as well as covalent interactions, although as described below this is not the case for all species. Arg-176, His-196, and Gly-222 interact with the substrate through van der waals forces. The tertiary structure of the protein is a cavity lined by the side chains of 25 residues, most of which represent β-turns between β-sheets or loops connecting α-helices. (8) One of the residues on each active site is contributed by the second monomer, and thus neither monomer could be catalytically active independently (8). In E. Coli and other studied prokaryotic species, thymidylate undergoes a conformational change from the open to the closed conformation upon binding the substrate dUMP, during which the C-terminus of the protein shifts by approximately 4 angstroms. (15). Figure 3 illustrates the proposed mechanism of catalysis for thymidylate synthase (2). The -SH group of the invariant residue Cys-195 is thought to provide the nucleophile that binds to the substrate dUMP during catalysis. In most species, a covalent bond forms between the 6-position of dUMP which then facilitates the binding of the cofactor, CH2H4folate (11). The attacking Cys-195 residue is thought to form a hydrogen bond with nearby Arg-215, which could lower the pKa of the thiol group and enhance its nucleophilic activity. Cys-195 also covalently binds to known inhibitors of the enzyme such as FdUMP. The active site also contains two invariant Arg residues (215 and 176) that are thought to serve as a binding site for the 5’-phopshate of dUMP, and the two sides of the active site are connected by conserved residues His-196 and Asn-226 (8). This ternary covalent complex, illustrated in Figure 1, then undergoes β-elimination and hydride hydride transfer, oxidizing the cofactor and methylating the substrate to regenerate the enzyme catalyst (8, 14). The site at which the cofactor binds is highly conserved, and an invariate His residue is thought to be crucial to cofactor binding (12). The proximity of Cys-195, Arg-215, and Arg-176 also provides a possible explanation for observed effects of binding independent phosphate groups rather than dUMP, as the two are believed to compete to bind on the active site (9). Binding of Pi has been observed to decrease the rate of enzyme inactivation by thiol reagents such as 5-fluorodeoxyuridine (FdUMP), and the proximity of the phosphate binding site to the catalytic thiol of Cys-195 is though to cause or contribute to this effect through steric effects or reduction of nucleophilicity (10).
Similarly, both monomeric units contain analogous binding sites for the enzyme's cofactor, CH2H4folate. Several of the same residues are observed in both sites, but interact through different forces. Asp-218 and Gly-222 here participate in hydrogen bonding with the cofactor, and Phe-80 interacts through electrostatic forces. The cofactor is stabilized through van der waals interactions with Ile-108, Trp-109, Leu-221, Phe-225, and Tyr-258. (14)
Although the structure of thymidylate synthase is highly conserved between species, some key differences are observed that, influence the mechanism of catalysis. Human thymidylate synthase is displays two stable ternary complex conformations, as it has been crystallized both in the closed conformation (16) but also has been observed to retain the open conformation change upon binding to dUMP. Retention of the open conformation does not alter the activity of the enzyme. The folate analogue tomudex is in fact observed to inhibit the human form of the enzyme ten times as strongly as the E. coli form, with a Ki of 4.6 x 10-6 for the human enzyme and a Ki of 4.6 x 10-7 in E.col. (14). Due to altered interaction with the glutamyl tail of folate (or, as in the experiment, the tolmudex analogue), which displace the complex by 1 angstrom away from its location in the closed conformation, retention of the open configuration prevents the formation of a covalent bond between the nucleophilic Cys-195 and C6 of dUMP. (14).
Additionally, mammalian thymidylate synthase displays an insert of 27 residues on the N-terminus not present in the prokaryotic enzyme. Removal of residues 7-29 resulted in a mutant that displayed activity equivilent to that of the wild type protein. This extension was initially believed to play a role only in the stability of the protein, but replacement of Val-3 with other residues in a separate experiment suggests that some catalytic role may be present as well, as replacement with Leu or Phe strongly compromised dUMP binding (13). This is thought to occur due to increased stabilization of the inactive conformation of TS in the case of the V3L mutant, and due to an altered location of substrate binding that prevents catalysis in the case of the V3F mutant (13).
An additional notable feature of thymidylate synthase is that it autoregulates translation of its own coding mRNA (5). Specific inhibition of thymidylate synthase mRNA was observed following interaction between the mRNA and the protein product, while the translation of other mRNA was unaffected by the presence of thymidylate synthase. When any of thymidylate synthases typical substrates were included, such as dUMP or 5,10-methylenetetrahydrofolate, translation of the coding mRNA proceeded as observed in the absence of thymidylate synthase. This discovery represented one of the first reported cases of mRNA autoregulation in eukaryotes, and as such provided new insight into molecular feedback and regulatory mechanisms (5). The protein domain at which thymidylate synthase interacts with its mRNA remains poorly understood.
Because dTMP is a DNA precursor, thymidylate synthase has generated a great deal of recent interest as a possible target in cancer treatment (5). Both thymidylate synthase and its substrate 5,10-methyenetetrafolate have been the targets of specifically-designed inhibitors intended to act as antitumor agents (6). 5-Fluorouracil (FU) and fluoropyrimidine prodrugs are frequently used as effective thymidylate synthase inhibitors, as shown in figure 2 (10). 5-fluorouracil is physiologically converted into fluorodeoxyuridine monophosphate (FdUMP), which acts as a suicide substrate by forming an inactive ternary complex with thymidylate synthase and 5,10-methylethylenetetrahydrofolate. This effectively inhibits thymidylate synthase thus induces the arrest of DNA synthesis (7).