Mutant dCTP Deaminase
Created by Katherine Lambertson
dCTP deaminase E138A (PDB ID 1XS4) is a mutant form of the wild-type dCTP deaminase from Escherichia coli (PDB ID 1XS1). It has a molecular weight of 127057.28 Da and an isoelectric point of 5.83, and it is a member of the dUTPase-like family (1). Although it does not display measurable function, it has played an important role in elucidating the mechanism of its biologically significant counterpart, wild-type dCTP deaminase. Wild-type dCTP deaminase catalyzes the deamination of deoxycytidine triphosphate (dCTP) to deoxyuridine triphosphate (dUTP) and ammonia. In Gram-negative bacteria, this reaction plays a crucial role in the biosynthesis of deoxythymidine triphosphate (dTTP), a compound which is essential for the synthesis of DNA. Following the dCTP deamination reaction, dUTPase hydrolyzes the product dUTP to produce deoxyuridine monophosphate (dUMP) and pyrophosphate. (1) Thymidylate synthetase methylates the dUMP product to form deoxythymidine monophosphate, or dTMP. Then, thymidylate kinase and nucleoside diphosphate kinase phosphorylate dTMP to yield dTTP. (2,3) While there are other pathways for the biosynthesis of dTTP in E. coli based on deoxyuridine diphosphate (dUDP) and deoxycytidine, this dCTP pathway is by far the most common. This pathway produces 75 to 80 percent of de novo thymidylate in E. coli. (4)
In order to carry out its role in this pathway, dCTP deaminase must bind certain ligands. One of these is the substrate dCTP, which binds to the active site of the dCTP deaminase E138A discussed here (1). The ability of the mutant to bind dCTP shows that the mutation E138A only affects the deaminase reaction, and not the enzyme’s ability to bind nucleotides. In fact, the way dCTP deaminase E138A binds nucleotides is similar to the way the wild-type enzyme binds them. (5) The methods differ only in the arrangement of water molecules around the 4-position of the nucleotide's pyrimidine ring (6). The structure of the wild-type dCTP deaminase in complex with dUTP (PDB ID 1XS1) is thus similar to the structure of dCTP deaminase E138A in complex with dCTP (PDB 1XS4), even though different nucleotides are bound. A comparison of tertiary structure using the DALI server gives a Z value as high as 33.0 (7). This is much greater than 2.0, indicating that these proteins have extremely similar tertiary structures.
Another important ligand is Mg2+. It binds to the active site of dCTP deaminase (both the E138A and wild-type forms) with dCTP. The ion and the substrate are generally considered to be in complex. The magnesium ion shields the negative charges of dCTP’s three phosphate groups, allowing the active site conformation to change: the C-terminal loop of one of the dCTP deaminase subunits can close over both the substrate and the active site, forming a sort of lid (1,6). The magnesium ion thus affects the binding of the substrate, and not the catalytic action of the enzyme. This is unique among nucleobase and nucleoside deaminases, since these enzymes usually require catalytic metal ions (1). In yeast cytosine deaminase, for instance, the metal ion (Zn2+) activates a water molecule to form a nucleophile (8). In wild-type E. coli dCTP deaminase, however, this role is carried out by the side chain of Glu-138 (1).
A third ligand that binds to dCTP deaminase is the product dUTP, which is bound to the active site of the wild-type dCTP deaminase discussed here. Though dCTP and dUTP are different nucleotides, there is little change in the dCTP deaminase structure when one binds instead of the other. In fact, for dCTP deaminase E138A the dCTP complex (PDB ID 1XS4) and the dUTP complex (PDB ID 1XS6) are virtually identical. (6) A comparison with the DALI server yields a Z value as high as 33.4 (7). As this is far greater than 2.0, it is indicative of significant similarity between these proteins’ tertiary structure.
dCTP deaminase binds other ligands, as well. Rather than acting as substrates, cofactors or products, these act as inhibitors. One such ligand is inorganic phosphate. It binds to the active site of the enzyme, acting as a competitive inhibitor. However, it also increases the cooperativity of dCTP binding, meaning that once inorganic phosphate binds to one active site of dCTP deaminase, the other active sites bind dCTP more effectively. The mechanism for this is still not entirely understood. (1) The mechanism of action for another inhibitor is much clearer. This inhibitor is dTTP, the final product of the dCTP metabolic pathway, and it binds to the active site of dCTP deaminase E138A. However, it does not simply block the active site. Once in complex with the enzyme it causes changes to the tertiary structure, resulting in a new conformation of dCTP deaminase E138A (PDB ID 2J4Q) termed the inactive conformation. Using this terminology, the original conformation of E138A dCTP deaminase (in complex with dCTP or dUTP) is called the active conformation. (6)
Before considering either conformation in detail, consider the basic structure of dCTP deaminase. The gene for the enzyme codes for one subunit which is 193 amino acids in length. This gene is copied multiple times, to form six subunits labeled Chains A-F. These subunits form two independent homotrimers, made of Chains A-C and Chains D-F. Within these proteins, the interaction of two subunits forms an active site. Because there are three such interactions per homotrimer, each has three active sites. Thus, the role of the subunit is the formation of the dCTP deaminase active site, and the creation of catalytic function. (1)
The subunits have a distinct secondary and tertiary structure, for both the mutant and wild-type enzymes. For secondary structure, each subunit has three alpha helices, α1 – α3, four 3/10 helices, γ1 – γ4, and14 beta strands, β1 – β14. The beta strands form three antiparallel beta sheets, named S1 (β1, β8, β12 and β10), S2 (β2, β3, β14 and β9) and S3 (β4 and β6). For tertiary structure, each subunit forms a distorted beta barrel, due to beta sheet S2 packing over beta sheet S1. (1)
The interaction between the subunits in the homotrimers is important, as it produces the distinctive quaternary structure of dCTP deaminase. When three out of the six subunits come together, they form a compact homotrimer which has an equilateral triangular face perpendicular to its three-fold axis. The homotrimer is maintained by specific interactions between different subunits’ residues. Consider chains A and C. Arg-115 from Chain C hydrogen bonds to Gln-182 from Chain A, working with other parts of the hydrogen bond network to lock subunits together across the active site. Similarly, Asp-193 from the C-terminal loop of Chain A forms a salt-bridge with Arg-110 of Chain C, after the C-terminal loop has closed over the active site and substrate. There are also many other interactions, which are responsible both for maintaining the homotrimer and for creating additional secondary structure across the subunits. This secondary structure is a beta sheet. When two subunits interact with each other, a mixed beta sheet termed S4 (β5 and β13 from one subunit, β7 from another) is formed. (1)
As mentioned previously, one of the other structures that results from the interaction of the subunits is the active site. This has been characterized extensively for the active conformation of dCTP deaminase E138A. More specifically,the active site constructed by Chains A and C has been characterized (1). The following applies to the primary form addressed in this paper, the mutant enzyme in complex with dCTP, and to its nearly identical counterpart, the mutant enzyme in complex with dUTP. Both are in the active conformation.
The three phosphates of the nucleotide in the active site are octahedrally coordinated to a magnesium ion, which shields the negative charges. The α-phosphate interacts with Arg-126 and Ser-111 from Chains A and C, respectively; the β-phosphate interacts with Ser-112 and Arg-110 from Chain C; and the γ-phosphate interacts with Lys-178 and Tyr-171, from the folded over C-terminal loop of Chain A. The deoxyribose ring and the pyrimidine ring have interactions of their own, in another region of the active site. Deoxyribose stacks with Trp-131, while the pyrimidine ring maintains hydrophobic interactions with Ile-135. Both of these residues are from Chain A, and seem to have special significance. The tryptophan may prevent ribose compounds from binding, as its large hydrophobic side chain presumably blocks the –OH group of ribose. The isoleucine represents a common feature of this enzyme family, the dUTPase-like family. In a dCTP deaminase amino acid sequence alignment, the corresponding residue always has stacking properties that match those of isoleucine – that is, the amino acid is isoleucine, valine, leucine or tryptophan. (1)
Other residues interact with the pyrimidine ring in the active site, as well. The oxygen at Position 2 on the pyrimidine ring forms a hydrogen bond with Gln-182 from Chain A, and also hydrogen bonds with the amino backbone of Val-136 from Chain A and the Arg-115 from Chain C. All three of these residues prove important during catalysis. In addition to these interactions, the carbonyl backbone of the Val-136 hydrogen bonds to the nitrogen at Position 3 in dUTP, and to the nitrogens at Positions 3 and 4 in dCTP. (1)
The deoxyribose also interacts with other residues. The oxygen at Position 3 on the ring forms two hydrogen bonds with Asp-128. One hydrogen bond is to an oxygen of the side chain, and the other is to the backbone amino group. (1)
The structure of this active site leads directly to its function, the deamination of dCTP. Consider the following mechanism, summarized in part in. Hydrogen bonds to the amino group of Val-136 and the side chain of Gln-182 hold the pyrimidine ring of dCTP in place. In addition to dCTP, the active site contains two important water molecules. The firstof these water molecules acts as hydrogen bond donor with the side chain of Glu-138 from Chain A (an alanine in the mutant form of the enzyme), and the carbonyl backbone of Ala-124, also from Chain A. The same water molecule also acts as a hydrogen bond acceptor with the side chain of Ser-111. Ser-111 is firmly held in place by hydrogen bonds to Arg-115. This seems significant, as both residues are invariant in dCTP deaminase sequences, and additional research has proved this to be the case. (1,5) Arg-115 has a lower pKa than Ser-111, and its close proximity to Ser-111 keeps the serine alcohol group from being deprotonated when the water stabilized by the residue becomes a hydroxide (5).
This hydroxide is formed when Glu-138 activates the first water molecule by deprotonating it. This activation is extremely important, both because it enables the reaction and because it makes the enzyme unique, as a deaminase that does not require a metal catalyst. The E138A mutant’s lack of activity makes the catalytic importance of the residue especially clear. The alanine cannot activate the water molecule, and thus the reaction cannot proceed.
However, in wild-type dCTP deaminase, the glutamate is present, and it can activate the water molecule. Following the activation, the water goes on to act as a nucleophile and form a tetrahedral reaction intermediate. It attacks the carbon at Position 4 of the pyrimidine ring, and expels the amino group. The amino group extracts a proton from the second water molecule mentioned earlier, which bifurcatedly coordinates to Glu-138 and to the backbone carbonyl of Val-136. The dCTP releases ammonia, forming dUTP. The enzyme neutralizes the hydroxide formed by the proton extraction, and returns its amino acids to their original state. dUTP is released, and a new dCTP may bind. (1)
All of this, from the description of the active site to the description of the mechanism, applies to the active conformation of dCTP deaminase. When dTTP binds and the enzyme assumes its inactive form, the tertiary structure of the active site changes and the enzyme can no longer catalyze the deamination reaction (although the E138A form could not do this in the active form, either). First, there are significant changes in the structure of the C-terminal loop. When dTTP binds, the C-terminal loop (defined as the final 20 amino acid residues) becomes disordered and does not appear in electron density maps. Other portions of the active site also change. Alpha helix 2 (α2, residues 55-65) and a portion of beta strand 5 (β5) form a lip of the active site, and this moves into the region the C-terminal loop would have occupied were dCTP or dUTP to bind. This suggests that either the absence of the defined C-terminal loop causes the lip to move, or the movement of the lip causes the C-terminal loop not to fold over the active site into a definite structure. A smaller loop which contains active site residues 120-125 also moves, presumably to accommodate the thymidine moiety of dTTP. Two things happen to these residues. First, His-121 seems to flip. This has grave consequences, because the flipping may expel the catalytic water molecule hydrogen bonded to the side chain of Glu-138 and the backbone carbonyl of Ala-124 in the active conformation of dCTP deaminase. This would certainly account for the loss of activity. Second, Ala-124 no longer seems to interact with the pyrimidine ring as it did in the active conformation. It moves from Position 4 of the pyrimidine ring, where it interacted either with the amino group in dCTP or the carbonyl in dUTP. (6)
Two other residues also move in the conformation change. These are Val-122, from one subunit in the active site, and Thr-123, from the other subunit in the active site. Because of the change in position of residues 120-125, these residues must also move, to avoid clashing with the loop. This suggests that these two residues may drive a concerted conformation change, causing each subunit to go from the active conformation to the inactive conformation when dTTP binds. (6)
Having discussed the function and structure of E. coli dCTP deaminase E138A in detail, it will be interesting to compare it with other, similar proteins. As has been mentioned, dCTP deaminase is part of the dUTPase like family. In addition to dCTP deaminase, this family contains trimeric dUTPases and bifunctional dCTP deaminase-dUTPases (1). Below, dCTP deaminase is compared to both a dUTPase from E. coli, and a bifunctional dCTP deaminase-dUTPase from Mycobacterium tuberculosis.
dUTPase hydrolyzes dUTP to produce dUMP and pyrophosphate (1). The enzyme addressed here, E. coli dUTPase in complex with dUMP•Mg2+ (PDB ID 1SEH), carries out this same function. It was compared to dCTP deaminase E138A complexed with dCTP (PDB ID 1XS4) using the PSI-BLAST align tool, and an E value of 8 × 10-5 was determined. This is much less than 0.05, indicating there is significant overlap between these two enzymes’ primary structures. They have a percent similarity of 46%. Then, dUTPase was compared to dCTP deaminase using the DALI server. A Z value of 10.3 was determined, specifically for Chain A (7). This is much greater than 2.0, the lowest value to indicate significant similarities. There is thus considerable overlap in these two proteins' tertiary structures.
These similarities manifest within the two proteins in several ways. dUTPase carries out substrate hydrolysis with a nucleophilic attack by a water molecule, which is activated by a conserved aspartate residue, Asp-90 (9). This is remarkably similar to the reaction mechanism determined for dCTP deaminase. The role of the magnesium ion in dUTPase is also similar. Substrate binding in a catalytically competent formation is assisted by interaction of the Mg2+ with the triphosphate moiety of dUTP (9). In addition to these mechanistic similarities, there are easily observed structural similarities. dUTPase is a trimer, with active sites located in clefts between neighboring monomers. The C-terminus of the third subunit interacts with the active site formed by the cleft between two other subunits. (9) In dCTP deaminase, the C-terminus also plays a role in forming the active site (1). However, only two subunits are involved, rather than three.
This leads to discussion of the differences between these two proteins. The most obvious difference is in function, and is a direct result of key differences in structure. One of the differences that leads to dCTP deaminase function is the presence of Arg-126 in dCTP deaminase. This residue occupies the position that the catalytic water molecule in dUTPase would hold, and thus dUTPase function (the hydrolysis of the phosphate chain) is precluded. There are also other differences in structure, both secondary and tertiary. dUTPase is shorter than dCTP deaminase, and it thus lacks alpha helix 2 (α2), beta strands 4-6 (β4 – β6), and 3/10 helix 2 (γ2). dCTP deaminase also has a different fold in its N-terminus, in comparison to dUTPase. (1)
Now, consider the bifunctional dCTP deaminase-dUTPase from M. tuberculosis, in its apo form (PDB ID 2QLP). As its name suggests, it carries out the functions of both dCTP deaminase and dUTPase, first deaminating dCTP to product dUMP and then hydrolyzing dUTP to product dUMP. Since this is the apo form, the function cannot really be carried out. Still, the structure is that of a functioning protein, and this bifunctional enzyme was thus compared to dCTP deaminase E138A complexed with dCTP. The PSI-BLAST align tool produced an E value of 2 × 10-33, indicating significant similarity in primary structure: the proteins have a percent similarity of 43%. Next, a comparison was done using the DALI server. This produced a Z value as great as 22.1. Some chains had lower values than others, but none went below 21.8 (7). Thus, there is considerable similarity in the tertiary structure of these two proteins.
These proteins are known to share 34 residues. They also share other features related to tertiary structure. Both proteins form homotrimers whose centers are composed of alpha helices and a distorted beta barrel. Additionally, both form active sites from two subunits, rather than three. They react similarly to dTTP inhibition, as well, switching from an active conformer to an inactive one. In each, a small loop at the active site moves. (1,10)
However, the behavior of the C-terminal loop is different for the bifunctional protein. When dTTP binds and the inactive conformation forms, the C-terminal loop does not become disordered. Rather, it still binds over the active site, to form a lid. Another difference is the monofunctionality of dCTP deaminase – it lacks dUTPase activity due to the substitution of Ala-148 for Gln-148, the substitution of His-125 for a conserved glycine residue, and the positioning of Arg-126, which prevents the catalytic dUTPase water molecule from binding. (1,10)