The dUTPase of white spot syndrome virus assembles its active sites in a noncanonical manner

Abstract

dUTPases are essential enzymes for maintaining genome integrity and have recently been shown to play moonlighting roles when containing extra sequences. Interestingly, the trimeric dUTPase of white spot syndrome virus (wDUT) harbors a sequence insert at the position preceding the C-terminal catalytic motif V (pre-V insert), rarely seen in other dUTPases. However, whether this extra sequence endows wDUT with additional properties is unknown. Herein, we present the crystal structures of wDUT in both ligand-free and ligand-bound forms. We observed that the pre-V insert in wDUT forms an unusual β-hairpin structure in the domain-swapping region and thereby facilitates a unique orientation of the adjacent C-terminal segment, positioning the catalytic motif V onto the active site of its own subunit instead of a third subunit. Consequently, wDUT employs two-subunit active sites, unlike the widely accepted paradigm that the active site of trimeric dUTPase is contributed by all three subunits. According to results from local structural comparisons, the active-site configuration of wDUT is similar to that of known dUTPases. However, we also found that residues in the second-shell region of the active site are reconfigured in wDUT as an adaption to its unique C-terminal orientation. We also show that deletion of the pre-V insert significantly reduces wDUT's enzymatic activity and thermal stability. We hypothesize that this rare structural arrangement confers additional functionality to wDUT. In conclusion, our study expands the structural diversity in the conserved dUTPase family and illustrates how sequence insertion and amino acid substitution drive protein evolution cooperatively.

Introduction

dUTPase (EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and PP_i, playing essential roles in cellular nucleotide metabolism (1). First, the enzyme supplies dUMP to synthesize dTTP, a DNA building block. Second, the enzyme lowers the dUTP/dTTP ratio in the cell, thus preventing dUTP misincorporation into genome DNA; deficiency of the enzyme would result in too high a uracil content in DNA and could finally lead to genome instability and thymineless cell death due to overdosed DNA repair activity (2, 3). Because of their essential roles, dUTPases have been proposed as potential drug targets for the therapy of cancer (4, 5) and infectious diseases (6).

dUTPases have been intensively studied structurally and enzymatically (1). According to their structure and oligomerization state, dUTPases are classified into three families: homotrimeric, homodimeric, and monomeric. The homotrimeric and monomeric dUTPases consist mainly of β-pleated sheets and are evolutionarily related (7), whereas the homodimeric dUTPases are all-α proteins and evolutionarily distinct from the other two families (8). Among the three, the homotrimeric family comprises the majority of dUTPases and has been the best studied. So far, a number of trimeric dUTPase structures are available, with those of Escherichia coli (eDUT)² (9) and humans (hDUT) (10) as prototypes. The trimeric dUTPase has a 3-fold symmetry with the C terminus of each subunit swapped. Seven β-strands from one subunit plus one β-strand from the swapped C-terminal arm of the neighboring subunit form a β-barrel with a jellyroll fold, as a repeating structural unit to assemble the trimer. A striking feature of trimeric dUTPase is that each of its three active sites is formed by five conserved motifs from all three subunits (10), which is rare among enzymes. In such a three-subunit active site, motifs I, II, and IV from one subunit and motif III from another are preformed, whereas the C-terminal P-loop–like motif V from the third subunit is rather flexible in the absence of substrate and undergoes a disorder-to-order transition upon ligand binding, hence capping the active site (11–14). Regarding the catalytic mechanism, previous studies have revealed several interesting features of the enzyme. For example, motif V is not required for substrate binding, but it is essential for organizing the active site into a catalytically competent conformation (11), by contributing several key residues, such as a “Phe-lid” (10) and an arginine finger (15). Interestingly, besides the key individual residues, a proper amino acid interacting network within the active site is also important for catalysis. Mutation of a single residue that does not directly interact with the substrate could disrupt the interacting network and thus significantly reduce enzyme activity (16).

Recently, the finding that in addition to their involvement in nucleotide metabolism, dUTPases can have moonlighting functions in various cellular processes, such as immune system modulation (17, 18), pathogenicity island transferring (19–22), and apoptosis (23), has raised a lot of interest. These “moonlighting” dUTPases often bear an extra sequence insert, called motif VI (24). For example, in the dUTPase of Mycobacterium smegmatis, next to the motif V is a 5-residue insert, deletion of which did not affect the enzyme's activity but was lethal for bacterial viability by an unknown mechanism (25). In staphylococcal phages, motif VI at the central domain is not required for enzyme activity but critical for transferring the pathogenicity island (22). The rat dUTPase contains an N-terminal motif VI, important for interacting with the peroxisome proliferator–activated nuclear receptor α (26). These examples show that active evolution occurs in the putatively conserved dUTPase family, where sequence insertion is a major driving force.

White spot syndrome virus (WSSV) is a deadly pathogen for crustacean animals (27), causing severe annual economic loss in the aquaculture industry. It is a large DNA virus and the sole member of the genus Whispovirus and the family Nimaviridae. So far, its replication mechanism is not understood, and no effective treatment is available (28). Genome sequence analysis uncovered that the ORF wsv112 encodes a dUTPase, although the host can provide dUTPase for viral DNA synthesis (29). The viral dUTPase contains 461 amino acid residues; the N-terminal 171 residues comprise a typical homotrimeric dUTPase domain that shows competitive enzyme activity on its own (30), whereas the C-terminal 290 residues form a region of unknown function. Interestingly, sequence alignment reveals that the N-terminal dUTPase domain harbors an extra 20-residue insert with reference to hDUT, which makes its sequence length between motif IV and V significantly longer than in any other known trimeric dUTPase (Fig. 1); this insert is defined as pre-V insert (sequence insert preceding the catalytic motif V). We propose that the unusual pre-V insert and the long C-terminal tail might endow WSSV dUTPase with additional functions. Otherwise, a short version of the enzyme would be more economical for the virus, if the role of the viral dUTPase is only to complement the host dUTPase for more enzymatic activity. To provide more clues for its potential new functions and also facilitate antiviral drug design targeting this enzyme, we subjected WSSV dUTPase to a structural study.

Figure 1.

Sequence alignment of trimeric dUTPases that have structure solved. FIV, feline immunodeficiency virus; EIAV, equine infectious anemia virus; PBCV, Paramecium bursaria chlorella virus; VACV, vaccinia virus; BSUB, B. subtilis; MTUB, M. tuberculosis; ECOL, E. coli; SCER, Saccharomyces cerevisiae; ATHA, Arabidopsis thaliana; DMEL, Drosophila melanogaster; HSAP, human. Strictly and relatively conserved residues are highlighted in red and yellow boxes, respectively. The secondary structure elements of wDUT and hDUT are shown above and below the alignment, respectively. Positions of motifs I–V and the pre-V insert are indicated. The position of the conserved arginine/lysine on motif IV of classic dUTPases is indicated by a blue triangle, whereas the position of its function-subrogate residue in wDUT (Arg²⁴) is indicated by a red triangle.

Here we present the crystal structures of the N-terminal dUTPase domain of WSV112 (residues 1–171, hereafter referred to as WSSV dUTPase (wDUT), unless otherwise specified), in both its free and ligand-bound forms. Surprisingly, wDUT employs two-subunit active sites, challenging the well-accepted concept that active sites of trimeric dUTPases are contributed by all three subunits. We show how this novel structural form is realized as a combined result of sequence insert and amino acid substitution.

Results

wDUT contains an unusual β-hairpin structural motif in the swapping region

The wDUT crystal grew in space group P2₁2₁2₁ with six molecules in one asymmetric unit, forming two trimers. Although our protein construct comprises 171 residues of wDUT, we were only able to model residues 3–150 for four monomers and 3–149 for the other two monomers. The remaining C-terminal residues of all monomers lacked defined electron density, supposedly due to disorder. Most modeled residues showed definite electron density as well as a good quality of geometry, whereas several residues in loop regions and at surfaces exhibited relatively weaker electron density for side chains. Ramachandran plot analysis marked residue Gly⁸⁵ in all monomers as an outlier, slightly out of the allowed region. However, electron density clearly showed that this residue was correctly modeled. Such an “outlier” conformation is supposed to be necessary for function, as a Ramachandran outlier was also observed for a residue (Ala⁷⁵) in the same region in hDUT (10).

The wDUT homotrimer has 3-fold symmetry, with a narrow central channel (Fig. 2A). PISA analysis showed that the accessible surface area buried in the interface is 9060 Ų, corresponding to about half of the accessible surface area of all subunits (19,300 Ų), indicating a stable trimer in solution, which is in agreement with the estimation from the gel filtration experiment (data not shown). All of the monomers in the asymmetric unit share essentially identical structures, with a root mean square deviation (RMSD) of 0.2–0.8 Å for all Cα positions when superposed in a pairwise manner (Fig. S1). Compared with residues located in the main body of the protein, residues Tyr¹³⁴–Glu¹⁴¹ that protrude from the main body exhibit larger RMSD in Cα positions, reflecting their conformational flexibility. The monomer of wDUT adopts the typical dUTPase fold (Fig. 2A). The N-terminal region (residues Ser³–Arg¹²³) forms a β-barrel core; β1, β3, β6, β5b, β4, β7, and β2 form a distorted β-barrel, with β2b, β6b, β5a, and α1 capping the barrel. From residue Tyr¹²⁴ on, the polypeptide traces outward from the body and contributes a β-strand to arm the adjacent subunit. Up to residue Val¹³³, the structure of wDUT resembles those of known dUTPases. Then follows the extra pre-V insert (residues Tyr¹³⁴–Asn¹⁵³), which unexpectedly forms a β-hairpin structure instead of a loop (Fig. 2 (A and B) and Fig. S2). In detail, residues Tyr¹³⁴–Asn¹³⁶ continue the β8 strand, making this strand longer than that of any known trimeric dUTPase. Residues Asn¹³⁶–Gly¹⁴⁰ form a tight 5-residue turn, with the backbone oxygen of Asn¹³⁶ accepting one hydrogen bond from the backbone nitrogen of Gly¹⁴⁰, matching the criteria of an α-turn. Residues Gly¹⁴⁰–Lys¹⁵⁰ form β9, a long β-strand that folds onto β8 in an antiparallel manner, with extensive interstrand hydrogen bonding. Within β9, a single-residue bulge occurs at the position of Ile¹⁴⁵. In summary, whereas “classic” dUTPase structures contain one β-strand in the swapping region, wDUT contains a β-hairpin structural motif (including β8, the α-turn, and β9). As a notable result, the C-terminal orientation of wDUT is reversed in comparison with that of classic dUTPases.

Figure 2.

Overall structure of ligand-free wDUT. A, top view (left) and side view (right) of the trimer. Secondary structure elements are indicated in one monomer. The pre-V insert is highlighted in red, and the β-hairpin in the swapping region is indicated with a rectangle. The first residue of the swapping C terminus (Tyr¹²⁴), the first residue of the pre-V insert (Tyr¹³⁴), and the last modeled residue (Lys¹⁵⁰) are indicated. B, stereo view of the β-hairpin in the swapping region.

The C terminus of classic dUTPase carrying the catalytic motif V would reach the third subunit to cap the active site, after a disorder-to-order transition upon substrate binding (11–14). Because of its direction reversal caused by the β-hairpin, the C-terminal region of wDUT would have to fold back again beyond Lys¹⁵⁰ for the C terminus to reach the third subunit in the classic manner. Such a “detour” path would require at least about 25 residues in the C-terminal region following the β-hairpin, even in an extended conformation (Fig. S3). However, only 20 residues are present in the remaining C-terminal region; therefore, there must be an “out-of-reach” difficulty in this scenario. It is very likely that the C-terminal segment of wDUT (which is not defined by electron density) continues in the current direction to reach the active site of its own subunit (Fig. S3). To test this hypothesis, a crystal structure with ordered C-terminal region would be desirable.

The C-terminal region of wDUT takes a novel path to reach the active site of its own subunit in the complex structure

Previous studies have shown that ligand binding can induce ordering of the C-terminal region in dUTPases. Co-crystallization of wDUT with dUTP induced the C-terminal ordering of the enzyme in the crystal structure. (However, the bound ligand is not dUTP, as discussed below). Crystal of the enzyme-ligand complex grew in space group P2₁, different from the P2₁2₁2₁ space group of the free wDUT, with six monomers forming two trimers in one asymmetric unit. The structure of the first 150 residues, including the β-hairpin in the swapping region, is similar to that of the ligand-free form (Fig. 3). The C-terminal residues beyond Lys¹⁵⁰, which are invisible in the ligand-free structure, appeared with clear electron density (Fig. S4). However, the degree of order of the C-terminal region is not the same for all monomers. The C-terminal regions in four monomers show a closed conformation to cap the active site of their own monomer; among these, three exhibit excellent and the fourth one exhibits weaker but discernible electron density. The C-terminal region of the fifth monomer is partially ordered (up to Arg¹⁵⁸); however, it folds onto the neighboring trimer, which seems physiologically irrelevant. The last monomer lacks any electron density at all for the C-terminal region. This asymmetric ordering is commonly seen in dUTPase structures, reflecting the mobile nature of the C-terminal region. Nevertheless, superposing all monomers in the asymmetric unit shows that the C-terminal regions with closed conformation overlaid well (Fig. S5). Considering that these monomers are located in different crystallographic environments, the observed conformation is unlikely to be a packing artifact but rather reflects a physiologically relevant state in solution.

Figure 3.

Overall structure of the wDUT-dU-PP_i-Mg²⁺ complex in a top (left) and side view (right). Secondary structure elements are indicated in one monomer. The C-terminal regions that become ordered upon ligand binding are in boldface type. The pre-V insert is highlighted in red. dU and PP_i are shown as sticks and indicated. The first and last residues of the pre-V insert (Tyr¹³⁴ and Asn¹⁵³, respectively), motif V, and the C-terminal residue (Asn¹⁷¹) are indicated.

In the closed conformation, the C-terminal tail of wDUT continues to reach the active site of its own subunit (Fig. 3) instead of reaching the third subunit as seen in classic dUTPases, supporting our previous hypothesis. This novel orientation is attributed to the chain reversal effect of the β-hairpin in the swapping region. The C-terminal tail covers a rather flat surface of the trimer body and exhibits a meandering conformation (Fig. 4A) and finally brings the catalytic motif V onto the active site. Most interactions between the tail and the trimer body take place in the active-site region, where the ligands interact extensively with both motif V in the C-terminal region and other motifs on the trimer body to stick the two parts together. The details of ligand-protein interactions will be discussed below. Serving as an anchoring point, Lys¹⁵⁰ forms hydrogen bonds with residues Pro²⁸ and Asp¹²⁶ from the neighboring subunit and somehow restricts the bending direction of the following C-terminal region. Mutation of Lys¹⁵⁰ to alanine or glutamate caused slight reduction of both the catalytic rate and the substrate-binding affinity (Table 1 and Fig. S6). In addition, the backbone oxygen of Asn¹⁵⁵ forms a hydrogen bond with the guanidinium group of Arg²⁴ (neighboring subunit); the backbone nitrogen of Ala¹⁵⁷ forms a hydrogen bond with the backbone oxygen of Ala²⁶ (neighboring subunit). Meanwhile, there are several water molecules located in a small pocket between the tail and the body (covered by Lys¹⁵⁰–Gln¹⁵⁶), contributing indirect hydrogen bonds to fix the tail as well. On the whole, the interaction between the C-terminal tail and the main body is rather weak, explaining the mobile nature of the C terminus.

Figure 4.

The C-terminal tail takes a novel path to reach the active site in the wDUT-dU-PP_i-Mg²⁺ structure. A, the conformation of the C-terminal tail (schematic) and its interactions with the trimer body (transparent surface). Amino acid residues and ligands are displayed as sticks, and their carbons are in subunit-based color and white, respectively. Water molecules are displayed as red balls. Hydrogen bonds are displayed as red dotted lines. The interactions mediated by the water molecules that are covered by Lys¹⁵⁰–Gln¹⁵⁶ are extensive but are not shown here for clarity. B, superposing wDUT and hDUT monomers shows divergence of their C-terminal regions. Residues at divergent points and motifs V are indicated. C, superposing wDUT and hDUT trimers reveals convergence of the latter parts of their C-terminal regions. For clarity, only the wDUT trimer is shown as a surface, and its bound ligands are shown as sticks to indicate the active-site position. Residues at the convergent point are indicated.

Table 1.

Enzyme kinetics data

Every data point was repeated three times, and S.E. was calculated by the fitting software. Relative efficiency is defined as enzyme efficiency (k_cat/K_m) divided by that of WT. ND, not determined.

Enzyme	kcat	Km	kcat/Km	Relative efficiency
	s−1	μm	m−1 s−1	%
wDUTWT	7.4 ± 0.2	1.7 ± 0.2	4.4 × 106	100
wDUTR24T	0.45 ± 0.02	2.9 ± 0.3	1.6 × 105	3.6
wDUTD34N	0.92 ± 0.06	13 ± 2	7.1 × 104	1.6
wDUTR71E	ND	ND	ND	ND
wDUTD88N	ND	ND	ND	ND
wDUTS115R	2.6 ± 0.1	4.5 ± 0.5	5.8 × 105	13
wDUTK150A	5.9 ± 0.1	2.5 ± 0.2	2.4 × 106	54
wDUTK150E	5.5 ± 0.1	2.6 ± 0.2	2.1 × 106	49
wDUTR158A	3.2 ± 0.1	2.6 ± 0.2	1.2 × 106	28
wDUTR158E	2.6 ± 0.1	2.6 ± 0.1	1.0 × 106	23
wDUTR158W	3.1 ± 0.1	3.2 ± 0.2	9.7 × 105	22
wDUTV160D	7.4 ± 0.2	2.0 ± 0.2	3.7 × 106	85
wDUTR161K	0.053 ± 0.002	5.4 ± 0.7	9.8 × 103	0.23
wDUTF166Y	1.2 ± 0.1	5.0 ± 0.2	2.4 × 105	5.5
wDUTD170G	6.2 ± 0.2	2.1 ± 0.3	3.0 × 106	68
wDUTD170L	0.53 ± 0.01	7.1 ± 0.4	7.5 × 104	1.7
wDUTR24T/S115R	0.51 ± 0.01	6.7 ± 0.6	7.6 × 104	1.8
wDUTR24T/S115R/D170G	3.1 ± 0.1	4.2 ± 0.4	7.4 × 105	17
wDUTΔ134−153	0.67 ± 0.01	21 ± 1	3.2 × 104	0.73
wDUTΔ161−170	ND	ND	ND	ND
wDUTΔ170−171	1.7 ± 0.1	2.8 ± 0.2	6.1 × 105	14
eDUTWT	9.3 ± 0.2	0.93 ± 0.11	1.0 × 107	100
eDUTR116S	2.7 ± 0.1	5.2 ± 0.7	5.2 × 105	5.2
eDUTT22R/R116S	3.3 ± 0.2	13 ± 2	2.5 × 105	2.5

On the other hand, several hydrogen bonds within the tail itself are important in maintaining the C-terminal conformation (Fig. 4A). Particularly interesting interactions are from Arg¹⁵⁸, which is located just before catalytic motif V. It forms hydrogen bonds with Val¹⁶⁰, Arg¹⁶¹, and Ser¹⁶⁸, introducing a bending conformation to correctly position motif V onto the active site (Fig. 4A). Mutation of Arg¹⁵⁸ to alanine, glutamate, or tryptophan removed these interactions and interfered with motif V positioning, reflected by an about 2.5-fold reduction of k_cat, whereas dUTP binding affinity in these mutants was slightly decreased (Table 1 and Fig. S6). As a negative control, mutation of Val¹⁶⁰, whose side chain points toward the solvent region, to an aspartate showed little effect on enzyme activity (Table 1 and Fig. S6). Based on these mutagenesis results, we conclude that the C-terminal conformation observed in the crystal structure is most likely physiologically relevant, and perturbation of the conformation by site mutation (outside the motif V region) reduces the enzyme activity to a certain extent.

Superposing monomers of wDUT and hDUT (with an RMSD of 1.14 Å for 123 aligned Cα atoms) clearly shows that their C-terminal tails divert into different orientations at a certain point on the swapped β-strand, which is residue Tyr¹³⁴ in wDUT or Gln¹⁴⁶ in hDUT (Fig. 4B). Whereas the C terminus of hDUT proceeds forward to wrap the third subunit, the C terminus of wDUT takes a go-and-return mode and reaches the subunit to which it belongs. Interestingly, superposing wDUT and hDUT trimers demonstrates that the latter portions of their C-terminal tails converge again after residue wDUT Val¹⁶⁰ or hDUT Glu¹⁵² (Fig. 4C). As a result, motif V at the C terminus caps the active site in essentially the same manner in both enzymes, independently of whether the C-terminal tail reaches the active site of its own subunit (in wDUT) or the third subunit (in hDUT).

wDUT employs noncanonical two-subunit active sites

The ordering of motif V in the complex structure enables us to see a complete active site, which is situated at the intersubunit cleft. Distinct from other dUTPases, the C-terminal region of wDUT is reversed by the β-hairpin in the swapping region and thus orients toward its own subunit. As a remarkable result, the active site of wDUT is actually formed by two subunits only: the catalytic motifs I, II, and IV from one subunit and motifs III and V from the other (Fig. 5A and Fig. S7). This is at variance with the well-accepted concept that the active site of trimeric dUTPase is formed by five conserved motifs from all of its three subunits (Fig. S7). Thus, this complex structure provides definite evidence for the presence of two-subunit active sites in trimeric dUTPases.

Figure 5.

The active site of wDUT shares structural similarity with that of other dUTPases. A, the active site of wDUT is composed of residues from two subunits. Amino acid residues are colored in a subunit-based manner, with the conserved motifs indicated in parentheses. The Mg²⁺ ion is shown as a green ball, and the water molecules are shown as red balls. Hydrogen bonds are indicated as red dotted lines. B, superimposition of the active sites of wDUT (green) and hDUT (wine). C, superimposition of the active sites of wDUT (green) and bDUT (magenta).

At the active site, we can see clear electron density for ligands. After careful inspection, we modeled this electron density as dU, PP_i, and Mg²⁺ (Fig. S4). The appearance of dU seemed confusing, because we co-crystallized the protein with dUTP. We found that wDUT cannot further hydrolyze the dUMP product to yield dU under standard assay conditions; however, a small amount of dU can be spontaneously formed from dUMP after a certain period of time, which may result from the instability of dUMP itself (Fig. S8). Thus, dU in the crystal seems to be a degradation product formed during the crystallization process. In the complex structure, the binding of ligands is not equivalent in all active sites. dU is found in all six active sites, PP_i in five active sites, and Mg²⁺ in four active sites that also contain dU and PP_i. In the following, we present one complete active site that contains all of the above-mentioned ligands to elucidate details of protein-ligand interactions.

dU is deeply buried in the active-site pocket (Fig. 5A and Fig. S9). The uracil ring is sandwiched between Ile⁸⁷ of motif III and the “Phe-lid” Phe¹⁶⁶ of motif V. It forms hydrogen bonds with the main-chain oxygen and nitrogen of Lys⁹⁶ and with a conserved structural water molecule, thereby mimicking Watson–Crick pairing. The deoxyribose moiety is stabilized in position by stacking with Tyr⁹¹, which is at the bottom of the pocket and can discriminate against ribose. The deoxyribose moiety also forms hydrogen bonds with the main-chain nitrogen of Asp⁸⁸, the side-chain hydroxyl of Ser⁷², and a water molecule.

The PP_i molecule roughly occupies the position where the β- and γ-phosphate groups of the dUTP substrate are located and extensively interacts with the residues at the active site (Fig. 5A and Fig. S9). Its first phosphate group forms hydrogen bonds with Ser⁷² of motif II and Gly⁷³. The second phosphate group forms extensive hydrogen bonds with the residues of motif V, including Gly¹⁶⁷, Ser¹⁶⁸, and Thr¹⁶⁹, as well as Arg¹⁶¹, which can act as an arginine finger to coordinate the nucleotide substrate (15). Both phosphates form hydrogen bonds with Arg⁷¹, which is supposed to play a key role in neutralizing the charge developed in the catalytic process. Moreover, two oxygens of PP_i and four water molecules coordinate a Mg²⁺ in an octahedral configuration. Via water-mediated hydrogen bonding, PP_i also interacts with several other residues at the active site, such as the catalytic residue Asp⁸⁸ of motif III and Asp³⁴, a residue important for maintaining the right interaction network at the active site (16).

Independently of the distinct manner of assembly, the active sites of wDUT and hDUT actually share high structural similarity (Fig. 5B). Superposing all five catalytic motifs gives an RMSD of 0.42 Å for all atoms, with small conformational alterations for individual residues. With respect to ligands, dU in wDUT is imposed well onto the uracil moiety of dUPNPP, whereas the PP_i occupies the corresponding position of the β- and γ-phosphate groups of dUPNPP. However, the Mg²⁺ positions are obviously different; in wDUT, it is coordinated by the PP_i group, whereas in hDUT, it is coordinated by all of the α-, β-, and γ-phosphates of dUPNPP. Accordingly, the water positions differ a lot. In fact, the binding of dU, PP_i, and Mg²⁺ was previously observed in the Bacillus subtilis dUTPase (bDUT; PDB code 4B0H), where these ligands together with AlF₃ were intentionally added to the crystallization buffer to mimic the transition state (14). When the active site of wDUT is superposed onto that of bDUT, the geometries of the dU, PP_i, Mg²⁺, and water molecules around them are almost identical (Fig. 5C), indicating that the conformation we see in the active site of wDUT is probably a mimic of the pseudo transition state, as described in the bDUT study (14).

To test the roles of individual active-site residues in catalysis, we carried out a mutagenesis study of wDUT (Table 1 and Fig. S6). Deletion of motif V (wDUT^Δ161–170) abolished the catalytic power, demonstrating its crucial role that has been well characterized in other dUTPases (31, 32). Mutating the putative catalytic residue Asp⁸⁸ to an asparagine, thereby removing its role as a general base in catalysis, also eliminated the enzyme's activity. Mutating Arg⁷¹, which is supposed to neutralize the negative charge developed in the catalytic process, to a glutamate diminished the enzymatic activity to an undetectable level. Replacing Arg¹⁶¹, which may function as an arginine finger to organize the active-site conformation, with a lysine caused an about 140-fold reduction of the catalytic rate. Change of the “Phe-lid” Phe¹⁶⁶ to tyrosine reduced the catalytic efficiency by 20-fold. Mutation of Asp³⁴ to asparagine deteriorated both the catalytic rate and dUTP binding to result in an about 50-fold reduction of catalytic efficiency, supporting the critical role of Asp³⁴ in maintaining a proper interaction network at the active site. Taken together, the mutagenesis results and the structural analysis support the critical roles of these residues in catalysis, as shown in previous studies, suggesting that wDUT shares a similar catalytic mechanism with the known trimeric dUTPases, despite using a different way to assemble the active site.

Residues in the second-shell region of the active site are reconfigured in wDUT as an adaption to its unique C-terminal orientation

In addition to the pre-V insert, sequence alignment revealed that a conserved arginine/lysine residue of motif IV (corresponding to Arg¹²⁸ in hDUT) in the known trimeric dUTPases is replaced by a serine in wDUT (residue Ser¹¹⁵) (Fig. 1). The role of this conserved arginine/lysine in dUTPase catalysis has not been well investigated. In the hDUT-dUPNPP structure (PDB code 2HQU), Arg¹²⁸ does not contact the substrate; instead, it forms two hydrogen bonds with Asp⁴⁹, a residue playing a key role in maintaining ordered interactions at the active site (16). It also forms a hydrogen bond with Thr¹⁶¹, contributing to the ordering of the C terminus (Fig. 6A). To test its role, we carried out a mutagenesis study (Table 1 and Fig. S6). Because hDUT was difficult to produce, it was only used for structural analysis, whereas eDUT was used for mutagenesis and enzyme kinetics. Mutation of this conserved arginine in eDUT (Arg¹¹⁶) to a serine reduces both the catalytic rate and binding affinity to cause a 20-fold reduction of catalytic efficiency, indicating that the hydrogen-bonding interactions provided by this arginine play a significant role in catalysis. This result also shows that not only a proper interaction network within the active site (16), but also that within the second shell of the active site, is important for dUTPase activity.

Figure 6.

Residues in the second-shell region of the active site are reconfigured in wDUT. A, the second-shell region around the conserved Arg¹²⁸ in hDUT. B, the second-shell region around Arg²⁴ in wDUT, in an aligned view of A. C–E, the conformations of Arg²⁴ in the free wDUT (C), wDUT^D88N/R158E-dUTP-Mg²⁺ (D), and wDUT-dU-PP_i-Mg²⁺ (E), shown in aligned views. The σA-weighted 2F_o − F_c electron density maps (contoured at 1σ level) are shown as gray mesh. For all panels, hydrogen bonds are shown as red dotted lines. Residues are in subunit-based colors. Ligands are shown as sticks.

Puzzlingly, whereas the eDUT^R116S mutant suffers from substantial activity loss, wDUT exhibits activity comparable with that of the wild-type eDUT (Table 1 and Fig. S6), even if the conserved arginine/lysine is also substituted by a serine. To address the discrepancy, we inspected the region around Ser¹¹⁵ in the wDUT complex structure and identified another arginine, residue Arg²⁴, which is located at a position in front of motif I and not conserved in dUTPases (its counterpart residue is Thr³⁹ in hDUT or Thr²² in eDUT) (Figs. 1 and 6B). Its guanidinium moiety sits at the corresponding position of the guanidinium moiety of hDUT Arg¹²⁸ and forms hydrogen bonds with Asp³⁴, whose orientation is supposed to be critical for enzyme activity (16). Arg²⁴ also interacts with the C-terminal Thr¹⁶⁹ through a water-mediated hydrogen bond. Moreover, Arg²⁴ forms a hydrogen bond with residue Asn¹⁵⁵, stabilizing the closed conformation of the C-terminal tail that takes a novel orientation. Mutating Arg²⁴ to threonine significantly reduced the catalytic rate and moderately decreased dUTP binding affinity to result in a 30-fold reduction of catalytic efficiency (Table 1 and Fig. S6), supporting its important role in catalysis. Therefore, Arg²⁴ subrogates the conserved arginine/lysine to maintain a proper interaction network in the second-shell region of wDUT (Fig. 6B). Why wDUT adopts an arginine configuration different from other dUTPase was unclear. We wondered whether this reconfiguration is related to the unusual structural organization of wDUT.

First, we checked whether wDUT can use a classic arginine configuration. We mutated Ser¹¹⁵ of wDUT to an arginine residue, as in other dUTPases. The mutant wDUT^S115R showed a catalytic efficiency of only about 13% of that of the wild type (Table 1 and Fig. S6). Probably, Arg¹¹⁵ is too close to Arg²⁴ in space, resulting in an improper hydrogen-bonding network and thus a rather low enzymatic activity. Accordingly, we prepared a double mutant, replacing Ser¹¹⁵ and Arg²⁴ of wDUT to an arginine and a threonine, respectively, thereby mimicking the residue combination in hDUT. Surprisingly, the catalytic efficiency of wDUT^R24T/S115R is even lower than that of the single mutant, only about 1% of that of the wild type (Table 1 and Fig. S6). Apparently, a proper interaction network was not built either in this double mutant. In the vicinity of Ser¹¹⁵ and Arg²⁴, wDUT contains residue Asp¹⁷⁰, which is usually a glycine residue at the same position in the known dUTPases (except for Mason–Pfizer monkey virus, where it is also an aspartate (Fig. 1)). In the wDUT complex structure, Asp¹⁷⁰ forms a salt bridge with Arg⁷¹ (Fig. 6B). Deleting Asp¹⁷⁰-Asn¹⁷¹ or mutating Asp¹⁷⁰ to leucine greatly decreased the catalytic efficiency, whereas mutating Asp¹⁷⁰ to the common glycine only slightly decreased enzyme activity (Table 1 and Fig. S6). In wDUT^R24T/S115R, Asp¹⁷⁰ could alternatively form a salt bridge with Arg¹¹⁵ to seriously disturb the interaction network, thereby leading to a very low enzymatic activity. Therefore, we prepared a triple mutant, wDUT^{R24T/S115R/D170G}, to mimic the classic dUTPase better. Its catalytic efficiency is about 20% of that of the wild type, much higher than that of the single or double mutant, suggesting that a relatively well-functioning interaction network in this region is restored. Nevertheless, the enzyme's power was not restored to the level of the wild type. Compared with the wild-type wDUT, the hydrogen bond between Arg²⁴ and Asn¹⁵⁵ (Fig. 4A), which facilitates the ordering of the C-terminal region, is missing in wDUT^{R24T/S115R/D170G}, probably explaining the inferior activity of the triple mutant. We conclude that the wDUT arginine configuration is superior to the classic arginine configuration for wDUT; besides maintaining a proper hydrogen-bonding network at the second shell of the active site, the wDUT arginine configuration can facilitate the ordering of the C-terminal region in its noncanonical orientation, which cannot be provided by the classic configuration.

Second, we checked whether the wDUT arginine configuration can be used by classic dUTPase. We prepared the eDUT^T22R/R116S mutant to mimic the wDUT arginine configuration. Surprisingly, this double mutant exhibited a catalytic efficiency of only 2.5% of that of the wild-type eDUT (Table 1 and Fig. S6). Apparently, Arg²² in eDUT^T22R/R116S is not capable of subrogating the conserved arginine Arg¹¹⁶ as wDUT Arg²⁴ does. Because we were not able to obtain a crystal structure of eDUT^T22R/R116S for a direct structural explanation, we attempted to find a clue from the wDUT structures. Whereas Arg²⁴ makes hydrogen bonds with Asp³⁴ and Thr¹⁶⁹ in the wDUT-dU-PP_i-Mg²⁺ structure (Fig. 6B), it is located at the surface of the protein in the ligand-free structure, showing various conformations in all monomers that are located in different crystallographic environments. In all cases, its side chain points away from the active position (Fig. 6C). Obviously, the interaction network in this region is not preformed in the ligand-free wDUT. To achieve full catalytic power, Arg²⁴ must be ordered to form a proper hydrogen-bonding network in the second-shell region. Is its ordering induced by ligand binding? Within our project, we also determined the structure of the wDUT^D88N/R158E-dUTP-Mg²⁺ complex, where dUTP clearly bound to the active site, but the C-terminal region was still disordered and not defined by electron density. In this structure, Arg²⁴ deviates from its proper position as well (Fig. 6D), suggesting that ligand binding cannot directly induce Arg²⁴ ordering. Indeed, it is the ordering of the C-terminal region upon ligand binding and its subsequent interaction with Arg²⁴ that are required for positioning Arg²⁴ into a functional state (Fig. 6E). In contrast, in eDUT^T22R/R116S, Arg²² could be always in a disordered state even if the C-terminal segment becomes ordered upon ligand binding, as the C-terminal segment takes the classic route and cannot interact with Arg²² to induce its ordering. This gives a plausible explanation for the low activity of eDUT^T22R/R116S. Therefore, whereas the wDUT arginine configuration is optimal in wDUT due to the novel C-terminal orientation, it is not a good option for classic dUTPases. From the above analysis, we conclude that reconfiguration of the residues in the second-shell region of the active site in wDUT facilitates the ordering of its C-terminal region in the novel orientation, which in turn promotes a proper interaction network in the second-shell region.

Deletion of the pre-V insert significantly reduces the enzymatic activity and thermal stability of wDUT

From the above analysis, the pre-V insert (¹³⁴YINETTGERTIIDSSSKKDN¹⁵³; Fig. 1) is the key factor to endow wDUT with a new structural form. We wondered whether deletion of this extra sequence could convert wDUT into a classic form. We prepared a truncated version, wDUT^Δ134–153, which was supposed to take a classic form, restricted by the length of the C terminus. However, its enzymatic efficiency was extremely low, less than 1% of that of wild type (Table 1 and Fig. S6). In this respect, the pre-V insert is different from the known motifs VI, which are dispensable for the dUTPase activity (22, 25). As shown above, the residue configuration in the second shell also plays a significant role in enzyme activity. We propose that the enzymatic activity might be improved by introducing the classic arginine configuration into wDUT^Δ134–153. Unfortunately, we could not test this hypothesis, because the mutant protein could not be produced in a soluble form. Meanwhile, we also monitored the thermal stability of wDUT^Δ134–153. Its T_m values were about 29 and 23 °C lower than that of wild type, or about 19 and 15 °C lower than that of eDUT, estimated from the Thermofluor assay and the thermal inactivation assay, respectively (Fig. S10). The reduced thermal stability may be partially responsible for the low activity of wDUT^Δ134–153 as well. To convert wDUT into a well-functioning classic form, more amino acid substitutions should be introduced to counteract the adverse effect of the pre-V deletion on both enzyme activity and thermal stability.

Discussion

In this report, we present the crystal structures of wDUT in both ligand-free and ligand-bound forms. wDUT shares a similar structure fold with the known trimeric dUTPases, but its C-terminal segment takes an alternative orientation, which is largely attributed to a sequence insert, called the pre-V insert in this study. This insert forms a β-hairpin reversing the C-terminal orientation and resulting in a noncanonical two-subunit active site in wDUT, in contrast to the three-subunit active site in classic dUTPases (Fig. S7). Despite the unusual structural organization, wDUT has a local structure at the active site similar to that of the classic dUTPases, implicating a similar catalytic mechanism. Nevertheless, wDUT reconfigures the residues in the second-shell region of the active site as an adaption to its novel C-terminal orientation. We also show that deletion of the pre-V insert solely is not able to convert wDUT back to a fully functional classic form. It is worthy of mention that the full-length dUTPase of WSSV contains a 290-residue C-terminal tail continuing motif V. This tail may not change the essential structural features of the dUTPase domain as observed in this study; however, it can modulate the dUTPase activity via affecting motif V organization.

Sequence insertion and deletion (indel), along with amino acid substitution, is a major way for protein evolution, even more powerful than the latter, to achieve novel structure and function (33, 34). Recent studies show that a sequence insert, often named motif VI, is an important factor in acquiring new functions in the dUTPase family (24). This extra motif can be located at the N-terminal, the middle, or the C-terminal position of dUTPase. wDUT is unique by harboring a 20-residue insert preceding the C-terminal motif V, the pre-V insert. Although a five-residue insert at a similar position is reported in Mycobacterium tuberculosis dUTPase (25), such a “long” insert is absent in any other trimeric dUTPases. This pre-V insert can be regarded as a motif VI of wDUT; however, it is distinct from other known motifs VI by having an obvious structural role. It forms a β-hairpin, dramatically affecting the orientation of the following C-terminal region and resulting in a two-subunit active site in wDUT. In general, most inserts in proteins just loop out in situ with limited local structure alteration. Only in few characterized structures can a sequence insert cause dramatic changes in the neighboring region to obviously drive protein evolution (35). An outstanding example is found for myosin VI. Compared with other myosin molecules, myosin VI contains an extra sequence insertion forming a helix, which changes the direction of the following level arm and introduces an unusual backstroke function (36). Our study adds another showcase, being the first report that a short sequence insert plays a role in reorganizing the active site.

A remarkable discovery of this study is that wDUT adopts a noncanonical two-subunit active site. The trimeric dUTPases have been intensively studied for decades, and it is widely accepted that their active sites are formed by all three subunits. It is therefore surprising to find an exception in wDUT. Indeed, the isolated dUTPase domain of Mason–Pfizer monkey virus (MPMV) was suggested to be an exception to the rule as well (37). The initial orientation of the C-terminal region observed in the MPMV dUTPase structure and a calculation suggested that this dUTPase uses a two-subunit active site. However, the complete active site was not shown, because motif V is disordered even in the ligand-bound crystal structure. The hypothesis was supported by molecular dynamics and other experiments. Here we present definite structural evidence to support the presence of a two-subunit active site in the trimeric dUTPase family. Notably, the structural solutions to form a two-subunit active site are distinct in WSSV and MPMV dUTPases. In MPMV dUTPase, it results from a shortage of residues between motif IV and V and a lack of the N-terminal β-strand that is necessary for domain swapping (Fig. S11A). By contrast, in wDUT, it is attributed to a sequence insert, which forms a β-hairpin to reverse the following C terminus (Fig. S11B). Comparison of the two structures side by side shows that the missing C-terminal region of MPMV dUTPase might take a similar route as in wDUT to reach the active site (Fig. S11). For the moment, the two-subunit active site form seems a rare variation in the dUTPase family. However, this form may become more populated when more dUTPase sequences and structures are studied.

How nature evolves such a new structural form is suggested in our study. Despite using distinct solutions to organize their active sites, wDUT and the classic dUTPases share similar overall and active-site structures, suggesting that the former evolved from the latter. Obviously, the pre-V insert plays a key role in the evolution, by forming the β-hairpin to reverse the C-terminal orientation, finally leading to a two-subunit active site. Besides the sequence insert, we found that a conserved arginine/lysine located in motif IV of the classic dUTPases is functionally substituted by another arginine (residue Arg²⁴) in wDUT (Figs. 1 and 6). We show that such a reconfiguration in the second-shell region of the active site is an adaption to the novel C-terminal orientation of wDUT and required for maintaining full enzyme activity. Eventually, the evolution of wDUT should be considered the combined result of sequence insertion and amino acid substitution.

Why wDUT adopts this unusual structural form is an interesting question. The catalytic power of wDUT is comparable with that of the traditional members of the dUTPase family. Apparently, the new structural form does not constitute a benefit for the virus regarding the dUTPase activity. Compared with the prototypic hDUT, wDUT contains a long β-hairpin, largely derived from its extra pre-V insert. It protrudes from the trimeric body as a bulge (Bulge 1 in Fig. 7A) and can be an excellent structural element to mediate intermolecular interactions (38). Furthermore, in the vicinity, there is another bulge formed by residues Pro¹⁴–Glu¹⁷ (Bulge 2 in Fig. 7A), which is also not commonly seen in other dUTPase structures. These two bulges together form an extended surface with negative charges (Fig. 7B), serving as a potential region to mediate intermolecular interaction. Interestingly, a recent study showed that pseudorabies virus UL50, a monomeric family dUTPase, can inhibit IFNα signaling independent of its enzymatic activity. In this protein, a sequence insert of about 30 residues at a position similar to that of the pre-V insert plays a key role (39). Analogously, by harboring the extra pre-V insert, wDUT might benefit the virus in certain aspects beyond its dUTPase activity. Nevertheless, the biological meaning of this new form remains to be investigated.

Figure 7.

Structural features of wDUT. A, superposition of the trimers of wDUT and hDUT reveals extra bulges in wDUT. Only one pair of bulges is indicated. B, charge distribution on the surface of wDUT. The positive, negative, and neutrally charged residues are colored blue, red, and white, respectively.

Last but not least, our structures would facilitate structure-based antiviral design against WSSV. WSSV is a serious pathogen in shrimp aquaculture, causing $1 billion economic loss every year since its emergence more than 20 years ago (28). Until now, there has been no efficient treatment available, and development of a suitable antiviral drug is an urgent task. A previous study shows that WSSV dUTPase can be related to virus replication and thus serve as a potential antiviral target (30). Our study shows that the active site of wDUT shares high similarity with those of known dUTPases, indicating that inhibitors designed to target the active site of general dUTPases should be applicable for the inhibition of wDUT as well. Notably, such inhibitors could have high cytosolic toxicity, by acting on host dUTPase. On the other hand, unique structural features of wDUT can be explored to design specific inhibitors. For example, the C-terminal tail of wDUT binds the trimer body at a surface completely different from that of classic dUTPases; several small pockets present at this surface can serve as potential “hot spots” for drug design.

In summary, our study discovers a novel structural form of dUTPase and demonstrates how sequence insertion and amino acid substitution in concert drive protein evolution. The structural information is valuable for antiviral drug design against the white spot syndrome virus.

Experimental procedures

Gene cloning and protein production

The DNA sequence encoding wDUT (comprising the N-terminal 171 residues of WSV112) was amplified by PCR with primers 5′-TACTTCCAATCCAATGCCATGGACTCATCTGCATCTGTC-3′ (forward) and 5′- TTATCCACTTCCAATGCTATCAGTTATCTGTAGATCCAAAT-3′ (reverse), using the WSSV genomic DNA as template. The PCR product was inserted into a modified pET30 vector using a ligation-independent cloning protocol (40). The DNA encoding eDUT was amplified by PCR with primers 5′-CATGCCATGGCGATGAAAAAAATCGACGTTAAG-3′ (forward) and 5′-CCGCTCGAGTCACTGACGACCAGAGTGACCAAA-3′ (reverse), using E. coli (K12-MG1655) genomic DNA as template. The PCR product was inserted into vector pETM11 (EMBL) using NcoI and XhoI sites. Site-directed mutagenesis of wDUT or eDUT was performed using the QuikChange method (Stratagene). All recombinant proteins were expected to contain an N-terminal His₆ tag cleavable by tobacco etch virus protease (TEV), and the final protein products carry a few residues derived from the vector sequence (SNA for wDUT/mutants or GAMA for eDUT/mutants). Similar protocols were used for protein production, as described below.

The sequence-verified gene construct was transformed into the E. coli strain Rossetta (DE3). The culture was grown in Luria–Bertani medium with vigorous shaking at 37 °C until A₆₀₀ reached 0.8, and then 0.2 mm isopropyl β-d-l-thiogalactopyranoside was added to induce protein overexpression, with further incubation at 16 °C for 16 h. Cells were harvested by centrifugation and lysed by sonication in buffer containing 50 mm Tris-HCl, pH 8.0, 500 mm NaCl. The lysate was clarified by centrifugation, and the supernatant was applied onto a nickel-chelating Sepharose affinity chromatography column (GE Healthcare). After washing with washing buffer (50 mm Tris-HCl, pH 8.0, 500 mm NaCl, 10 mm imidazole), the protein was eluted with elution buffer (50 mm Tris-HCl, pH 8.0, 500 mm NaCl, 500 mm imidazole). Then the protein solution was changed to 50 mm Tris-HCl, pH 8.0, 500 mm NaCl using a PD-10 column (GE Healthcare) for TEV digestion. The TEV-digested protein was reloaded onto the nickel column to remove the uncleaved protein and TEV. The flow-through containing untagged protein was collected, concentrated, and finally purified with size-exclusion chromatography using a HiLoad 16/60 Superdex 200 column (GE Healthcare) equilibrated in buffer that was optimized according to protein usage. For protein that was used for crystallization, 20 mm Tris-HCl, pH 8.0, 500 mm NaCl, and 1 mm DTT was applied to wDUT, whereas 20 mm Tris-HCl, pH 8.0, 150 mm NaCl, and 1 mm DTT was applied to wDUT^D88N/R158E. When protein was used for the enzymatic assay, the buffer was 10 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm MgCl₂, 1 mm DTT. The purified protein was concentrated to about 10 mg/ml, as determined by absorbance at 280 nm. Protein was either used freshly or frozen at −80 °C for later use.

Crystallization and structure determination

Crystallization was conducted with the sitting-drop vapor-diffusion method at 20 °C. Crystals of free wDUT were grown in drops containing 1 μl of protein solution (6.7 mg/ml in a buffer containing 20 mm Tris-HCl, pH 8.0, 500 mm NaCl, and 1 mm DTT) and 1 μl of reservoir solution containing 30% (w/v) PEG 1500. Crystals of the wDUT-dU-PP_i-Mg²⁺ complex were grown in drops containing 1 μl of protein solution (10 mg/ml protein + 10 mm dUTP + 8 mm MgCl₂ incubated for 1 h at 4 °C) and 1 μl of reservoir solution (0.2 m sodium acetate, 0.1 m Tris-HCl, pH 8.1, 28% (w/v) PEG 3350). The solution (0.2 m sodium acetate, 0.1 m Tris-HCl, pH 8.1, 35% (w/v) PEG 3350) was used as cryoprotectant. Crystals of wDUT^D88N/R158E-dUTP-Mg²⁺ were grown in a drop containing 1 μl of protein solution (10 mg/ml protein + 10 mm dUTP + 8 mm MgCl₂ incubated for 1 h at 4 °C) and 1 μl of reservoir solution of 25% (w/v) PEG 1500. 35% (w/v) PEG 1500 was used as cryoprotectant.

Crystals were frozen by plunging into liquid nitrogen. The diffraction data were collected at 100 K on beamline BL17U1 (41) at the Shanghai Synchrotron Radiation Facility and were processed using autoPROC (42, 43) or XDS (44).

The ligand-free structure of wDUT was solved by molecular replacement using the Phaser program (45), with the trimeric dUTPase of chlorella virus (PDB code 3C3I) as a search model (46). Structure refinement of atomic coordinates, B-factors, and TLS parameters with Refmac5 (47) or autoBUSTER (48) and model building with Coot (49) were carried out alternatively. For the ligand-bound forms, the structures were solved by molecular replacement, using the refined ligand-free structure as a search model. Ligands were modeled at late stage of refinement, and the geometry restraints of ligands were generated by the GRADE server (http://grade.globalphasing.org).³ Refinement and model building were similar to that for the ligand-free structure. Crystallographic statistics are summarized in Table S1.

Sequence alignment was performed on the ESPript server (50). Protein-protein interactions were analyzed with PISA (51), whereas protein-ligand interactions were analyzed with LigPlot+ (52). The MolProbity server (53) and other CCP4 programs (54) were used for model analysis as well. Structural alignments were performed with Align (55). If not specified, ligand-bound DUTs that have an ordered C terminus are used in structural comparisons. Figures for structures were prepared using PyMOL (Schrödinger, LLC).

Enzyme kinetics measurement

The enzyme activities of dUTPases and their mutants were measured by a phenol red indicator assay (56), in which protons released in the dUTPase reaction were detected. The measurements were performed in 1 mm HEPES, pH 7.5, buffer containing 150 mm KCl, 40 μm phenol red, 5 mm MgCl₂, and 1–120 μm dUTP on a UV-1801 spectrophotometer (Rayleigh) with 10-mm path length cuvettes at 20 °C. Absorbance was recorded at 559 nm. The Michaelis–Menten equation was fitted to the steady-state curves using Origin version 8.0 (OriginLab Corp.).

HPLC assay

HPLC analysis of the dUTPase reaction products was performed on a Wufeng HPLC system equipped with a 5-μm WondaCract ODS-2 C18 column (GL Sciences) and a Wonda guard column (GL Sciences). Reactions were started by adding 25 mm enzyme (final concentration) into reaction buffer (10 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm MgCl₂, 1 mm DTT, 1 mm dUTP) with a final volume of 0.2 ml. After incubation at 20 °C for 30 min, the reaction solution was cleared by centrifugation at 13,800 × g for 5 min at 4 °C, and 20 μl of supernatant was analyzed on the HPLC system using 0.1 m KH₂PO₄ (pH 6.0) and 12% (v/v) methanol as mobile phase at a flow rate of 0.4 ml/min. The run was performed isocratically for 25 min at 20 °C, and the eluent was monitored by absorbance at 254 nm. dUTP, dUMP, and dU (all from Sigma) were used as controls, where no enzyme was added. To test dUMP stability, 1 mm dUMP was incubated in 0.2 ml of reaction buffer at 20 °C for 24 h.

Thermal stability assays

Thermofluor assay

The Thermofluor assay (57) was performed on a quantitative PCR instrument (CFX96, Bio-Rad). The fluorescence (excitation wavelength at 498 nm and emission wavelength at 610 nm) of the protein solution (100 μm protein in 10 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm MgCl₂, 1 mm DTT, plus 10× SYPRO Orange dye (Sigma) in a final volume of 25 μl) was measured as a function of temperature at a climbing rate of 1 °C/min from 25 to 95 °C. The data were analyzed using the Boltzmann equation in Origin version 8.0 (OriginLab Corp.).

Thermal inactivation assay

The thermal inactivation assay was conducted in the following manner. Protein samples were incubated for 10 min in buffer (10 mm HEPES, pH 7.5, 150 mm NaCl, 5 mm MgCl₂, 1 mm DTT) at various temperatures (37.5–80 °C) in a PCR instrument (Thermal cycler 2720, Applied Biosystems). After being cooled on ice for 5 min and centrifuged at 13,800 × g for 5 min at 4 °C, enzyme activity was measured at 20 °C with the phenol red indicator assay to acquire the initial velocity.

Accession numbers

The coordinates and diffraction data of free wDUT, wDUT-dU-PP_i-Mg²⁺, and wDUT^D88N/R158E-dUTP-Mg²⁺ have been deposited in the PDB with accession numbers 5Y5O, 5Y5P, and 5Y5Q, respectively.

Author contributions

K. Z. and Q. M. designed, performed, and analyzed the experiments and wrote the paper. F. L. contributed new reagent. Q. M. conceived and supervised the study. All authors reviewed the results and approved the final version of the manuscript.