The Monomeric dUTPase from Epstein-Barr Virus Mimics Trimeric dUTPases

Tarbouriech, Nicolas; Buisson, Marlyse; Seigneurin, Jean‐Marie; Cusack, Stephen; Burmeister, W.P.

doi:10.1016/j.str.2005.06.009

Cited by 53 publications

(74 citation statements)

References 52 publications

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“…1 An additional enzymatic activity, deamination of dCTP (deoxycytidine triphosphate) to dUTP, may also be carried out by bifunctional dUTPases in Archaea [2][3][4][5] and also in Mycobacterium tuberculosis (M. tuberculosis), which also has a monofunctional dUTPase. 6 The dUTPases comprise two structurally different classes: 7 first, the β sheet−type dUTPases, possessed by most organisms, employ a one-metal ion catalytic mechanism, [8][9] and usually form a homotrimer 10 or a monomer, 11 while the second class, the α helical-type dUTPases, possessed by kinetoplastids, 12 form dimers and employ a two-metal ion catalytic mechanism. [13][14] In β sheet-type trimeric dUTPases, each active site is constituted from motifs of all three protomers, which are all required to interact for achieving proper enzymatic activity.…”

Section: Introductionmentioning

confidence: 99%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

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Most enzymes present a catalytic mechanism where one or more proton transfer events occur coupled tightly together with the enzymatic chemical reaction. We show here that inactivating mutations decouple this proton transfer step from the phosphate cleavage reaction in dUTPase. Homotrimeric dUTPase enzymes catalyze the hydrolysis of dUTP to dUMP and pyrophosphate, using largely similar structural and functional groups as most AAA+ enzymes. dUTPases typically use a single Mg 2+ ion as a cofactor in the active site that is formed by direct protein-protein contacts including all three protomers. Here we focus on the C-terminal arm structural motif, which has sequence and functional similarities to P-loop motifs, and is required for catalysis. In this work, we have studied the functional roles of the C-terminal arm in ligand binding and catalysis by using QM/MM (quantum mechanics/molecular mechanics) calculations in conjunction with site-directed mutagenesis experiments. We also present a new method to assess the metal ion coordination symmetry during the catalytic reaction. Using this new implementation, we identified that the coordination symmetry follows a consistent pattern in the three systems studied, reaching the most symmetrical state near the transition states. We found that the phosphate cleavage proceeds with a concerted bimolecular (A N D N ) mechanism with a loose dissociative transition state, and that it is coupled with a proton transfer step involving a unanimously conserved Asp residue. We show that the main mechanistic effect of the lack of the C-terminal arm is to decouple the phosphate cleavage from the subsequent proton transfer step, resulting in a highbarrier altered reaction pathway.

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Section: Introductionmentioning

confidence: 99%

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

et al. 2015

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“…Following the presumed gene duplication event, numerous mutations occurred rendering the two copies highly dissimilar. Interestingly, in this protein, the dUTPase fold is still preserved and the active site architecture is also very reminiscent of the situation found in trimeric dUTPases [40,45]. Yet, structural investigation of Epsein-Barr virus dUTPase enabled the hypothesis suggesting a unique conformational dynamics of the C-terminal arm of monomeric dUTPases, with the involvement of a disulfide bond that limits movements of this segment [40].…”

Section: All-b Dutpasementioning

confidence: 87%

“…[100,102] sequence conservation. Although human and EpsteinBarr viral dUTPases show only 19% identity [30,39,40], the same fold is displayed by the protein.…”

Section: All-b Dutpasementioning

confidence: 94%

“…The interest in these enzymes is highly motivated on the one hand by the peculiar active site architecture and, on the other hand, by the possibility of using these dUTPases as targets for developing drugs against neoplastic, as well as infectious diseases [31,[56][57][58][59][60][61][62][63][64][65] [100]. All-b dUTPases also include monomeric dUTPases [40]. recognition and sharp distinction at both the sugar and at the base level is ensured not only by the side chain of residues but, very importantly, by the protein backbone.…”

Section: All-b Dutpasementioning

confidence: 99%

“…In all cases where data are available, there is clear evidence for a mechanism initiated by nucleophilic attack on the a-phosphorus atom by a water or hydroxide molecule that is accommodated by the strictly conserved aspartate residue in motif 3 (Figs 2 and 3A) [31,39,40,52,66,67]. The other coordinating partners of the nucleophilic catalytic water are usually some other water molecules that are held in place by additional conserved residues.…”

Section: All-b Dutpasementioning

confidence: 99%

See 2 more Smart Citations

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool

2014

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The occurrence of modified bases in DNA is attributed to some major factors: incorporation of altered nucleotide building blocks and chemical reactions or radiation effects on bases within the DNA structure. Several enzyme families are involved in preventing the incorporation of noncanonical bases playing a 'sanitizing' role. The catalytic mechanism of action of these enzymes has been revealed for a number of representatives in clear structural and kinetic detail. In this review, we focus in detail on those examples where clear evidence has been produced using high-resolution structural studies. Comparing the protein fold and architecture of the enzyme active sites, two main classes of sanitizing deoxyribonucleoside triphosphate pyrophosphatases can be assigned that are distinguished by the site of nucleophilic attack. In enzymes associated with attack at the a-phosphorus, it is shown that coordination of the c-phosphate group is also ensured by multiple interactions. By contrast, enzymes catalyzing attack at the b-phosphorus atom mainly coordinate the a-and the b-phosphate only. Characteristic differences are also observed with respect to the role of the metal ion cofactor (Mg 2+ ) and the coordination of nucleophilic water. Using different catalytic mechanisms embedded in different protein folds, these enzymes present a clear example of convergent evolution.

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Targeting Epstein‐Barr Virus dUTPase, an Immunomodulatory Protein Using Antiviral, Anti‐Inflammatory, and Neuroprotective Phytochemicals

Tiwari

Murmu

Indari

et al. 2022

Chemistry & Biodiversity

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Although primary infection of Epstein-Barr virus is generally non-lethal, viral reactivation is often associated with fatal outcomes. Regardless, there is no FDA-approved treatment available for this omnipresent viral infection. The current investigation targets viral maintenance and reactivation by inhibiting the functioning of viral deoxyuridine-triphosphatase (dUTPase) using phytochemicals. The EBV-dUTPase is essential for maintaining nucleotide balance and thus, plays a vital role in the viral replication cycle. Additionally, the protein has shown neuroinflammatory effects on the host. To selectively target the protein and possibly alter its activity, we utilized a virtual screening approach and screened 45 phytochemicals reported to have antiviral, anti-inflammatory, and neuroprotective properties. The analysis revealed several phytochemicals bound to the target protein with high affinity. In-silico ADMET and Lipinski's rule analysis predicted favorable druggability of Dehydroevodiamine (DHE) among all the phytochemicals. Further, we corroborated our findings by molecular dynamic simulation and binding affinity estimation. Our outcomes ascertained a stable binding of DHE to EBV-dUTPase primarily through electrostatic interactions. We identified that the protein-ligand binding involves the region around His71, previously reported as a potent drug target site. Conclusively, the phytochemical DHE showed a promising future as a drug development candidate against EBV-dUTPase.

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The Monomeric dUTPase from Epstein-Barr Virus Mimics Trimeric dUTPases

Cited by 53 publications

References 52 publications

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

Preventive DNA repair by sanitizing the cellular (deoxy)nucleoside triphosphate pool

Targeting Epstein‐Barr Virus dUTPase, an Immunomodulatory Protein Using Antiviral, Anti‐Inflammatory, and Neuroprotective Phytochemicals

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