The Flexible Motif V of Epstein-Barr Virus Deoxyuridine 5′-Triphosphate Pyrophosphatase Is Essential for Catalysis

Freeman, Lucy; Buisson, Marlyse; Tarbouriech, Nicolas; Heyden, Angéline Van der; Labbé, Pierre; Burmeister, W.P.

doi:10.1074/jbc.m109.019315

Cited by 30 publications

(39 citation statements)

References 32 publications

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“…The measured binding affinities and catalytic rates of the T138Stop and H145A enzymes are consistent with the essential role of the C-terminal arm in other dUTPase proteins investigated. 17,25,[27][28][29][30][31][32] To better understand the underlying energetic and structural causes determining the catalytic activity, we carried out QM/MM calculations of the reaction mechanism for proton transfer and phosphate cleavage in the WT and the two mutant (H145A and T138Stop) enzyme complexes. We recently identified a one-step associative A N D N catalytic mechanism for the hydrolysis at the α-phosphate, involving also a coupled proton transfer step from the nucleophilic water to the catalytic Asp83 residue.…”

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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“…Surprisingly, the active site architecture with bound substrate in the crystal structure of the C-terminally truncated inactive enzyme is identical to that of the wild-type (WT) (7). On the basis of activity measurements on full length and enzymatically truncated Escherichia coli enzymes, it was concluded that ordering of the flexible C terminus upon the active site, which occurs only in the presence of the gamma phosphate (γ-P) containing substrate analog dUPNPP is responsible for dUDP/dUTP discrimination (3,5).…”

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confidence: 97%

“…The sequence of the C-terminal motif V shares a limited number of features with those of the P-loop motifs (Fig. 1A) (3, 4) present in a large number of ATPase and GTPase enzyme families known as P-loop NTPases including kinases, cytoskeleton and DNA motors, membrane pumps, and transporters.It has been shown that the C terminus of dUTPase is necessary for dUTP hydrolysis but not for nucleotide binding or structural integrity (3,(5)(6)(7). Surprisingly, the active site architecture with bound substrate in the crystal structure of the C-terminally truncated inactive enzyme is identical to that of the wild-type (WT) (7).…”

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confidence: 99%

“…It has been shown that the C terminus of dUTPase is necessary for dUTP hydrolysis but not for nucleotide binding or structural integrity (3,(5)(6)(7). Surprisingly, the active site architecture with bound substrate in the crystal structure of the C-terminally truncated inactive enzyme is identical to that of the wild-type (WT) (7).…”

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confidence: 99%

“…S1B). The mutations were intended to partially (Ser160Ala/Thr161Ala termed hDUT ST∕AA , and Arg153Lys termed hDUT R∕K ) or completely (Thr151Stop termed hDUT armless ) disrupt the secondary interaction network between the γ-P and the protein as also shown in an earlier work by Freeman et al (7). These mutant enzymes were then subjected to kinetic and ligand binding analysis.…”

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confidence: 99%

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Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination

Pecsi

Szabó

Adams

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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We investigated the potential (d)NDP/(d)NTP discrimination mechanisms in nucleotide pyrophosphatases. Here, we report that dUTPase, an essential nucleotide pyrophosphatase, uses a C-terminal P-loop-like sequence in a unique mechanism for substrate discrimination and efficient hydrolysis. Our spectroscopy and transient kinetics results on human dUTPase mutants combined with previous structural studies indicate that (i) H-bond interactions between the γ-phosphate and the P-loop-like motif V promote the catalytically competent conformation of the reaction center at the α-phosphate group; (ii) these interactions accelerate the chemical step of the kinetic cycle and that (iii) hydrolysis occurs very slowly or not at all in the absence of the γ-phosphate-motif V interactions, i.e., in dUDP, dUDP.BeFx, or in the motif V-deleted mutant. The physiological role of dUTPase is to set cellular dUTP∶dTTP ratios and prevent injurious uracil incorporation into DNA. Based upon comparison with related pyrophosphate generating (d)NTPases, we propose that the unusual use of a P-loop-like motif enables dUTPases to achieve efficient catalysis of dUTP hydrolysis and efficient discrimination against dUDP at the same time. These specifics might have been advantageous on the appearance of uracil-DNA repair. The similarities and differences between dUTPase motif V and the P-loop (or Walker A sequence) commonly featured by ATP-and GTPases offer insight into functional adaptation to various nucleotide hydrolysis tasks.NTP hydrolysis | nucleotide discrimination | dUTP pyrophosphatase | Walker A motif | evolutionary adaptation I t is an intriguing question how pyrophosphate generating nucleotide hydrolases distinguish between (d)NDP and (d)NTP both containing the α-β phosphoanhydride bond to be hydrolyzed. In the present paper, we investigate two fundamental questions related to the nucleotide pyrophosphatase enzymatic activity exhibited by the enzyme dUTPase and by other pyrophosphatases: (i) the mechanism of discrimination between nucleoside di-and triphosphate ligands; (ii) the potential contribution of a P-loop-like motif to such discrimination. The enzyme dUTPase naturally evoked these questions as it specifically performs the hydrolysis of dUTP between the α-β phosphates with no further coupled reactions and it contains a P-loop-like motif (Fig. 1A). In addition, the structural comparison between Mg:dUDP-and Mg:dUTP analog-bound enzymes does not offer a straightforward explanation to why dUDP is not hydrolyzed because the scissile bond and the nucleophile adopt the same conformation in both (Fig. 1B and in stereo in Fig. S1A, the sole Mg:dUDPcomplexed structure is superposed with a human Mg:dUPNPP complex).dUTPase hydrolyzes dUTP to yield dUMP (a precursor for dTTP biosynthesis) and pyrophosphate (PP i ). The action of dUTPase is the only known direct mechanism to minimize uracil incorporation into DNA (1). Most dUTPases are homotrimers and confer three active sites. The substrate in each active site is bound by conserved sequence motif...

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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 Flexible Motif V of Epstein-Barr Virus Deoxyuridine 5′-Triphosphate Pyrophosphatase Is Essential for Catalysis

Cited by 30 publications

References 32 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

Nucleotide pyrophosphatase employs a P-loop-like motif to enhance catalytic power and NDP/NTP discrimination

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

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