Kinetic properties and inhibition of the dimeric dUTPase‐dUDPase from <i>Leishmania major</i>

Hidalgo-Zarco, Fernando; Camacho, Ana; Bernier-Villamor, Vı́ctor; Nord, Johan; Ruíz-Pérez, Luis M.; González-Pacanowska, Dolores

doi:10.1110/ps.48801

Cited by 37 publications

(24 citation statements)

References 27 publications

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“…The bifunctional dUDP/dUTPase enzymes generate dUMP from both dUDP and dUTP with comparable k cat ∕K M values in the order of 10 6 s −1 M −1 by a mechanism similar to that of DNA polymerases (28,29). We analyzed the available structures of these enzymes and discovered that the γ-P of bound dUTP is only coordinated by water molecules and is largely exposed to the bulk solvent ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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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Section: Resultsmentioning

confidence: 99%

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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“…[18][19][20] In addition to the key C-terminal arm, the requirement of divalent metal ion cofactors for efficient catalysis is also shared by AAA+ (ATPases associated with diverse cellular activities) enzymes in general, commonly a single Mg 2+ coordinating the phosphate groups of the substrate. Substitution of this Mg 2+ with Ca 2+ generally results in substantial loss of activity, [21][22] with only a few exceptions reported (e.g., for human endogenous retrovirus (HERV-K) dUTPases). 23 The P loop-like C-terminal arms, the Mg 2+ ion bound triphosphate nucleotides, and the binding interface of the remaining two protomers, − all contribute to the resulting three active sites of the native dUTPase complex.…”

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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“…These enzymes act as dimers and are able to hydrolyze not only dUTP but also dUDP (15), whereas dUDP has been shown to be an inhibitor of the trimeric enzyme in E. coli (16). They are, in addition, subject to product inhibition by dUMP (17).…”

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confidence: 99%

The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine

Hemsworth

Moroz

Fogg

et al. 2011

Journal of Biological Chemistry

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Members of the Leishmania genus are the causative agents of the life-threatening disease leishmaniasis. New drugs are being sought due to increasing resistance and adverse side effects with current treatments. The knowledge that dUTPase is an essential enzyme and that the all ␣-helical dimeric kinetoplastid dUTPases have completely different structures compared with the trimeric ␤-sheet type dUTPase possessed by most organisms, including humans, make the dimeric enzymes attractive drug targets. Here, we present crystal structures of the Leishmania major dUTPase in complex with substrate analogues, the product dUMP and a substrate fragment, and of the homologous Campylobacter jejuni dUTPase in complex with a triphosphate substrate analogue. The metal-binding properties of both enzymes are shown to be dependent upon the ligand identity, a previously unseen characteristic of this family. Furthermore, structures of the Leishmania enzyme in the presence of dUMP and deoxyuridine coupled with tryptophan fluorescence quenching indicate that occupation of the phosphate binding region is essential for induction of the closed conformation and hence for substrate binding. These findings will aid in the development of dUTPase inhibitors as potential new lead antitrypanosomal compounds.Various members of the genus Leishmania cause leishmaniasis, which threatens ϳ350 million people worldwide and gives rise to about two million clinical cases each year, of which ϳ25% are of the fatal visceral form (1). The disease is largely endemic to developing countries, and current treatments are expensive and can result in undesirable side effects for the patient (1). This, combined with the increasing drug resistance that is developing in the Leishmania species, means that new and novel anti-parasitic drug targets are urgently required.Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) 2 represents such a target. dUTPases catalyze the hydrolysis of dUTP to dUMP and pyrophosphate (2). This provides the starting material for the synthesis of dTMP by thymidylate synthase and in addition maintains the ratio of dTTP:dUTP in the cell at a high enough level to prevent excessive misincorporation of dUMP into the genome during DNA replication (3). dUTPase activity is essential as was shown by gene knock-outs in Escherichia coli and Saccharomyces cerevisiae. Inhibition of dUTPase activity results in futile cycles of DNA repair that ultimately cause the fragmentation of DNA and cell death (4, 5). dUTPase activity also appears to be essential in L. major (6) and the related parasite Trypanosoma brucei, where an RNAi approach was used to knock down dUTPase expression, resulting in decreased cell proliferation and growth in both procyclic and bloodstream forms of the organism (7). dUTPases have been characterized extensively both biochemically and structurally in many species. Most organisms, including humans, have trimeric dUTPases with structures largely consisting of ␤-pleated sheet (8). Three active sites are formed at the trimer interfaces by...

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Kinetic properties and inhibition of the dimeric dUTPase‐dUDPase from Leishmania major

Cited by 37 publications

References 27 publications

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

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

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

The Crystal Structure of the Leishmania major Deoxyuridine Triphosphate Nucleotidohydrolase in Complex with Nucleotide Analogues, dUMP, and Deoxyuridine

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