Kinetic properties and inhibition of the dimeric dUTPase-dUDPase from<i>Campylobacter jejuni</i>

Musso-Buendia, Juan Alexander; Vidal, Antonio E.; Kasinthan, Ganasan; Nguyen, Corinne; Carrero-Lérida, Juana; Ruíz-Pérez, Luis M.; Wilson, K.S.; Johansson, Nils Gunnar; Gilbert, Ian H.; González-Pacanowska, Dolores

doi:10.1080/14756360801915476

Cited by 10 publications

(1 citation statement)

References 23 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%