Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Varga, Balázs; Barábas, O.; Kovari, J.; Tóth, Judit; Hunyadi‐Gulyás, Éva; Klement, Éva; Medzihradszky, Katalin F.; Tölgyesi, F.; Fidy, Judit; Vértessy, Beáta G.

doi:10.1016/j.febslet.2007.09.005

Cited by 66 publications

(143 citation statements)

References 21 publications

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“…In G-proteins and myosins, its most likely role in catalysis is to orient the nucleotide and the attacking water molecule for efficient nucleophilic attack on the γ-P as well as charge stabilization (31,33). In dUTPase, the γ-P coordination of the P-loop-like motif is also necessary for orientation of the catalytic apparatus and stabilization of the associative type transition state (17,18). The completely different topology and the partly similar function of these loops indicates convergent evolution of phosphate binding motifs in a larger group of nucleotide hydrolases including PPi generating diphosphatases.…”

Section: Resultsmentioning

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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Section: Resultsmentioning

confidence: 99%

“…Proteins were expressed and purified as described previously (17). Site-directed mutagenesis was performed by the QuikChange method (Stratagene) and verified by sequencing of both strands.…”

Section: Methodsmentioning

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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“…[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: 95%

“…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%

“…Studies on all-b dUTPases present potentially the most in-depth enzyme kinetic and structure-based evidence for the mechanism of action leading to nucleophilic attack on nucleotide a-phosphate. The key features of this mechanism include (a) a conserved acidic residue (Asp) coordinating the nucleophile in an in-line position to a-phosphorus and deprotonating it before attack; (b) triphosphate chain positioned for catalysis by a single Mg 2+ cofactor; (c) charge-stabilization of the bridging oxygen between a-and b-phosphate by a conserved Ser residue; and (d) tight c-phosphate coordination upon catalysis performed by a mobile glycine-rich P-loop [8,29,30,32,39,[51][52][53]65,67,69]. The catalytic mechanism of ITPases exhibits high similarities in all of these structural solutions, with a Lys performing the role described previously [35] and providing an alternative solution for c-phosphate coordination compared to the P-loop motif [35,79,81].…”

Section: Enzymes With Different Fold Employ Different Catalytic Mechamentioning

confidence: 99%

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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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The Stl repressor from Staphylococcus aureus is an efficient inhibitor of the eukaryotic fruitfly dUTPase

et al. 2017

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DNA metabolism and repair is vital for the maintenance of genome integrity. Specific proteinaceous inhibitors of key factors in this process have high potential for deciphering pathways of DNA metabolism and repair. The dUTP ase enzyme family is responsible for guarding against erroneous uracil incorporation into DNA . Here, we investigate whether the staphylococcal Stl repressor may interact with not only bacterial but also eukaryotic dUTP ase. We provide experimental evidence for the formation of a strong complex between Stl and Drosophila melanogaster dUTP ase. We also find that dUTP ase activity is strongly diminished in this complex. Our results suggest that the dUTP ase protein sequences involved in binding to Stl are at least partially conserved through evolution from bacteria to eukaryotes.

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Active site closure facilitates juxtaposition of reactant atoms for initiation of catalysis by human dUTPase

Cited by 66 publications

References 21 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

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

The Stl repressor from Staphylococcus aureus is an efficient inhibitor of the eukaryotic fruitfly dUTPase

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