Human dUTP pyrophosphatase: uracil recognition by a β hairpin and active sites formed by three separate subunits

Mol, Clifford D.; Harris, Jonathan M.; McIntosh, Evan M.; Tainer, John A.

doi:10.1016/s0969-2126(96)00114-1

Cited by 174 publications

(308 citation statements)

References 71 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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“…A wealth of data and in-depth analyses are available on thymidylate synthase and dihydrofolate reductase, but our knowledge on dUTPases is lagging behind. For human dUTPase, a series of thorough studies (18, 20 -23) addressed cellular level and regulation of physiological isoforms, and a crystal structure was also published (24). However, no enzymological characterization is yet available for any eukaryotic dUTPase.…”

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confidence: 99%

“…Despite limited sequence homology, the dUTPase trimeric fold seems to be well conserved from bacteria to man and even in retroviruses. Active site architecture also follows a common pattern where the three active sites of the homotrimer, located in clefts between neighboring subunits, are built in a fashion of basically 3-fold symmetry recruiting conserved motifs from all subunits (24,(27)(28)(29). Two successive ␤-strands and their connecting turn constitute the core of the active site that accommodates the uracil and deoxyribose rings of the substrate dUTP.…”

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confidence: 99%

“…Phosphate recognition is provided conserved motifs of the neighboring subunit. The third subunit contributes its C-terminal fifth conserved motif, the ordering of which upon the active site induces the catalytically competent closed conformation (24,29,30). Despite the overall similarities, the available data suggest the existence of marked differences between prokaryotic and eukaryotic dUTPases.…”

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confidence: 99%

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Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Kovari

Barábas

Takács

et al. 2004

Journal of Biological Chemistry

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dUTPase is responsible for preventive DNA repair via exclusion of uracil. Developmental regulation of the Drosophila enzyme is suggested to be involved in thymine-less apoptosis. Here we show that in addition to conserved dUTPase sequence motifs, the gene of Drosophila enzyme codes for a unique Ala-Pro-rich segment. Kinetic and structural analyses of the recombinant protein and a truncation mutant show that the Ala-Pro segment is flexible and has no regulatory role in vitro. The homotrimer enzyme unfolds reversibly as a trimeric entity with a melting temperature of 54°C, 23°C lower than Escherichia coli dUTPase. In contrast to the bacterial enzyme, Mg 2؉ binding modulates conformation of fly dUTPase, as identified by spectroscopy and by increment in melting temperature. A single well folded, but inactive, homotrimeric core domain is generated through three distinct steps of limited trypsinolysis. In fly, but not in bacterial dUTPase, binding of the product dUMP induces protection against proteolysis at the tryptic site reflecting formation of the catalytically competent closed conformer. Crystallographic analysis argues for the presence of a stable monomer of Drosophila dUTPase in crystal phase. The significant differences between prototypes of eukaryotic and prokaryotic dUTPases with respect to conformational flexibility of the active site, substrate specificity, metal ion binding, and oligomerization in the crystal phase are consistent with alteration of the catalytic mechanism and hydropathy of subunit interfaces.

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“…Furthermore, most dUTPase active enzymes are homo-trimeric complexes (e.g. the human or the E. coli enzyme), where three active sides are built by five highly conserved motifs from each monomeric chain (Larsson et al, 1996 b ;Mol et al, 1996). In contrast, the herpesviral enzymes appear to be monomeric (Caradonna & Adamkiewicz, 1984 ;McGeoch, 1990).…”

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confidence: 99%

Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) ORF54 encodes a functional dUTPase expressed in the lytic replication cycle.

Kremmer¹,

Sommer²,

Holzer³

et al. 1999

Journal of General Virology

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Human dUTP pyrophosphatase: uracil recognition by a β hairpin and active sites formed by three separate subunits

Cited by 174 publications

References 71 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

Altered Active Site Flexibility and a Structural Metal-binding Site in Eukaryotic dUTPase

Kaposi's sarcoma-associated herpesvirus (human herpesvirus-8) ORF54 encodes a functional dUTPase expressed in the lytic replication cycle.

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