Dimeric dUTPases Research Articles

HDX and Native Mass Spectrometry Reveals the Different Structural Basis for Interaction of the Staphylococcal Pathogenicity Island Repressor Stl with Dimeric and Trimeric Phage dUTPases.

The dUTPase enzyme family plays an essential role in maintaining the genome integrity and are represented by two distinct classes of proteins; the β-pleated homotrimeric and the all-α homodimeric dUTPases. Representatives of both trimeric and dimeric dUTPases are encoded by Staphylococcus aureus phage genomes and have been shown to interact with the Stl repressor protein of S. aureus pathogenicity island SaPIbov1. In the present work we set out to characterize the interactions between these proteins based on a range of biochemical and biophysical methods and shed light on the binding mechanism of the dimeric φNM1 phage dUTPase and Stl. Using hydrogen deuterium exchange mass spectrometry, we also characterize the protein regions involved in the dUTPase:Stl interactions. Based on these results we provide reasonable explanation for the enzyme inhibitory effect of Stl observed in both types of complexes. Our experiments reveal that Stl employs different peptide segments and stoichiometry for the two different phage dUTPases which allows us to propose a functional plasticity of Stl. The malleable character of Stl serves as a basis for the inhibition of both dimeric and trimeric dUTPases.

Deinococcus radiodurans DR2231 is a two-metal-ion mechanism hydrolase with exclusive activity on dUTP.

Structural data are available in the PDB under the accession numbers 5HVA, 5HWU, 5HX1, 5HYL, 5I0J, 5HZZ, 5I0M.

Crystal structure of the MazG-related nucleoside triphosphate pyrophosphohydrolase from Thermotoga maritima MSB8.

The MazG family proteins, which are highly conserved in bacteria, are nucleoside triphosphate pyrophosphohydrolases that hydrolyze all canonical nucleoside triphosphates, and are also involved in removing noncanonical nucleoside triphosphates to prevent their incorporation into DNA or RNA. The primary structure of TM0360 from Thermotoga maritima MSB8 suggested that TM0360 is a MazG-related nucleoside triphosphate pyrophosphohydrolase. The crystal structure of the TM0360 protein was determined by the MAD technique at 2.0Å resolution. The asymmetric unit contains an intact dimer molecule. The overall structure of TM0360 is similar to the known structures of the dimeric MazG protein and dUTPases. The putative NTP binding pocket in TM0360, identified by considering the probable NTP-interacting residues and structural features, suggested that TM0360 resembles the C-terminal domain of Escherichia coli MazG, although TM0360 may be a truncated paralog of the N-terminal domain of T. maritima MazG (TM0913), according to its primary structure. The putative function of TM0360 is discussed, based on structural homology.

On the catalytic mechanism of dimeric dUTPases

The Tritryps Trypanosoma brucei, Trypanosoma cruzi and Leishmania donovani are responsible for great morbidity and mortality in developing countries. Their dimeric dUTPases are members of the all-α NTP pyrophosphohydrolase family and represent promising drug targets due to their essential nature and markedly different structural and biochemical properties compared with the trimeric human enzyme. In the present paper we describe the structure of the T. brucei enzyme in open and closed conformations. Furthermore, we probe the reaction mechanism through the binding of transition state mimics both in solution and in the crystal. 31P-NMR and tryptophan fluorescence quenching in the presence of AlF3 and MgF3- identified which phosphate is subject to nucleophilic attack by a water molecule. The structures in complex with two transition state analogues confirm that the nucleophilic attack occurs on the β-phosphate in contrast with the α-phosphate in the trimeric enzymes. These results establish the structural basis of catalysis of these important housekeeping enzymes and has ramifications for the wider all-α NTP pyrophosphohydrolase family.

Structural and Functional Insights into DR2231 Protein, the MazG-like Nucleoside Triphosphate Pyrophosphohydrolase from Deinococcus radiodurans

Deinococcus radiodurans is among the very few bacterial species extremely resistant to ionizing radiation, UV light, oxidizing agents, and cycles of prolonged desiccation. The proteome of D. radiodurans reflects the evolutionary pressure exerted by chronic exposure to (nonradioactive) forms of DNA and protein damage. A clear example of this adaptation is the overrepresentation of protein families involved in the removal of non-canonical nucleoside triphosphates (NTPs) whose incorporation into nascent DNA would promote mutagenesis and DNA damage. The three-dimensional structure of the DR2231 protein has been solved at 1.80 Å resolution. This protein had been classified as an all-α-helical MazG-like protein. The present study confirms that it holds the basic structural module characteristic of the MazG superfamily; two helices form a rigid domain, and two helices form a mobile domain and connecting loops. Contrary to what is known of MazG proteins, DR2231 protein shows a functional affinity with dUTPases. Enzymatic and isothermal calorimetry assays have demonstrated high specificity toward dUTP but an inability to hydrolyze dTTP, a typical feature of dUTPases. Co-crystallization with the product of hydrolysis, dUMP, in the presence of magnesium or manganese cations, suggests similarities with the dUTP/dUDP hydrolysis mechanism reported for dimeric dUTPases. The genome of D. radiodurans encodes for all enzymes required for dTTP synthesis from dCMP, thus bypassing the need of a dUTPase. We postulate that DR2231 protein is not essential to D. radiodurans and rather performs "house-cleaning" functions within the framework of oxidative stress response. We further propose DR2231 protein as an evolutionary precursor of dimeric dUTPases.

Structure of a putative NTP pyrophosphohydrolase: YP_001813558.1 from Exiguobacterium sibiricum 255-15.

The crystal structure of a putative NTPase, YP_001813558.1 from Exiguobacterium sibiricum 255-15 (PF09934, DUF2166) was determined to 1.78 Å resolution. YP_001813558.1 and its homologs (dimeric dUTPases, MazG proteins and HisE-encoded phosphoribosyl ATP pyrophosphohydrolases) form a superfamily of all-α-helical NTP pyrophosphatases. In dimeric dUTPase-like proteins, a central four-helix bundle forms the active site. However, in YP_001813558.1, an unexpected intertwined swapping of two of the helices that compose the conserved helix bundle results in a `linked dimer' that has not previously been observed for this family. Interestingly, despite this novel mode of dimerization, the metal-binding site for divalent cations, such as magnesium, that are essential for NTPase activity is still conserved. Furthermore, the active-site residues that are involved in sugar binding of the NTPs are also conserved when compared with other α-helical NTPases, but those that recognize the nucleotide bases are not conserved, suggesting a different substrate specificity.

Depletion of dimeric all-α dUTPase induces DNA strand breaks and impairs cell cycle progression in Trypanosoma brucei

The enzyme deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) is responsible for the control of intracellular levels of dUTP thus controlling the incorporation of uracil into DNA during replication. Trypanosomes and certain eubacteria contain a dimeric dUTP-dUDPase belonging to the recently described superfamily of all-α NTP pyrophosphatases which bears no resemblance with typical eukaryotic trimeric dUTPases and presents unique properties regarding substrate specificity and product inhibition. While the biological trimeric enzymes have been studied in detail and the human enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies, little is known regarding the biological function of dimeric proteins. Here, we show that in Trypanosoma brucei, the dimeric dUTPase is a nuclear enzyme and that down-regulation of activity by RNAi greatly reduces cell proliferation and increases the intracellular levels of dUTP. Defects in growth could be partially reverted by the addition of exogenous thymidine. dUTPase-depleted cells presented hypersensitivity to methotrexate, a drug that increases the intracellular pools of dUTP, and enhanced uracil-DNA glycosylase activity, the first step in base excision repair. The knockdown of activity produces numerous DNA strand breaks and defects in both S and G2/M progression. Multiple parasites with a single enlarged nucleus were visualized together with an enhanced population of anucleated cells. We conclude that dimeric dUTPases are strongly involved in the control of dUTP incorporation and that adequate levels of enzyme are indispensable for efficient cell cycle progression and DNA replication.

Dimeric dUTPases, HisE, and MazG belong to a New Superfamily of all-α NTP Pyrophosphohydrolases with Potential “House-cleaning” Functions

Structure-guided analysis of the new dimeric dUTPase family revealed its sequence relationship to the phage T4 dCTPase, phosphoribosyl-ATP pyrophosphatase HisE, NTP pyrophosphatase MazG, and several uncharacterized protein families, including the human protein XTP3TPA (RS21-C6), which is overexpressed in embryonic and cancer cells. Comparison with the recently determined structure of a MazG-like protein from Sulfolobus solfataricus supported the unification of these enzymes in one superfamily of all-alpha NTP pyrophosphatases, suggesting that dimeric dUTPases evolved from a tetrameric MazG-like ancestor by gene duplication. Analysis of the structure of the Sulfolobus MazG points to 2-hydroxyadenosine (isoguanosine) triphosphate, a product of oxidative damage of ATP, as the most likely substrate. We predict that uncharacterized members of this superfamily perform "house-cleaning" functions by hydrolyzing abnormal NTPs and are functionally analogous to the structurally unrelated hydrolases of the Nudix superfamily. We outline probable tertiary and quaternary structures of the all-alpha NTP pyrophosphatase superfamily members.

The Crystal Structure of a Complex of Campylobacter jejuni dUTPase with Substrate Analogue Sheds Light on the Mechanism and Suggests the “Basic Module” for Dimeric d(C/U)TPases

The crystal structure of the dUTPase from the important gastric pathogen Campylobacter jejuni has been solved at 1.65 A spacing. This essential bacterial enzyme is the second representative of the new family of dimeric dUTPases to be structurally characterised. Members of this family have a novel all-alpha fold and are unrelated to the all-beta dUTPases of the majority of organisms including eukaryotes such as humans, bacteria such as Escherichia coli, archaea like Methanococcus jannaschii and animal viruses. Therefore, dimeric dUTPases can be considered as candidate drug targets. The X-ray structure of the C.jejuni dUTPase in complex with the non-hydrolysable substrate analogue dUpNHp allows us to define the positions of three catalytically significant phosphate-binding magnesium ions and provides a starting point for a detailed understanding of the mechanism of dUTP/dUDP hydrolysis by dimeric dUTPases. Indeed, a water molecule present in the structure is ideally situated to act as the attacking nucleophile during hydrolysis. A comparison of the dUTPases from C.jejuni and Trypanosoma cruzi reveals a common fold with certain distinct features, both in the rigid and mobile domains as defined in the T.cruzi structure. Homologues of the C.jejuni dUTPase have been identified in several other bacteria and bacteriophages, including the dCTPase of phage T4. Sequence comparisons of these proteins define a new superfamily of d(C/U)TPases that includes three distinct enzyme families: (1) dUTPases in trypanosomatides, C.jejuni and several other Gram-negative bacteria, (2) predicted dUTPases in various Gram-positive bacteria and their phages, and (3) dCTP/dUTPases in enterobacterial T4-like phages. All these enzymes share a basic module that consists of two alpha-helices from the rigid domain, two helices from the mobile domain and connecting loops. These results in concert with a number of conserved residues responsible for interdomain cross-talk provide valuable insight towards rational drug design.