Homotrimeric dUTPases; Structural Solutions for Specific Recognition and Hydrolysis of dUTP

Persson, Rebecca; Cedergren-Zeppezauer, Eila; Wilson, K. S.

doi:10.2174/1389203013381035

Cited by 61 publications

(58 citation statements)

References 27 publications

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“…Therefore, the nature of the interaction with 65% non-polar and 35% polar residues with 79 bridging water molecules for M. jannaschii DCD-DUT may not be typical for dCTP deaminases. A similar trend is observed in the dUTPases, wherein the character of the subunit interactions varies from exclusively hydrophobic (E. coli dUTPase) to alternating layers of positively and negatively charged residues that contain numerous water molecules (human dUTPase) (30).…”

Section: Discussionsupporting

confidence: 62%

“…His 128 is substituted by an asparagine residue in some dCTP deaminases, a replacement that may preserve the hydrogen bonding abilities of the histidine residue. There is a very narrow pocket formed by motif 3 in homotrimeric dUTPases, where the O4 atom of uracil is hydrogen bonded to a water molecule that is held in place by hydrogen bonds to main chain atoms (11,30). In DCD-DUT, the His 128 side chain occupies the position equivalent to this water molecule, but, nevertheless, binding of uracil is possible (Fig.…”

Section: Discussionmentioning

confidence: 99%

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Structure of the Bifunctional dCTP Deaminase-dUTPase from Methanocaldococcus jannaschii and Its Relation to Other Homotrimeric dUTPases

Johansson

Björnberg

Nyman

et al. 2003

Journal of Biological Chemistry

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The bifunctional dCTP deaminase-dUTPase (DCD-DUT) from Methanocaldococcus jannaschii catalyzes the deamination of the cytosine moiety in dCTP and the hydrolysis of the triphosphate moiety forming dUMP, thereby preventing uracil from being incorporated into DNA. The crystal structure of DCD-DUT has been determined to 1.88-Å resolution and represents the first known structure of an enzyme catalyzing dCTP deamination. The functional form of DCD-DUT is a homotrimer wherein the subunits are composed of a central distorted ␤-barrel surrounded by two ␤-sheets and four helices. The trimeric DCD-DUT shows structural similarity to trimeric dUTPases at the tertiary and quaternary levels. There are also additional structural elements in DCD-DUT compared with dUTPase because of a longer primary structure. Four of the five conserved sequence motifs that create the active sites in dUTPase are found in structurally equivalent positions in DCD-DUT. The last 25 C-terminal residues of the 204-residuelong DCD-DUT are not visible in the electron density map, but, analogous to dUTPases, the C terminus is probably ordered, closing the active site upon catalysis. Unlike other enzymes catalyzing the deamination of cytosine compounds, DCD-DUT is not exploiting an enzyme-bound metal ion such as zinc or iron for nucleophile generation. The active site contains two water molecules that are engaged in hydrogen bonds to the invariant residues Ser 118 , Arg 122 , Thr 130 , and Glu 145 . These water molecules are potential nucleophile candidates in the deamination reaction.dUMP is the origin of thymidine precursors for DNA in all organisms. It is the substrate of thymidylate synthase that catalyzes the formation of dTMP (1). The main pathway for dUMP generation differs markedly between different organisms. dUMP is produced on the monophosphate level by the action of dCMP deaminase in eukaryotes and Gram-positive bacteria, whereas in Gram-negative bacteria such as Escherichia coli and some Archaea, dCTP is deaminated and dephosphorylated. dCTP deaminase (EC 3.5.4.13) and dUTPase (EC 3.6.1.23) catalyze the two consecutive steps where dUMP is formed in E. coli (dCTP 3 dUTP 3 dUMP). The hyperthermophilic archaeon Methanocaldococcus jannaschii produces a bifunctional dCTP deaminase-dUTPase (DCD-DUT) 1 with the ability to catalyze both reactions ( Fig. 1) (2, 3). The gene that encodes this enzyme was first annotated as coding for a dCTP deaminase, but enzymatic studies revealed the bifunctionality, which implies that dUTP is never released from the enzyme.dUTP is also formed upon spontaneous deamination of dCTP, and the reaction catalyzed by dUTPase is therefore essential in keeping the cellular concentration of dUTP low to suppress misincorporation of uracil into DNA. If such an event would happen, DNA is restored by an inherent repair mechanism. However, when dUTPase is deficient, the organism will not survive (4, 5). The dUTPases exist in different oligomeric forms. The homotrimeric form of dUTPases is by far the most common, but there are also mo...

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Section: Discussionsupporting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Structure of the Bifunctional dCTP Deaminase-dUTPase from Methanocaldococcus jannaschii and Its Relation to Other Homotrimeric dUTPases

Johansson

Björnberg

Nyman

et al. 2003

Journal of Biological Chemistry

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“…This enzyme is highly regulated in order to maintain the balance of deoxyribonucleotide pools (3)(4)(5). dUTP is also formed by ribonucleotide reductase, but because of the toxicity of this nucleotide (6,7), all organisms express an enzyme with dUTPase activity that hydrolyzes dUTP to dUMP (8).…”

mentioning

confidence: 99%

Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

Oehlenschlæger

Løvgreen

Reinauer

et al. 2015

Appl Environ Microbiol

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Analysis of the genome of Bacillus halodurans strain C125 indicated that two pathways leading from a cytosine deoxyribonucleotide to dUMP, used for dTMP synthesis, were encoded by the genome of the bacterium. The genes that were responsible, the comEB gene and the dcdB gene, encoding dCMP deaminase and the bifunctional dCTP deaminase:dUTPase (DCD:DUT), respectively, were both shown to be expressed in B. halodurans, and both genes were subject to repression by the nucleosides thymidine and deoxycytidine. The latter nucleoside presumably exerts its repression after deamination by cytidine deaminase. Both comEB and dcdB were cloned, overexpressed in Escherichia coli, and purified to homogeneity. Both enzymes were active and displayed the expected regulatory properties: activation by dCTP for dCMP deaminase and dTTP inhibition for both enzymes. Structurally, the B. halodurans enzyme resembled the Mycobacterium tuberculosis enzyme the most. An investigation of sequenced genomes from other species of the genus Bacillus revealed that not only the genome of B. halodurans but also the genomes of Bacillus pseudofirmus, Bacillus thuringiensis, Bacillus hemicellulosilyticus, Bacillus marmarensis, Bacillus cereus, and Bacillus megaterium encode both the dCMP deaminase and the DCD:DUT enzymes. In addition, eight dcdB homologs from Bacillus species within the genus for which the whole genome has not yet been sequenced were registered in the NCBI Entrez database. The biosynthetic route to the nucleotide dUMP, the precursor of dTTP, as shown in Fig. 1, differs between organisms and sets it apart from the rest of nucleotide metabolism (1). This is in agreement with the general belief that uracil was the original DNA base-pairing partner for adenine and that the biosynthesis of dTTP from dUMP arose later in evolution (2). The deoxyribonucleotides dGTP, dATP, and dCTP are all produced in a pathway that goes through the multisubstrate enzyme ribonucleotide reductase. This enzyme is highly regulated in order to maintain the balance of deoxyribonucleotide pools (3-5). dUTP is also formed by ribonucleotide reductase, but because of the toxicity of this nucleotide (6, 7), all organisms express an enzyme with dUTPase activity that hydrolyzes dUTP to dUMP (8).The direct formation of dUMP via ribonucleotide reductase (Fig. 1) is not an efficient process, as the ribonucleotide reductase has a reduced affinity for UDP compared to the affinities of three other canonical ribonucleotides (9-12). Instead, most of the dUMP arises from deamination of cytosine deoxyribonucleotides (13-15). Enteric bacteria synthesize an enzyme with dCTP deaminase activity that converts dCTP into dUTP (EC 3.5.4.13), which accounts for 70 to 80% of the dUMP production (15). The dCTP deaminase is a homotrimeric enzyme structurally related to the trimeric dUTPase and part of the dCTP deaminase/dUTPase superfamily (16-18). The genomes of many Gram-positive bacteria and all eukaryotes encode a dCMP deaminase (EC 3.5.4.12) that shares no resemblance to the enteric dCTP de...

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“…The substrate in each active site is bound by conserved sequence motifs from all three subunits. Therefore, although each subunit contains all necessary residues for substrate binding, trimer formation is indispensable to bring these residues in proximity for the cognate binding site (1,2). dUTPase, similar to many other nucleotide binding proteins, contains a conserved loop motif (motif V) to coordinate the phosphate chain of the protein.…”

mentioning

confidence: 99%

“…contributed equally to this work. 2 To whom correspondence may be addressed. E-mail: tothj@enzim.hu or vertessy@ enzim.hu.…”

mentioning

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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Homotrimeric dUTPases; Structural Solutions for Specific Recognition and Hydrolysis of dUTP

Cited by 61 publications

References 27 publications

Structure of the Bifunctional dCTP Deaminase-dUTPase from Methanocaldococcus jannaschii and Its Relation to Other Homotrimeric dUTPases

Structure of the Bifunctional dCTP Deaminase-dUTPase from Methanocaldococcus jannaschii and Its Relation to Other Homotrimeric dUTPases

Bacillus halodurans Strain C125 Encodes and Synthesizes Enzymes from Both Known Pathways To Form dUMP Directly from Cytosine Deoxyribonucleotides

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

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