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

Moroz, Olga V.; Harkiolaki, Maria; Galperin, Michael Y.; Vagin, A.A.; González-Pacanowska, Dolores; Wilson, K.S.

doi:10.1016/j.jmb.2004.07.050

Cited by 60 publications

(96 citation statements)

References 58 publications

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“…There were 52 878 data to 1.65 Å resolution and the data were artificially extended to 1 Å . A low-homology model, Trypanosoma cruzi dUTPase (PDB code 1ogk), was used to solve the structure by molecular replacement with 4 Å data (Moroz et al, 2004).…”

Section: Density Enhancementmentioning

confidence: 99%

ACORN: a review

Yao

Dodson

Wilson

et al. 2006

Acta Crystallogr D Biol Crystallogr

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The ACORN system was originally developed as a means of ab initio solution of protein structures when atomic resolution data were available. The first step is to obtain a starting set of phases, which must be at least slightly better than random. These may be calculated from a fragment of the structure, which can be anything from a single metal atom to a complete molecular-replacement model. A number of standard procedures are available in ACORN to orientate and position such a fragment. The fragment provides initial phases that give the first of a series of maps that are iteratively refined by a dynamic density-modification (DDM) process. Another FFTbased procedure is Sayre-equation refinement (SER), which modifies phases better to satisfy the Sayre equation. With good-quality atomic resolution data, the final outcome of applying DDM and SER is a map similar in appearance to that found from a refined structure, which is readily interpreted by automated procedures. Further development of ACORN now enables structures to be solved with less than atomic resolution data. A critical part of this development is the artificial extension of the data from the observed limit to 1 Å resolution. These extended reflections are allocated unit normalized structure amplitudes and then treated in a similar way to observed reflections except that they are downweighted in the calculation of maps. ACORN maps, especially at low resolution, tend to show C atoms less well, in particular C atoms which fall within the first diffraction minimum of their three neighbours. Two new density-modification procedures (DDM1 and DDM2) and a density-enhancement procedure (ENH) have been devised to counter this problem. It is demonstrated that high-quality maps showing individual atoms can be produced with the new ACORN. ACORN has also been demonstrated to be very effective in refining phase sets derived from physical processes such as those using anomalous scattering or isomorphous derivative data. Future work will be directed towards applying ACORN to resolutions down to 2 Å .

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Section: Density Enhancementmentioning

confidence: 99%

ACORN: a review

Yao

Dodson

Wilson

et al. 2006

Acta Crystallogr D Biol Crystallogr

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“…6 The dUTPases comprise two structurally different classes: 7 first, the β sheet−type dUTPases, possessed by most organisms, employ a one-metal ion catalytic mechanism, [8][9] and usually form a homotrimer 10 or a monomer, 11 while the second class, the α helical-type dUTPases, possessed by kinetoplastids, 12 form dimers and employ a two-metal ion catalytic mechanism. [13][14] In β sheet-type trimeric dUTPases, each active site is constituted from motifs of all three protomers, which are all required to interact for achieving proper enzymatic activity. [15][16] These dUTPases have a C-terminal arm structural motif that has sequence similarity to P-loop motifs 17 also present in triphosphate hydrolysis catalyzing enzymes including kinases, motor proteins, and membrane pumps.…”

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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“…There are reasons to believe that the catalytic mechanism of DR2231 protein is similar, if not identical, to that of C. jejuni dUTPase (6). The orientation of the product relative to the metal-binding site is very similar to that observed in the structure of C. jejuni dUTPase complexed with dUpNHpp.…”

Section: Discussionmentioning

confidence: 83%

“…Like other bacteria, D. radiodurans seems to lack entirely monomeric, trimeric, and archeal dUTPases, as has been pointed out by Moroz et al (6). Recently, the crystal structures of dUTPases from Campylobacter jejuni and Trypanosoma 2 To whom correspondence may be addressed.…”

mentioning

confidence: 86%

“…Through sequence analysis restricted to the active site motif, Moroz et al (6,7) identified a "basic module" of the dUTPase/ dCTPase family in the genomes of several Gram-positive bacteria and respective phages. This basic module, consisting of only five active site-forming helices, is conserved in two other families: the nonspecific NTP-PPase MazG and phosphoribosyl-ATP pyrophosphatase HisE.…”

mentioning

confidence: 99%

See 1 more Smart Citation

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

Gonçalves¹,

Sanctis

McSweeney

2011

Journal of Biological Chemistry

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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-␣-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.

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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

Cited by 60 publications

References 58 publications

ACORN: a review

ACORN: a review

Mutations Decouple Proton Transfer from Phosphate Cleavage in the dUTPase Catalytic Reaction

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

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