Crystal structure of dUTPase from equine infectious anaemia virus; active site metal binding in a substrate analogue complex 1 1Edited by K. Nagai

Dauter, Zbigniew; Persson, Rebecca; Rosengren, Anna; Nyman, Per Olof; Wilson, K.S.; Cedergren-Zeppezauer, Eila

doi:10.1006/jmbi.1998.2332

Cited by 77 publications

(93 citation statements)

References 78 publications

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“…57), and second, the low k cat also argues against the physiological significance of dTTP hydrolysis by dUTPase. Inspecting the active site of E. coli, retroviral and Drosophila dUTPases (27,28,31) provides a structural explanation for the increased affinity toward dTTP. In the bacterial enzyme, the Asn 84 side chain constructs an extra inward bulge toward the C-5 carbon atom of the uracil ring in the ␤-hairpin accommodating the base moiety of the substrate.…”

Section: Cloning Expression and Purification Of D Melanogaster Dutmentioning

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%

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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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Section: Cloning Expression and Purification Of D Melanogaster Dutmentioning

confidence: 99%

mentioning

confidence: 99%

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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“…This enzyme hydrolyzes dUTP into dUMP and pyrophosphate, thereby removing dUTP from the DNA biosynthetic pathway. Although several high to medium resolution x-ray studies of dUTPases from human (3), bacterial (4)(5)(6), and viral (7,8) sources have been reported, there is no understanding of the structural basis of the catalytic mechanism. The C-terminal portion of each subunit of this trimeric enzyme is partly disordered in crystals and does not contribute to the electron density map.…”

mentioning

confidence: 99%

“…Furthermore, the binding site of Mg 2ϩ in the active site has not been identified. In the crystal structure of the equine infectious anemia virus (EIAV) enzyme, a Sr 2ϩ ion bound to both the ␣-and ␤-phosphate groups of dUDP has been localized (7), but in a position that would sterically prevent nucleophilic attack by a hydrolytic water molecule. Nonetheless, the catalytic importance of the C-terminal residues and the dependence of dUTPase action on Mg 2ϩ have been amply demonstrated by studies of the enzyme in solution (9)(10)(11)(12).…”

mentioning

confidence: 99%

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

Mustafi

Békési

Vértessy

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The metal ion dependence of the catalytic activity of recombinant Escherichia coli dUTP pyrophosphatase (dUTPase), an essential enzyme preventing incorporation of uracil into DNA, has been investigated by steady-state kinetic, electron paramagnetic resonance, and electron nuclear double resonance methods. Values of k cat and kcat͞Km were 4.5 ؎ 0.1 s ؊1 and 0.49 ؎ 0.1 ؋ 10 6 M ؊1 ⅐s ؊1 in the absence of divalent metal ions, 14.7 ؎ 2.2 s ؊1 and 25.1 ؎ 7.4 ؋ 10 6 M ؊1 ⅐s ؊1 in the presence of Mg 2؉ or Mn 2؉ , and 24.2 ؎ 3.6 s ؊1 and 2.4 ؎ 0.7 ؋ 10 6 M ؊1 ⅐s ؊1 when supported by VO 2؉ or bis(acetylacetonato)oxovanadium(IV). Binding of VO 2؉ to the enzyme in the presence of dUDP, a nonhydrolyzable substrate analog, was specific and competitive with Mg 2؉ . Electron paramagnetic resonance spectra of the ternary enzyme-VO 2؉ -chelatedUDP complex revealed a pattern of 31 P superhyperfine coupling specifying two structurally equivalent phosphate groups equatorially coordinated to the VO 2؉ ion. Proton electron nuclear double resonance spectra revealed an equatorial acetylacetonate ligand, indicating that one of the organic ligands had been displaced. By molecular graphics modeling, we show that the diphosphate group of enzyme-bound dUDP is sterically accessible to a hemi-chelate form of VO 2؉ . We propose a similar location compatible with all kinetic and spectroscopic results to account for the reactivity of VO 2؉ and the VO 2؉ -chelate in dUTP hydrolysis. In this location the metal ion could promote an ordered conformation of the Cterminal fragment that is obligatory for catalysis but dynamically flexible in the free enzyme.C ontrary to ordinary expectation, the nucleic acid uracil can be incorporated into DNA in the cell because DNA polymerases do not distinguish between thymine and uracil. The most important cellular vehicle for hindering this process is the enzyme dUTP pyrophosphatase (dUTPase) (1, 2). This enzyme hydrolyzes dUTP into dUMP and pyrophosphate, thereby removing dUTP from the DNA biosynthetic pathway. Although several high to medium resolution x-ray studies of dUTPases from human (3), bacterial (4-6), and viral (7, 8) sources have been reported, there is no understanding of the structural basis of the catalytic mechanism. The C-terminal portion of each subunit of this trimeric enzyme is partly disordered in crystals and does not contribute to the electron density map. Furthermore, the binding site of Mg 2ϩ in the active site has not been identified. In the crystal structure of the equine infectious anemia virus (EIAV) enzyme, a Sr 2ϩ ion bound to both the ␣-and ␤-phosphate groups of dUDP has been localized (7), but in a position that would sterically prevent nucleophilic attack by a hydrolytic water molecule. Nonetheless, the catalytic importance of the C-terminal residues and the dependence of dUTPase action on Mg 2ϩ have been amply demonstrated by studies of the enzyme in solution (9-12). Because dUTPase has acquired increasing attention as a potential target enzyme in cancer and antiretroviral chemot...

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“…During the original structure solution for this protein (Dauter et al, 1999), an ARP/wARP protocol in which the model was converted to free atoms and then re®ned against the 2.0 A Ê data was pioneered (unrestrained ARP or uARP). This convincingly showed the power of ARP in re®nement of molecular-replacement solutions.…”

Section: Equine Infectious Anaemia Virus Dutpasementioning

confidence: 99%

ARP/wARP and molecular replacement

Perrakis¹,

Harkiolaki

Wilson

et al. 2001

Acta Crystallogr D Biol Crystallogr

482

378

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The aim of ARP/wARP is improved automation of model building and re®nement in macromolecular crystallography. Once a molecular-replacement solution has been obtained, it is often tedious to re®ne and rebuild the initial (search) model. ARP/wARP offers three options to automate that task to varying extents: (i) autobuilding of a completely new model based on phases calculated from the molecular-replacement solution, (ii) updating of the initial model by atom addition and deletion to obtain an improved map and (iii) docking of a structure onto a new (or mutated) sequence, followed by rebuilding and re®ning the side chains in real space. A few examples are presented where ARP/wARP made a considerable difference in the speed of structure solution and/or made possible re®nement of otherwise dif®cult or uninterpretable maps. The resolution range allowing complete autobuilding of protein structures is currently 2.0 A Ê , but for map improvement considerable advances over more conventional re®nement techniques are evident even at 3.2 A Ê spacing. Automatic refinement, building and rebuilding of models from molecularreplacement solutions.

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Crystal structure of dUTPase from equine infectious anaemia virus; active site metal binding in a substrate analogue complex 1 1Edited by K. Nagai

Cited by 77 publications

References 78 publications

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

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

Catalytic and structural role of the metal ion in dUTP pyrophosphatase

ARP/wARP and molecular replacement

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