PDBsum entry 2we3

Hydrolase

PDB id: 2we3

Contents

Protein chain

240 a.a. *

Ligands

DUT

SO4 ×2

Metals

_MG

Waters ×84

* Residue conservation analysis

PDB id:

2we3

Name:

Hydrolase

Title:

Ebv dutpase inactive mutant deleted of motif v

Structure:

Deoxyuridine 5'-triphosphate nucleotidohydrolase. Chain: a. Fragment: residues 1-256. Synonym: dutp pyrophosphatase, dutpase. Engineered: yes

Source:

Human herpesvirus 4. Epstein-barr virus. Organism_taxid: 10377. Strain: b95-8. Expressed in: escherichia coli. Expression_system_taxid: 511693.

Resolution:

2.00Å R-factor: 0.215 R-free: 0.272

Authors:

L.Freeman,M.Buisson,N.Tarbouriech,W.P.Burmeister

Key ref:

L.Freeman et al. (2009). The flexible motif V of Epstein-Barr virus deoxyuridine 5'-triphosphate pyrophosphatase is essential for catalysis. J Biol Chem, 284, 25280-25289. PubMed id: 19586911 DOI: 10.1074/jbc.M109.019315

Date:

27-Mar-09 Release date: 07-Jul-09

Protein chain

P03195  (DUT_EBVB9) -  Deoxyuridine 5'-triphosphate nucleotidohydrolase from Epstein-Barr virus (strain B95-8)

Seq: 278 a.a.

Struc: 240 a.a.

Enzyme reactions

• Enzyme class: E.C.3.6.1.23 - dUTP diphosphatase.
• Reaction: dUTP + H2O = dUMP + diphosphate + H⁺

dUTP (Bound ligand (Het Group name = DUT) corresponds exactly) + H2O = dUMP + diphosphate + H(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1074/jbc.M109.019315

J Biol Chem 284:25280-25289 (2009)

PubMed id: 19586911

The flexible motif V of Epstein-Barr virus deoxyuridine 5'-triphosphate pyrophosphatase is essential for catalysis.

L.Freeman, M.Buisson, N.Tarbouriech, A.Van der Heyden, P.Labbé, W.P.Burmeister.

ABSTRACT

Deoxyuridine 5'-triphosphate pyrophosphatases (dUTPases) are ubiquitous enzymes essential for hydrolysis of dUTP, thus preventing its incorporation into DNA. Although Epstein-Barr virus (EBV) dUTPase is monomeric, it has a high degree of similarity with the more frequent trimeric form of the enzyme. In both cases, the active site is composed of five conserved sequence motifs. Structural and functional studies of mutants based on the structure of EBV dUTPase gave new insight into the mechanism of the enzyme. A first mutant allowed us to exclude a role in enzymatic activity for the disulfide bridge involving the beginning of the disordered C terminus. Sequence alignments revealed two groups of dUTPases, based on the position in sequence of a conserved aspartic acid residue close to the active site. Single mutants of this residue in EBV dUTPase showed a highly impaired catalytic activity, which could be partially restored by a second mutation, making EBV dUTPase more similar to the second group of enzymes. Deletion of the flexible C-terminal tail carrying motif V resulted in a protein completely devoid of enzymatic activity, crystallizing with unhydrolyzed Mg(2+)-dUTP complex in the active site. Point mutations inside motif V highlighted the essential role of lid residue Phe(273). Magnesium appears to play a role mainly in substrate binding, since in absence of Mg(2+), the K(m) of the enzyme is reduced, whereas the k(cat) is less affected.

Selected figure(s)

Figure 1.
EBV dUTPase sequence and structure.A, sequence alignment of different dUTPases with known structures and the one from C. glutamaticum. Residue numbers are given above the sequence for the human enzyme and below the sequences for the mycobacterial and EBV enzymes. The linker region is highlighted with cyan letters. The five conserved motifs are highlighted. Key residues of motif V are printed in red. A pink background highlights residues of motif V where side chain hydroxyl and main chain amide contact γ-phosphate in the crystal structure (13, 14). Green background, main chain amide interaction with β- and γ-phosphate; residues that have been deleted in the ΔV mutant are underlined. The conserved residues interacting with the motif V arginine of human-like dUTPases are printed in blue; those interacting with the motif V arginine of mycobacterium-like dUTPases are shown in magenta. B, structure of EBV dUTPase in complex with α,β-imido-dUTP and Mg^2+ (Protein Data Bank entry 2bt1). The catalytic residue Asp^76 is shown together with Asp^131 next to it. The four visible sequence motifs of the active site are colored according to Fig. 1A, and the loop connecting the two domains (residues 114–125) is colored in cyan. The bound α,β-imido-dUTP molecule is shown as sticks and colored according to atom types. C, the ΔV structure (pink) in complex with dUTP (sticks, atom colors) and Mg^2+. The disordered part of the connecting loop is located between the two arrows. Superposed is the dUMP-bound WT dUTPase structure (Protein Data Bank entry 2bsy; gray, connecting loop in cyan, motif I in green, disordered part dotted).

Figure 4.
Structure of the active site with bound ligands.A, stereoview of dUTP, Mg^2+, and its coordinating (green lines) water molecules bound to the active site of EBV dUTPase ΔV mutant (pink). The arginine residue of a neighbor symmetry mate (Arg^37) is shown in orange. B, M. tuberculosis dUTPase structure with bound α,β-imido-dUTP and the arginine and the histidine lid residues of motif V (Protein Data Bank entry 2py4) (14). Important hydrogen bonds and salt bridges are shown in the color of their partner molecule. C, F[o] − F[c] electron density contoured at 2.5 σ level. dUTP, Mg^2+, and water molecules have been left out of the structure factor calculation. D, structure of the EBV dUTPase ΔV mutant Mg^2+-dUTP complex. The accessible surface is shown colored according to the electrostatic potential of the protein (from negative (red) to positive (blue)) calculated with apbs (34).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2009, 284, 25280-25289) copyright 2009.

Figures were selected by the author.