PDBsum entry 1seh

Hydrolase

PDB id: 1seh

Contents

Protein chain

140 a.a. *

Ligands

UMP

TRS

Waters ×194

* Residue conservation analysis

PDB id:

1seh

Name:

Hydrolase

Title:

Crystal structure of e. Coli dutpase complexed with the product dump

Structure:

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

Source:

Escherichia coli. Organism_taxid: 562. Gene: dut. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Hexamer (from PDB file)

Resolution:

1.47Å R-factor: 0.159 R-free: 0.184

Authors:

O.Barabas,J.Kovari,V.Pongracz,M.Wilmanns,B.G.Vertessy

Key ref:

O.Barabás et al. (2004). Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase. J Biol Chem, 279, 42907-42915. PubMed id: 15208312 DOI: 10.1074/jbc.M406135200

Date:

17-Feb-04 Release date: 07-Sep-04

Protein chain

P06968  (DUT_ECOLI) -  Deoxyuridine 5'-triphosphate nucleotidohydrolase from Escherichia coli (strain K12)

Seq: 152 a.a.

Struc: 140 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

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

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

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

reference

DOI no: 10.1074/jbc.M406135200

J Biol Chem 279:42907-42915 (2004)

PubMed id: 15208312

Structural insights into the catalytic mechanism of phosphate ester hydrolysis by dUTPase.

O.Barabás, V.Pongrácz, J.Kovári, M.Wilmanns, B.G.Vértessy.

ABSTRACT

dUTPase is essential to keep uracil out of DNA. Crystal structures of substrate (dUTP and alpha,beta-imino-dUTP) and product complexes of wild type and mutant dUTPases were determined to reveal how an enzyme responsible for DNA integrity functions. A kinetic analysis of wild type and mutant dUTPases was performed to obtain relevant mechanistic information in solution. Substrate hydrolysis is shown to be initiated via in-line nucleophile attack of a water molecule oriented by an activating conserved aspartate residue. Substrate binding in a catalytically competent conformation is achieved by (i) multiple interactions of the triphosphate moiety with catalysis-assisting Mg2+, (ii) a concerted motion of residues from three conserved enzyme motifs as compared with the apoenzyme, and (iii) an intricate hydrogen-bonding network that includes several water molecules in the active site. Results provide an understanding for the catalytic role of conserved residues in dUTPases.

Selected figure(s)

Figure 3.
FIG. 3. Identification of the nucleophile water. A, simulated annealed omit electron density map, restricted to exclusively show the exact position of the catalytic water molecule in the wild type dUTPase: , -imino-dUTP:Mg2+ structure. The figure also shows the hydrogen-bonding network involving the phosphate chain in this complex structure. In addition to the catalytic water, Mg2+-coordinating waters, W1, W2, W4, W15, and W21, also participate in the primary hydrogen-bonding interactions. B, superimposed structures of wild type (dark tones) and Asp90 Asn mutant (light tones) dUTPase: , -imino-dUTP:Mg2+ complexes. Note that the only remarkable difference between the superimposed structures is the disappearance of W[cat] from the mutant complex. Atomic color code: carbon, dark/light gray; oxygen, dark/light red (pink); phosphorus, dark/light orange (yellow); nitrogen, dark/light blue; magnesium, dark/light purple. C, superimposed structures of Asp90 Asn mutant dUTPase: dUTP:Mg2+ (dark tones) and Asp90 Asn mutant dUTPase: , -imino-dUTP:Mg2+ (light tones) complexes. Note the close identity in the positions of the nucleotide ligands. D, apoenzyme retains a water closely corresponding to the W[cat] position. 3-Fold superimposition of the apoenzyme (green carbons and water, otherwise standard atom coloring), enzyme-substrate (dark tones), and enzyme-product (light tones) structures. Note the position of the catalytic water from the apoenzyme to the enzyme-substrate and enzyme-product complexes. E, F, and G, simulated annealed omit electron density maps for the substrates in wild type E. coli dUTPase: , -imino-dUTP:Mg2+, the Asp90 Asn E. coli dUTPase: , -imino-dUTP: Mg2+, and the Asp90 Asn E. coli dUTPase:dUTP:Mg2+ structures, respectively. Maps are restricted to show the nucleotide ligand, the Mg2+, the three water molecules coordinating to the metal ion, as well as the catalytic water, if present.

Figure 4.
FIG. 4. Interaction mapping in enzyme-substrate (A), and enzyme-product complexes (B). Interactions are shown only for the phosphate chain moiety of the ligand. Due to the close similarity of the nucleotide interactions in the three enzyme-substrate complexes determined in the present study (cf. Fig. 3 and Table I), the map was selected to show the actual distances as found in the wild type dUTPase: , -imino-dUTP: Mg2+ (X = N) complex where W[cat] is also present. In the Asp90 Asn mutant dUTPase: , -imino-dUTP:Mg2+ (X = N) and Asp90 Asn mutant dUTPase:dUTP: Mg2+ (X = O) complex, the only significant differences are that (i) W[cat] is absent and Asp90O 2 becomes AsnN 2 and (ii) in the Asp90 Asn mutant dUTPase:dUTP: Mg2+ (X = O) complex, the X-Ser72O interaction is absent. Changes in all other distances are within ±0.2 Å.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 42907-42915) copyright 2004.

Figures were selected by an automated process.