PDBsum entry 2qlp

Hydrolase

PDB id: 2qlp

Contents

Protein chains

(+ 0 more) 161 a.a. *

Ligands

1PE ×7

Waters ×120

* Residue conservation analysis

PDB id:

2qlp

Name:

Hydrolase

Title:

Bifunctional dctp deaminase:dutpase from mycobacterium tuberculosis, apo form

Structure:

Deoxycytidine triphosphate deaminase. Chain: a, b, c, d, e, f. Synonym: dctp deaminase. Engineered: yes

Source:

Mycobacterium tuberculosis. Organism_taxid: 83332. Strain: h37rv. Gene: dcd. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.47Å R-factor: 0.232 R-free: 0.295

Authors:

S.Christophersen,P.Harris,M.Willemoes

Key ref:

S.S.Helt et al. (2008). Mechanism of dTTP inhibition of the bifunctional dCTP deaminase:dUTPase encoded by Mycobacterium tuberculosis. J Mol Biol, 376, 554-569. PubMed id: 18164314 DOI: 10.1016/j.jmb.2007.11.099

Date:

13-Jul-07 Release date: 19-Feb-08

Protein chains

P9WP17  (DCDB_MYCTU) -  dCTP deaminase, dUMP-forming from Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv)

Seq: 190 a.a.

Struc: 161 a.a.

Enzyme reactions

• Enzyme class: E.C.3.5.4.30 - dCTP deaminase (dUMP-forming).
• Reaction: dCTP + 2 H2O = dUMP + NH4⁺ + diphosphate
• Cofactor: Mg(2+)

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

reference

DOI no: 10.1016/j.jmb.2007.11.099

J Mol Biol 376:554-569 (2008)

PubMed id: 18164314

Mechanism of dTTP inhibition of the bifunctional dCTP deaminase:dUTPase encoded by Mycobacterium tuberculosis.

S.S.Helt, M.Thymark, P.Harris, C.Aagaard, J.Dietrich, S.Larsen, M.Willemoes.

ABSTRACT

Recombinant deoxycytidine triphosphate (dCTP) deaminase from Mycobacterium tuberculosis was produced in Escherichia coli and purified. The enzyme proved to be a bifunctional dCTP deaminase:deoxyuridine triphosphatase. As such, the M. tuberculosis enzyme is the second bifunctional enzyme to be characterised and provides evidence for bifunctionality of dCTP deaminase occurring outside the Archaea kingdom. A steady-state kinetic analysis revealed that the affinity for dCTP and deoxyuridine triphosphate as substrates for the synthesis of deoxyuridine monophosphate were very similar, a result that contrasts that obtained previously for the archaean Methanocaldococcus jannaschii enzyme, which showed approximately 10-fold lower affinity for deoxyuridine triphosphate than for dCTP. The crystal structures of the enzyme in complex with the inhibitor, thymidine triphosphate, and the apo form have been solved. Comparison of the two shows that upon binding of thymidine triphosphate, the disordered C-terminal arranges as a lid covering the active site, and the enzyme adapts an inactive conformation as a result of structural changes in the active site. In the inactive conformation dephosphorylation cannot take place due to the absence of a water molecule otherwise hydrogen-bonded to O2 of the alpha-phosphate.

Selected figure(s)

Figure 3.
Fig. 3. Structure of M. tuberculosis dCTP deaminase:dUTPase. Ribbon view of the trimer generated of three A subunits viewed along the 3-fold axis (a) and perpendicular to the 3-fold axis (b). The three subunits are shown in cyan, pink and green, respectively. The nucleotides in the three active sites are shown in yellow ball-and-stick representation. N- and C-termini are presented in blue and red, respectively. (c) Schematic view of the hydrogen-bonding network surrounding dTTP in the structure of the M. tuberculosis dCTP deaminase:dUTPase–dTTP complex. The active site is composed of residues from two subunits indicated by an asterisk (e.g., Ala*). The dotted lines represent hydrogen bonds between atoms. (d) Electron density maps presenting the nucleotide in subunit A. The 2F[obs] − F[calc] map contoured at 1 σ is represented in blue and the F[obs] − F[calc] map contoured at 3 σ is represented in green.

Figure 8.
Fig. 8. dTTP inhibition of the dUTPase reaction in dCTP deaminase:dUTPase. Close-up of the active site (ribbon view) from superposition of the apo form of M. tuberculosis dCTP deaminase:dUTPase (green) and the dTTP complex (pink) with the M. tuberculosis dUTPase–α,β-imido dUTP complex (orange, PDB ID 1SIX^24). From all three structures the side chains corresponding to Ser102, Asp119 and Gln148 are shown as well as Ala115 from the bifunctional enzyme. Nucleotides are presented in ball-and-stick representation and in standard atom colours except carbon, which is coloured according to the matching enzyme. Important water molecules are also shown in colours corresponding to the colour of the matching enzyme. Magnesium ions are grey. The active site is shown from two directions (a and b). A continuous arrow points to the nucleophilic water molecule, while a dotted arrow points to the water molecule that is hydrogen-bonded to O2 of the α-phosphate in the trimeric dUTPases. See the text for details. The panels were prepared with PyMol (DeLano Scientific).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 376, 554-569) copyright 2008.

Figures were selected by an automated process.