PDBsum entry 1ppx

Hydrolase

PDB id: 1ppx

Contents

Protein chain

129 a.a. *

Ligands

8OG

Metals

_MG

Waters ×6

* Residue conservation analysis

PDB id:

1ppx

Name:

Hydrolase

Title:

Solution structure of the mutt pyrophosphohydrolase complexed with mg(2+) and 8-oxo-dgmp, a tightly-bound product

Structure:

Mutator mutt protein. Chain: a. Synonym: 7,8-dihydro-8-oxoguanine-triphosphatase, 8-oxo-dgtpase, dgtp pyrophosphohydrolase. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Gene: mutt or b0099. Expressed in: escherichia coli. Expression_system_taxid: 562.

NMR struc:

20 models

Authors:

M.A.Massiah,V.Saraswat,H.F.Azurmendi,A.S.Mildvan

Key ref:

M.A.Massiah et al. (2003). Solution structure and NH exchange studies of the MutT pyrophosphohydrolase complexed with Mg(2+) and 8-oxo-dGMP, a tightly bound product. Biochemistry, 42, 10140-10154. PubMed id: 12939141 DOI: 10.1021/bi030105p

Date:

17-Jun-03 Release date: 26-Aug-03

Protein chain

P08337  (MUTT_ECOLI) -  8-oxo-dGTP diphosphatase from Escherichia coli (strain K12)

Seq: 129 a.a.

Struc: 129 a.a.

Enzyme reactions

• Enzyme class: E.C.3.6.1.55 - 8-oxo-dGTP diphosphatase.
• Reaction: 8-oxo-dGTP + H2O = 8-oxo-dGMP + diphosphate + H⁺
• Cofactor: Mg(2+)

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

Key reference

DOI no: 10.1021/bi030105p

Biochemistry 42:10140-10154 (2003)

PubMed id: 12939141

Solution structure and NH exchange studies of the MutT pyrophosphohydrolase complexed with Mg(2+) and 8-oxo-dGMP, a tightly bound product.

M.A.Massiah, V.Saraswat, H.F.Azurmendi, A.S.Mildvan.

ABSTRACT

To learn the structural basis for the unusually tight binding of 8-oxo-nucleotides to the MutT pyrophosphohydrolase of Escherichia coli (129 residues), the solution structure of the MutT-Mg(2+)-8-oxo-dGMP product complex (K(D) = 52 nM) was determined by standard 3-D heteronuclear NMR methods. Using 1746 NOEs (13.5 NOEs/residue) and 186 phi and psi values derived from backbone (15)N, Calpha, Halpha, and Cbeta chemical shifts, 20 converged structures were computed with NOE violations <or=0.25 A and total energies <or=450 kcal/mol. The pairwise root-mean-square deviations (RMSD) of backbone N, Calpha, and C' atoms for the secondary structured regions and for all residues of the 20 structures are 0.65 and 0.98 A, respectively, indicating a well-defined structure. Further refinement using residual dipolar coupling from 53 backbone N-H vectors slightly improved the RMSD values to 0.49 and 0.84 A, respectively. The secondary structures, which consisted of two alpha-helices and a five-stranded mixed beta-sheet, were indistinguishable from those of free MutT and of MutT in the quaternary MutT-Mg(2+)-(H(2)O)-AMPCPP-Mg(2+) complex. Comparisons of these three tertiary structures showed a narrowing of the hydrophobic nucleotide-binding cleft in the 8-oxo-dGMP complex resulting from a 2.5-4.5 A movement of helix I and a 1.5 A movement of helix II and loop 4 toward the cleft. The binding of 8-oxo-dGMP to MutT-Mg(2+) buries 71-78% of the surface area of the nucleotide. The 10(3.7)-fold weaker binding substrate analogue Mg(2+)-AMPCPP induced much smaller changes in tertiary structure, and MutT buried only 57% of the surface of the AMP moiety of AMPCPP. Formation of the MutT-Mg(2+)-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues of the enzyme by factors of 10(1)-10(6) as compared with the MutT-Mg(2+) and the MutT-Mg(2+)-dGMP complexes, suggesting a more compact structure when 8-oxo-dGMP is bound. The 10(4.6)-fold weaker binding of dGMP to MutT-Mg(2+) (K(D) = 1.8 mM) slowed the backbone exchange rates of only 20 residues and by smaller factors of approximately 10. Hence, the high affinity of MutT-Mg(2+) for 8-oxo-dGMP likely results from widespread ligand-induced conformation changes that narrow the nucleotide binding site and lower the overall free energy of the enzyme-product complex. Specific hydrogen bonding of the purine ring of 8-oxo-dGMP by the side chains of Asn-119 and Arg-78 may also contribute.