PDBsum entry 1ppx

Hydrolase

PDB id: 1ppx

Contents

Protein chain

129 a.a. *

Ligands

8OG

Metals

_MG

Waters ×6

* Residue conservation analysis

References listed in PDB file

Key reference

• Title: Solution structure and nh exchange studies of the mutt pyrophosphohydrolase complexed with mg(2+) and 8-Oxo-Dgmp, A tightly bound product.
• Authors: M.A.Massiah, V.Saraswat, H.F.Azurmendi, A.S.Mildvan.
• Ref.: Biochemistry, 2003, 42, 10140-10154. [DOI no: 10.1021/bi030105p]
• PubMed id: 12939141

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.

Secondary reference #1

• Title: Solution structure of the quaternary mutt-M2+-Ampcpp-M2+ complex and mechanism of its pyrophosphohydrolase action.
• Authors: J.Lin, C.Abeygunawardana, D.N.Frick, M.J.Bessman, A.S.Mildvan.
• Ref.: Biochemistry, 1997, 36, 1199-1211. [DOI no: 10.1021/bi962619c]
• PubMed id: 9063868

Secondary reference #2

• Title: Nmr studies of the conformations and location of nucleotides bound to the escherichia coli mutt enzyme.
• Authors: D.N.Frick, D.J.Weber, C.Abeygunawardana, A.G.Gittis, M.J.Bessman, A.S.Mildvan.
• Ref.: Biochemistry, 1995, 34, 5577-5586. [DOI no: 10.1021/bi00016a032]
• PubMed id: 7727419

Secondary reference #3

• Title: Solution structure of the mutt enzyme, A nucleoside triphosphate pyrophosphohydrolase.
• Authors: C.Abeygunawardana, D.J.Weber, A.G.Gittis, D.N.Frick, J.Lin, A.F.Miller, M.J.Bessman, A.S.Mildvan.
• Ref.: Biochemistry, 1995, 34, 14997-15005.
• PubMed id: 7578113