PDBsum entry 1qsl

Transferase/DNA

PDB id: 1qsl

Contents

Protein chain

601 a.a. *

DNA/RNA

Metals

_EU

Waters ×191

* Residue conservation analysis

PDB id:

1qsl

Name:

Transferase/DNA

Title:

Klenow fragment complexed with single-stranded substrate and europium (iii) ion

Structure:

5'-d( Gp Cp Tp Tp Ap Cp Gp C)-3'. Chain: b. Engineered: yes. DNA polymerase i. Chain: a. Fragment: klenow fragment. Synonym: pol i. Engineered: yes. Mutation: yes

Source:

Synthetic: yes. Other_details: chemically synthesized by standard phosphoramidite methodology. Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.20Å R-factor: 0.214 R-free: 0.255

Authors:

C.A.Brautigam,K.Aschheim,T.A.Steitz

Key ref:

C.A.Brautigam et al. (1999). Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment. Chem Biol, 6, 901-908. PubMed id: 10631518

Date:

22-Jun-99 Release date: 30-Jun-99

Protein chain

P00582  (DPO1_ECOLI) -  DNA polymerase I from Escherichia coli (strain K12)

Seq: 928 a.a.

Struc: 601 a.a.*

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

DNA/RNA chain

A-C-G-C 4 bases

Enzyme reactions

• Enzyme class: E.C.2.7.7.7 - DNA-directed Dna polymerase.
• Reaction: DNA(n) + a 2'-deoxyribonucleoside 5'-triphosphate = DNA(n+1) + diphosphate

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

Added reference

Chem Biol 6:901-908 (1999)

PubMed id: 10631518

Structural elucidation of the binding and inhibitory properties of lanthanide (III) ions at the 3'-5' exonucleolytic active site of the Klenow fragment.

C.A.Brautigam, K.Aschheim, T.A.Steitz.

ABSTRACT

BACKGROUND: Biochemical and biophysical experiments have shown that two catalytically essential divalent metal ions (termed 'A' and 'B') bind to the 3'-5' exonuclease active site of the Klenow fragment (KF) of Escherichia coli DNA polymerase I. X-ray crystallographic studies have established the normal positions in the KF 3'-5' exonuclease (KF exo) active site of the two cations and the single-stranded DNA substrate. Lanthanide (III) luminescence studies have demonstrated, however, that only a single europium (III) ion (Eu3+) binds to the KF exo active site. Furthermore, Eu3+ does not support catalysis by KF exo or several other two-metal-ion phosphoryl-transfer enzymes. RESULTS: A crystal structure of KF complexed with both Eu3+ and substrate single-stranded oligodeoxynucleotide shows that a lone Eu3+ is bound near to metal-ion site A. Comparison of this structure to a relevant native structure reveals that the bound Eu3+ causes a number of changes to the KF exo active site. The scissile phosphate of the substrate is displaced from its normal position by about 1 A when Eu3+ is bound and the presence of Eu3+ in the active site precludes the binding of the essential metal ion B. CONCLUSIONS: The substantial, lanthanide-induced differences in metal-ion and substrate binding to KF exo account for the inhibition of this enzyme by Eu3+. These changes also explain the inability of KF exo to bind more than one cation in the presence of lanthanides. The mechanistic similarity between KF exo and other two-metal-ion phosphoryl-transfer enzymes suggests that the principles of lanthanide (III) ion binding and inhibition ascertained from this study will probably apply to most members of this class of enzymes.