DNA topoisomerase (type III)

 

Topoisomerases catalyse the winding and unwinding of super-coiled DNA, a process vital during DNA replication, transcription and repair. Type I topoisomerases reversibly cut one strand of the DNA helix, and can only relax supercoils. Analysis of sequence and mutagenesis data indicates that the type Ia topoisomerases (those found in prokaryotes, and the example given here) may be related to the major family of type II topoisomerases (found in all life apart from some archaea). Type II topoisomerases cut both strands and can either relax or tighten super-coils, but require ATP hydrolysis for catalytic turnover. All topoisomerases recorded form an intermediate with a tyr-DNA covalent bond during catalysis.

 

Reference Protein and Structure

Sequence
P14294 UniProt (5.6.2.1) IPR005738 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli K-12 (Bacteria) Uniprot
PDB
1d6m - CRYSTAL STRUCTURE OF E. COLI DNA TOPOISOMERASE III (3.0 Å) PDBe PDBsum 1d6m
Catalytic CATH Domains
1.10.290.10 CATHdb 3.40.50.140 CATHdb (see all for 1d6m)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:5.6.2.1)

single-stranded DNA
CHEBI:9160ChEBI
→
single-stranded DNA
CHEBI:9160ChEBI
Alternative enzyme names: Omega-protein, Nicking-closing enzyme, Relaxing enzyme, Swivelase, Type I DNA topoisomerase, Untwisting enzyme, DNA topoisomerase I,

Enzyme Mechanism

Introduction

The Tyr328 acts as a nucleophile towards the phosphorous of the DNA molecule, forming a pentavalent transition state before eliminating the 5' DNA with concomitant deprotonation of Glu7. The DNA uncoils before Glu7 can deprotonate the 5'-OH, which initiates a nucleophilic attack upon the phosphorous atom. This again proceeds through a pentavalent transition state before eliminating Tyr328 and restoring the enzyme active site. The reaction requires Mg(II), which is bound by acidic glutamine residues. The metal's importance is shown by the detrimental effect to binding and activity brought about by mutation at these sites, however the role of the Mg as either structurally or catalytically essential is still uncertain.

Catalytic Residues Roles

UniProt PDB* (1d6m)
Glu7 Glu7A Acts as a general acid/base. hydrogen bond acceptor, hydrogen bond donor, metal ligand, proton acceptor, proton donor
Tyr328 Tyr328A Acts as the catalytic nucleophile. hydrogen bond acceptor, nucleofuge, nucleophile
Arg330, Lys8 Arg330A, Lys8A Stabilises the reactive intermediates and are essential for bringing the two halves back together again. hydrogen bond donor, electrostatic stabiliser
Asp103 Asp103A Forms part of the magnesium 1 binding site. metal ligand
Asp105, Asp103 Asp105A, Asp103A Forms part of the magnesium 2 binding site. metal ligand
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation, overall product formed, enzyme-substrate complex cleavage, intermediate terminated, intermediate collapse, native state of enzyme regenerated

References

  1. Changela A et al. (2001), Nature, 411, 1077-1081. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule. DOI:10.1038/35082615. PMID:11429611.
  2. Zhang Z et al. (2011), Proc Natl Acad Sci U S A, 108, 6939-6944. Crystal structure of a covalent intermediate in DNA cleavage and rejoining by Escherichia coli DNA topoisomerase I. DOI:10.1073/pnas.1100300108. PMID:21482796.
  3. Narula G et al. (2011), J Biol Chem, 286, 18673-18680. The Strictly Conserved Arg-321 Residue in the Active Site of Escherichia coli Topoisomerase I Plays a Critical Role in DNA Rejoining. DOI:10.1074/jbc.m111.229450. PMID:21478161.
  4. Bugreev DV et al. (2009), Biochemistry (Mosc), 74, 1467-1481. Structure and mechanism of action of type IA DNA topoisomerases. DOI:10.1134/s0006297909130045. PMID:20210704.
  5. Xiong B et al. (2008), Proteins, 71, 1984-1994. The type IA topoisomerase catalytic cycle: A normal mode analysis and molecular dynamics simulation. DOI:10.1002/prot.21876. PMID:18186484.
  6. Champoux JJ (2002), Trends Pharmacol Sci, 23, 199-201. A first view of the structure of a type IA topoisomerase with bound DNA. DOI:10.1016/s0165-6147(02)01997-1. PMID:12007989.
  7. Chen SJ et al. (1998), J Biol Chem, 273, 6050-6056. Identification of Active Site Residues in Escherichia coli DNA Topoisomerase I. DOI:10.1074/jbc.273.11.6050. PMID:9497321.

Step 1. Tyr328 acts as a nucleophile, attacking the phosphorous of the DNA molecule, which proceeds through a pentavalent transition state, before eliminating the 5' DNA, with concomitant deprotonation of Glu7.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys8A hydrogen bond donor, electrostatic stabiliser
Glu7A hydrogen bond donor
Tyr328A hydrogen bond acceptor
Arg330A hydrogen bond donor, electrostatic stabiliser
Glu7A metal ligand
Asp103A metal ligand
Asp105A metal ligand
Glu7A proton donor
Tyr328A nucleophile

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, enzyme-substrate complex formation, intermediate formation

Step 2. The DNA uncoils before Glu7 deprotonates the 5'-OH, which initiates a nucleophilic attack upon the phosphorous atom, which proceeds through a pentavalent transition state, before eliminating Tyr328.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Tyr328A hydrogen bond acceptor
Lys8A hydrogen bond donor, electrostatic stabiliser
Glu7A hydrogen bond acceptor
Arg330A hydrogen bond donor, electrostatic stabiliser
Glu7A metal ligand
Asp103A metal ligand
Asp105A metal ligand
Glu7A proton acceptor
Tyr328A nucleofuge

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall product formed, enzyme-substrate complex cleavage, intermediate terminated, intermediate collapse, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. Tyr328 acts as a nucleophile, attacking the phosphorous of the DNA molecule, which proceeds through a pentavalent transition state, before eliminating the 5' DNA, with concomitant deprotonation of Glu7.

Step 2. The DNA uncoils before Glu7 deprotonates the 5'-OH, which initiates a nucleophilic attack upon the phosphorous atom, which proceeds through a pentavalent transition state, before eliminating Tyr328.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Anna Waters, Craig Porter