Colicin-E9

 

Colicin E9 from Escherichia coli is a non-specific endonuclease. This extracellular toxin is secreted by strains of Escherichia coli to kill competing bacteria. It kills the cell by enzymatic cleavage of nucleic acids once it has entered the cell. Since colicin E9 is non-specific, it can hydrolyse both single and double stranded DNA.

 

Reference Protein and Structure

Sequences
P13479 UniProt
P09883 UniProt (3.1.-.-) IPR024575 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli (Bacteria) Uniprot
PDB
1fr2 - CRYSTAL STRUCTURE OF THE E9 DNASE DOMAIN WITH A MUTANT IMMUNITY PROTEIN IM9(E41A) (1.6 Å) PDBe PDBsum 1fr2
Catalytic CATH Domains
3.90.540.10 CATHdb (see all for 1fr2)
Cofactors
Zinc(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.1.21.1)

single-stranded DNA
CHEBI:9160ChEBI
+
water
CHEBI:15377ChEBI
→
2'-deoxynucleoside 3'-monophosphate(2-)
CHEBI:131705ChEBI
+
5'-end 2'-deoxyribonucleotide(2-) residue
CHEBI:136412ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Escherichia coli endonuclease I, DNA depolymerase, DNA endonuclease, DNA nuclease, DNAase, DNase, DNase I, Alkaline DNase, Alkaline deoxyribonuclease, Deoxyribonuclease (pancreatic), Deoxyribonuclease A, Deoxyribonucleic phosphatase, Dornava, Dornavac, Endodeoxyribonuclease I, Pancreatic DNase, Pancreatic deoxyribonuclease, Pancreatic dornase, Thymonuclease, dornase, Thymonuclease,

Enzyme Mechanism

Introduction

His 103 acts as a general base by abstracting a proton from a water molecule, activating it for nucleophilic attack on the scissile phosphorus atom. This leads to a penta-covalent intermediate being formed. The Zn ion activates a water molecule so it will donate a proton to the 3' hydroxyl leaving group. The intermediate then collapses forming the products.

Catalytic Residues Roles

UniProt PDB* (1fr2)
Glu548, Arg544 Glu100B, Arg96B Stabilize the histidine residues involved in metal coordination. electrostatic stabiliser
His575, His579, His550 His127B, His131B, His102B Coordinate the Zn ion metal ligand
His551 His103B Acts as a general base by deprotonating a water molecule, activating it for nucleophilic attack on the phosphorus atom. increase nucleophilicity, proton acceptor, proton donor
Arg453 Arg5B Stabilizes the negatively charged intermediate. electrostatic stabiliser
*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

bimolecular nucleophilic addition, proton transfer, overall reactant used, coordination to a metal ion, decoordination from a metal ion, overall product formed, heterolysis, inferred reaction step, native state of enzyme regenerated

References

  1. Pommer AJ et al. (2001), J Mol Biol, 314, 735-749. Mechanism and cleavage specificity of the H-N-H endonuclease colicin E9. DOI:10.1006/jmbi.2001.5189. PMID:11733993.
  2. Maté MJ et al. (2004), J Biol Chem, 279, 34763-34769. Structure-based analysis of the metal-dependent mechanism of H-N-H endonucleases. DOI:10.1074/jbc.M403719200. PMID:15190054.

Step 1. His551 activates a water molecule allowing the water to perform a nucleophilic attack on the scissile phosphate group. This leads to a penta-covalent intermediate being formed which is stabilized by Arg453. After this has occurred a second water molecule replaces His579 in its coordination to the Zn ion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu100B electrostatic stabiliser
Arg5B electrostatic stabiliser
His103B increase nucleophilicity
His102B metal ligand
His127B metal ligand
His131B metal ligand
Arg96B electrostatic stabiliser
His103B proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer, overall reactant used, coordination to a metal ion, decoordination from a metal ion

Step 2. The second water molecule is activated by the Zn ion allowing it to transfer a proton to the 3' hydroxyl leaving group. This proton transfer facilitates the collapse of the intermediate and the formation of the products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His102B metal ligand
His127B metal ligand
Arg5B electrostatic stabiliser
Arg96B electrostatic stabiliser
Glu100B electrostatic stabiliser

Chemical Components

overall product formed, proton transfer, heterolysis

Step 3. In this inferred reaction step His551 donates a proton to the hydroxide ion to regenerate the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His102B metal ligand
His127B metal ligand
His103B proton donor

Chemical Components

inferred reaction step, proton transfer, native state of enzyme regenerated
Download: Image, Marvin File

Step 1. His551 activates a water molecule allowing the water to perform a nucleophilic attack on the scissile phosphate group. This leads to a penta-covalent intermediate being formed which is stabilized by Arg453. After this has occurred a second water molecule replaces His579 in its coordination to the Zn ion.

Step 2. The second water molecule is activated by the Zn ion allowing it to transfer a proton to the 3' hydroxyl leaving group. This proton transfer facilitates the collapse of the intermediate and the formation of the products.

Step 3. In this inferred reaction step His551 donates a proton to the hydroxide ion to regenerate the active site.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, James Willey