Colicin-E7

 

The ColE7 endonuclease from Escherichia coli binds nucleic acids and hydrolyses a phosphodiester bond. It contains the His-metal finger motif and may function with a range of metals, although the physiological metal is thought to be Zn(II).

 

Reference Protein and Structure

Sequences
Q03708 UniProt
Q47112 UniProt (3.1.-.-) IPR024575 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia coli (Bacteria) Uniprot
PDB
1znv - How a His-metal finger endonuclease ColE7 binds and cleaves DNA with a transition metal ion cofactor (2.0 Å) PDBe PDBsum 1znv
Catalytic CATH Domains
3.90.540.10 CATHdb (see all for 1znv)
Cofactors
Zinc(2+) (1), Nickel(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.1.-.-)

RNA (poly(A))
CHEBI:8756ChEBI
+
water
CHEBI:15377ChEBI
→
RNA (poly(A))
CHEBI:8756ChEBI
+
RNA (poly(A))
CHEBI:8756ChEBI

Enzyme Mechanism

Introduction

A water nucleophile is polarised by His 545, which acts as a general base to generate hydroxide. An non-bridging oxygen of the scissile phosphodiester group coordinates to Zn(II), making the phosphorus more electrophilic. Hydroxide attacks the scissile phosphodiester bond. Zn(II) stabilises the charge build-up in the pentacovalent transition state. The transition state collapses upon protonation of the leaving group oxygen by Arg 447, with Zn(II) stabilising the charge on the terminal 3' hydroxyl cleavage product.

Catalytic Residues Roles

UniProt PDB* (1znv)
Arg447 ArgNone(5)B Stabilises the oxyanion intermediate and also protonates the 3' leaving group. proton acceptor, proton relay, electrostatic stabiliser, proton donor
His573, His569, His544 His573(131)B, His569(127)B, His544(102)B Forms Zinc binding site metal ligand
His545 Glu545(103)B His 545 acts as the general base catalyst, deprotonating water as it attacks as a nucleophile. proton acceptor, proton donor
*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 addition, coordination to a metal ion, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, decoordination from a metal ion, intermediate collapse, native state of enzyme regenerated, overall product formed

References

  1. Doudeva LG et al. (2006), Protein Sci, 15, 269-280. Crystal structural analysis and metal-dependent stability and activity studies of the ColE7 endonuclease domain in complex with DNA/Zn2+ or inhibitor/Ni2+. DOI:10.1110/ps.051903406. PMID:16434744.
  2. Németh E et al. (2014), J Biol Inorg Chem, 19, 1295-1303. Substrate binding activates the designed triple mutant of the colicin E7 metallonuclease. DOI:10.1007/s00775-014-1186-6. PMID:25156149.
  3. Németh E et al. (2014), Protein Sci, 23, 1113-1122. Fine tuning of the catalytic activity of colicin E7 nuclease domain by systematic N-terminal mutations. DOI:10.1002/pro.2497. PMID:24895333.
  4. Czene A et al. (2014), Metallomics, 6, 2090-2099. A new insight into the zinc-dependent DNA-cleavage by the colicin E7 nuclease: a crystallographic and computational study. DOI:10.1039/C4MT00195H. PMID:25179124.
  5. Czene A et al. (2013), J Biol Inorg Chem, 18, 309-321. The role of the N-terminal loop in the function of the colicin E7 nuclease domain. DOI:10.1007/s00775-013-0975-7. PMID:23334162.

Step 1. His545 abstracts a proton from a water which activates it to nucleophilically attack the phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His544(102)B metal ligand
His569(127)B metal ligand
His573(131)B metal ligand
ArgNone(5)B electrostatic stabiliser
Glu545(103)B proton acceptor

Chemical Components

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

Step 2. Arg447 protonates the leaving group oxygen which initiates an elimination from the oxyanion and results the cleavage if the phosphodiester bond. Arg447 is reprotonated by His545.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
ArgNone(5)B electrostatic stabiliser
His544(102)B metal ligand
His569(127)B metal ligand
His573(131)B metal ligand
Glu545(103)B proton donor
ArgNone(5)B proton acceptor, proton relay, proton donor

Chemical Components

proton transfer, ingold: unimolecular elimination by the conjugate base, proton transfer, decoordination from a metal ion, intermediate collapse, native state of enzyme regenerated, overall product formed
Download: Image, Marvin File

Step 1. His545 abstracts a proton from a water which activates it to nucleophilically attack the phosphate.

Step 2. Arg447 protonates the leaving group oxygen which initiates an elimination from the oxyanion and results the cleavage if the phosphodiester bond. Arg447 is reprotonated by His545.

Products.

Contributors

Jonathan T. W. Ng, Gemma L. Holliday, Charity Hornby