Exodeoxyribonuclease (lambda-induced)

 

The 5'-3' exonuclease enzymes hydrolyse DNA.DNA and RNA.DNA substrates in a structurally specific manner. These enzymes are necessary for efficient cellular DNA replication, with the uncatalysed reaction having a half life of 30 million years at room temperature. The enzymes enhance the rate by 10^15-10^17, allowing the reaction to proceed on a biologically useful time scale.

 

Reference Protein and Structure

Sequence
P06229 UniProt (3.1.11.-, 3.1.11.3) IPR002421 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia phage T5 Uniprot
PDB
1exn - T5 5'-EXONUCLEASE (2.5 Å) PDBe PDBsum 1exn
Catalytic CATH Domains
3.40.50.1010 CATHdb 1.10.150.20 CATHdb (see all for 1exn)
Cofactors
Magnesium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:3.1.11.3)

single-stranded DNA
CHEBI:9160ChEBI
+
water
CHEBI:15377ChEBI
→
5'-end 2'-deoxyribonucleotide(2-) residue
CHEBI:136412ChEBI
+
2'-deoxynucleoside 3'-monophosphate(2-)
CHEBI:131705ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: E. coli exonuclease IV, Escherichia coli exonuclease IV, Exodeoxyribonuclease IV, Exonuclease IV, Lambda exonuclease, Phage lambda-induced exonuclease,

Enzyme Mechanism

Introduction

There are several proposed mechanisms for the action of 5'-3' exonuclease, taking into account many possible mechanisms involving up to three catalytic divalent metal ions. The most accepted mechanism implies the involvement of two catalytic Mg(2+) ions. A water molecule is deprotonated by the conserved basic Lys 83, which remains deprotonated at pH 6 through interactions with a positively charged dicationic metal and the positively charged guanidinium group of Arg 86 (also a conserved residue). This water molecule then acts as a nucleophile towards the scissile phosphate. The pentavalent intermediate is stabilised by a second dicationic Mg while the scissile phosphate oxygen is coordinated to both metal centres. The collapse of the intermediate and reprotonation of the 3' leaving group from the Lys 83 gives the hydrolysis products. Crystallographic evidence suggests a two-metal-ion catalytic process in homologous enzymes, although this pdb doesn't show the Mg atoms implicated. Later kinetic evidence suggests the presence of three metal cations is necessary. It has also been proposed on evidence from mutagenesis reactions that the endo- and exonucleolytic cleavage mechanisms are distinct.

Catalytic Residues Roles

UniProt PDB* (1exn)
Asp153 Asp153(152)A Forms part of the binding site for both divalent metal ions. metal ligand
Asp155, Asp201, Asp204 Asp155(154)A, Asp201(200)A, Asp204(203)A Forms part of the second divalent metal ion binding site. metal ligand
Asp131, Asp26, Asp68, Glu128 Asp131(130)A, Asp26(25)A, Asp68(67)A, Glu128(127)A Forms part of the first divalent metal ion binding site. metal ligand
Arg86 Arg86(85)A The residue's side chain influences the pKa of the general base Lys 83, enhancing its ability to remain deprotonated at relatively low pH levels. electrostatic stabiliser
Lys83 Lys83(82)A The residue acts as a general base towards a water molecule, activating it for nucleophilic attack at the scissile phosphate. Its pKa is modified by interactions with the guanidinium group of Arg 86 and the positive charge of a dicationic Mg ion. The resulting protonated residue then acts as a general acid to the 3' leaving group. proton shuttle (general acid/base), 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

References

  1. Garforth SJ et al. (1999), Proc Natl Acad Sci U S A, 96, 38-43. Mutagenesis of conserved lysine residues in bacteriophage T5 5'-3' exonuclease suggests separate mechanisms of endoand exonucleolytic cleavage. DOI:10.1073/pnas.96.1.38. PMID:9874768.
  2. AlMalki FA et al. (2016), Nat Struct Mol Biol, 23, 640-646. Direct observation of DNA threading in flap endonuclease complexes. DOI:10.1038/nsmb.3241. PMID:27273516.
  3. Algasaier SI et al. (2016), J Biol Chem, 291, 8258-8268. DNA and Protein Requirements for Substrate Conformational Changes Necessary for Human Flap Endonuclease-1-catalyzed Reaction. DOI:10.1074/jbc.M115.698993. PMID:26884332.
  4. Exell JC et al. (2016), Nat Chem Biol, 12, 815-821. Cellularly active N-hydroxyurea FEN1 inhibitors block substrate entry to the active site. DOI:10.1038/nchembio.2148. PMID:27526030.
  5. Tomlinson CG et al. (2011), J Biol Chem, 286, 30878-30887. Neutralizing mutations of carboxylates that bind metal 2 in T5 flap endonuclease result in an enzyme that still requires two metal ions. DOI:10.1074/jbc.M111.230391. PMID:21734257.
  6. Syson K et al. (2008), J Biol Chem, 283, 28741-28746. Three Metal Ions Participate in the Reaction Catalyzed by T5 Flap Endonuclease. DOI:10.1074/jbc.m801264200. PMID:18697748.
  7. Yang W et al. (2006), Mol Cell, 22, 5-13. Making and Breaking Nucleic Acids: Two-Mg2+-Ion Catalysis and Substrate Specificity. DOI:10.1016/j.molcel.2006.03.013. PMID:16600865.
  8. Feng M et al. (2004), Nat Struct Mol Biol, 11, 450-456. Roles of divalent metal ions in flap endonuclease-substrate interactions. DOI:10.1038/nsmb754. PMID:15077103.
  9. Tock MR et al. (2003), EMBO J, 22, 995-1004. Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease. DOI:10.1093/emboj/cdg098. PMID:12606565.

Step 1.

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Catalytic Residues Roles

Residue Roles
Lys83(82)A proton shuttle (general acid/base), electrostatic stabiliser
Arg86(85)A electrostatic stabiliser
Asp26(25)A metal ligand
Asp68(67)A metal ligand
Glu128(127)A metal ligand
Asp131(130)A metal ligand
Asp153(152)A metal ligand
Asp155(154)A metal ligand
Asp201(200)A metal ligand
Asp204(203)A metal ligand

Chemical Components

Step 1.

Contributors

James W. Murray, Craig Porter, Gemma L. Holliday