Exodeoxyribonuclease (lambda-induced)

 

Lambda-exonuclease from Bacteriophage lambda is a 5'->3' exonuclease. It catalyses the hydrolysis of one strand of double stranded DNA in a 5' to 3' direction, leaving single stranded DNA. This is involved in DNA replication, recombination and repair. Once started on a reaction, lambda-exonuclease will continue to hydrolyse until either the DNA strand ends, or the enzyme dissociates into monomers.

 

Reference Protein and Structure

Sequence
P03697 UniProt (3.1.11.3) IPR019080 (Sequence Homologues) (PDB Homologues)
Biological species
Escherichia virus Lambda (Virus) Uniprot
PDB
1avq - TOROIDAL STRUCTURE OF LAMBDA EXONUCLEASE DETERMINED AT 2.4 ANGSTROMS (2.4 Å) PDBe PDBsum 1avq
Catalytic CATH Domains
3.90.320.10 CATHdb (see all for 1avq)
Cofactors
Magnesium(2+) (3)
Click To Show Structure

Enzyme Reaction (EC:3.1.11.3)

water
CHEBI:15377ChEBI
+
single-stranded DNA
CHEBI:9160ChEBI
→
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

The three-metal mechanism of EcoRV is the most likely mechanism for lambda-exonuclease. This is a dissociative hydrolysis mechanism where the O-P bond is broken before the nucleophilic of water upon the phosphate. The dissociation of the O-P bond is facilitated by protonation from a magnesium activated water molecule. And the nucleophilic attack is facilitated by Glu129 deprotonating a second water promoting the attack.

Catalytic Residues Roles

UniProt PDB* (1avq)
Glu129 Glu129(131)A Activates the nucleophilic water increase nucleophilicity, metal ligand, proton acceptor, proton donor
Asp109, Glu129, Glu93, Leu130 (main-C) Asp109(111)A, Glu129(131)A, Glu93(95)A, Leu130(132)A (main-C) Involved in coordinating the magnesium ions metal ligand
Glu102 Glu102(104)A Stabilizes a water molecule which coordinates the magnesium ion electrostatic stabiliser
Lys131 Lys131(133)A Stabilizes the nucleophilic water so its in the correct orientation to perform a nucleophilic attack 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

proton transfer, overall reactant used, overall product formed, heterolysis, bimolecular nucleophilic addition, native state of enzyme regenerated, proton relay

References

  1. Kovall RA et al. (1999), Curr Opin Chem Biol, 3, 578-583. Type II restriction endonucleases: structural, functional and evolutionary relationships. DOI:10.1016/s1367-5931(99)00012-5. PMID:10508668.
  2. Imhof P et al. (2009), Biochemistry, 48, 9061-9075. Catalytic mechanism of DNA backbone cleavage by the restriction enzyme EcoRV: a quantum mechanical/molecular mechanical analysis. DOI:10.1021/bi900585m. PMID:19678693.

Step 1. A magnesium activated water molecule donates a proton to the 5' hydroxyl group promoting its dissociation from the phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys131(133)A electrostatic stabiliser
Glu93(95)A metal ligand
Asp109(111)A metal ligand
Glu129(131)A metal ligand
Leu130(132)A (main-C) metal ligand
Glu102(104)A electrostatic stabiliser

Chemical Components

proton transfer, overall reactant used, overall product formed, heterolysis

Step 2. Glu129 rotates to become closer to a second water molecule. It then deprotonates the water molecule allowing the water to perform a nucleophilic attack on the phosphate. Lys131 is thought to be responsible for orientating the water molecule for nucleophilic attack.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu93(95)A metal ligand
Asp109(111)A metal ligand
Glu129(131)A metal ligand
Leu130(132)A (main-C) metal ligand
Glu102(104)A electrostatic stabiliser
Glu129(131)A increase nucleophilicity
Lys131(133)A electrostatic stabiliser
Glu129(131)A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton transfer

Step 3. Glu129 donates a proton to a phosphate oxygen regenrating the residue in its native state.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu93(95)A metal ligand
Asp109(111)A metal ligand
Glu129(131)A metal ligand
Leu130(132)A (main-C) metal ligand
Glu102(104)A electrostatic stabiliser
Lys131(133)A electrostatic stabiliser
Glu129(131)A proton donor

Chemical Components

proton transfer, native state of enzyme regenerated

Step 4. The leaving group hydroxyl acts as a proton relay deprotonating the phosphate group and protonating the metal coordinated hydroxide ion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu93(95)A metal ligand
Asp109(111)A metal ligand
Glu129(131)A metal ligand
Leu130(132)A (main-C) metal ligand
Glu102(104)A electrostatic stabiliser
Lys131(133)A electrostatic stabiliser

Chemical Components

proton relay, overall product formed
Download: Image, Marvin File

Step 1. A magnesium activated water molecule donates a proton to the 5' hydroxyl group promoting its dissociation from the phosphate.

Step 2. Glu129 rotates to become closer to a second water molecule. It then deprotonates the water molecule allowing the water to perform a nucleophilic attack on the phosphate. Lys131 is thought to be responsible for orientating the water molecule for nucleophilic attack.

Step 3. Glu129 donates a proton to a phosphate oxygen regenrating the residue in its native state.

Step 4. The leaving group hydroxyl acts as a proton relay deprotonating the phosphate group and protonating the metal coordinated hydroxide ion.

Products.

Introduction

In this associative mechanism Glu129 activates a water molecule for nucleophilic attack on the scissile phosphate. A penta-covalent intermediate is formed which collapses to form the products and restore the enzyme to its native state. See other mechanism for the references.

Catalytic Residues Roles

UniProt PDB* (1avq)
Glu129 Glu129(131)A Activates the water molecule for nucleophilic attack by deprotonating it. metal ligand, proton acceptor, proton donor
Asp109, Glu93, Leu130 (main-C) Asp109(111)A, Glu93(95)A, Leu130(132)A (main-C) Coordinate the magnesium ion. metal ligand
Glu102 Glu102(104)A Stabilizes a magnesium coordinating water molecule. electrostatic stabiliser
Lys131 Lys131(133)A Orientate the water molecule for nucleophillic attack. 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

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

References

Step 1. Glu129 deprotonates a water molecule this will activate it for nucleophillic attack. The deprotonating oxygen of Glu129 and the hydroxide will decoordinate from the magnesium ions after the proton transfer has occurred.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu102(104)A electrostatic stabiliser
Leu130(132)A (main-C) metal ligand
Glu129(131)A metal ligand
Asp109(111)A metal ligand
Glu93(95)A metal ligand
Lys131(133)A electrostatic stabiliser
Glu129(131)A proton acceptor

Chemical Components

proton transfer, decoordination from a metal ion

Step 2. The activated hydroxide ion performs a nucleophillic on the phosphate group forming a penta-covalent intermediate. Lys131 acts to orientate the hydroxide ion for the nucleophilic attack.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu93(95)A metal ligand
Asp109(111)A metal ligand
Leu130(132)A (main-C) metal ligand
Glu102(104)A electrostatic stabiliser
Lys131(133)A electrostatic stabiliser

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used

Step 3. The intermediate collapses forming the products. This is facilitated by the leaving group hydroxyl accepting a proton from a water molecule with the concomitant deprotonation of Glu129 by the same water molecule. Glu129 can now re-coordinate to the magnesium ion.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu93(95)A metal ligand
Asp109(111)A metal ligand
Leu130(132)A (main-C) metal ligand
Glu102(104)A electrostatic stabiliser
Lys131(133)A electrostatic stabiliser
Glu129(131)A proton donor

Chemical Components

overall product formed, coordination to a metal ion, heterolysis, native state of enzyme regenerated, proton transfer, proton relay
Download: Image, Marvin File

Step 1. Glu129 deprotonates a water molecule this will activate it for nucleophillic attack. The deprotonating oxygen of Glu129 and the hydroxide will decoordinate from the magnesium ions after the proton transfer has occurred.

Step 2. The activated hydroxide ion performs a nucleophillic on the phosphate group forming a penta-covalent intermediate. Lys131 acts to orientate the hydroxide ion for the nucleophilic attack.

Step 3. The intermediate collapses forming the products. This is facilitated by the leaving group hydroxyl accepting a proton from a water molecule with the concomitant deprotonation of Glu129 by the same water molecule. Glu129 can now re-coordinate to the magnesium ion.

Products.

Introduction

This is another dissociative mechanism where the first step is identical to the first proposal. These mechanisms differ because in this proposal Lys131 acts as a proton relay to activate the water molecule for nucleophilic attack. See first mechanism for the references.

Catalytic Residues Roles

UniProt PDB* (1avq)
Asp109, Glu129, Glu93, Leu130 (main-C) Asp109(111)A, Glu129(131)A, Glu93(95)A, Leu130(132)A (main-C) Coordinate the magnesium ion metal ligand
Glu102 Glu102(104)A Stabilizes a water molecule coordinated to the magnesium ion. electrostatic stabiliser
Lys131 Lys131(133)A Acts as a proton relay to activate the water mole clue for nucleophilic attack. proton relay, proton acceptor, electrostatic stabiliser, 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

heterolysis, overall product formed, overall reactant used, proton transfer, bimolecular nucleophilic addition, proton relay

References

Step 1. A magnesium activated water molecule donates a proton to the 5' hydroxyl group promoting its dissociation from the phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu102(104)A electrostatic stabiliser
Leu130(132)A (main-C) metal ligand
Glu129(131)A metal ligand
Asp109(111)A metal ligand
Glu93(95)A metal ligand
Lys131(133)A electrostatic stabiliser

Chemical Components

heterolysis, overall product formed, overall reactant used, proton transfer

Step 2. Lys131 acts as a proton relay to promote the nucleophilic attack of a water molecule on to the phosphate group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu93(95)A metal ligand
Asp109(111)A metal ligand
Glu129(131)A metal ligand
Leu130(132)A (main-C) metal ligand
Glu102(104)A electrostatic stabiliser
Lys131(133)A electrostatic stabiliser
Lys131(133)A proton relay
Lys131(133)A proton donor, proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, proton relay, overall product formed, proton transfer
Download: Image, Marvin File

Step 1. A magnesium activated water molecule donates a proton to the 5' hydroxyl group promoting its dissociation from the phosphate.

Step 2. Lys131 acts as a proton relay to promote the nucleophilic attack of a water molecule on to the phosphate group.

Products.

Contributors

Ellie Wright, Gemma L. Holliday, James Willey