Phospholipase A2 (prokaryotic/fungal)

 

The secreted phospholipase A2 (PLA2) from Streptomyces violaceoruber is the first identified prokaryotic PLA2. This Ca II dependent, lipolytic enzyme hydrolyses the 2-acyl ester bonds of 1,2-diacylglycero-3-phospholipids. The very high catalytic activities of secreted PLA2's relative to the aggregated substrates when compared to the monomeric substrates is due to interfacial activation.

 

Reference Protein and Structure

Sequence
Q6UV28 UniProt IPR015141 (Sequence Homologues) (PDB Homologues)
Biological species
Streptomyces violaceoruber (Bacteria) Uniprot
PDB
1it4 - Solution structure of the prokaryotic Phospholipase A2 from Streptomyces violaceoruber (solution nmr Å) PDBe PDBsum 1it4
Catalytic CATH Domains
1.20.90.10 CATHdb (see all for 1it4)
Cofactors
Water (3), Calcium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.1.1.4)

1,2-diacyl-sn-glycero-3-phosphocholine
CHEBI:57643ChEBI
+
water
CHEBI:15377ChEBI
→
hydron
CHEBI:15378ChEBI
+
fatty acid anion
CHEBI:28868ChEBI
+
1-O-acyl-sn-glycero-3-phosphocholine
CHEBI:58168ChEBI
Alternative enzyme names: Lecithinase A, Phosphatidase, Phosphatidolipase, Phospholipase A, Phospholipase A2,

Enzyme Mechanism

Introduction

Prokaryotic PLA2 is Ca II dependent and catalyses the hydrolysis of the 2-acyl ester bonds of 1,2-diacylglycero-3-phospholipids. According to the enzyme model of interfacial activation, the conformational change in PLA2 induced by phospholipid binding optimises the active site and enhances catalysis in the enzyme. A catalytic dyad is formed between His 93 and Asp 114, and the hydrogen bonding network extends from these catalytic residues to the C-terminal region. His 93 is hydrogen bonded to Wat 260 via the His N-delta-1 atom. His 93 abstracts a proton from this water molecule, activating it towards nucleophilic attack of the carbonyl carbon of the substrate. Asp 114, which forms a hydrogen bond with His 93 Ne2 neutralises the positive charge of His 93 created by the ester bond cleavage during catalysis. The calcium binding site is formed by Asp 94 carboxylate, Asp 72 carboxylate, Leu 73 carbonyl oxygen and three water molecules. The Ca II is neccessary for enzymatic activity and helps to stabilise the His 93.

Catalytic Residues Roles

UniProt PDB* (1it4)
Asp72, Asp94, Leu73 (main-N) Asp43A, Asp65A, Leu44A (main-N) Coordinate the calcium ion. metal ligand
His93 His64A The His 64 is hydrogen bonded to Wat 260 via the His N-delta-1 atom. His 64 abstracts a proton from this water molecule, activating it towards nucleophilic attack of the carbonyl carbon of the substrate.
The histidine is aslo hydrogen bonded to Asp 85, forming the catalytic dyad.
His 64 interacts electrostatically with the Ca II cofactor for stabilisation.		 proton acceptor, electrostatic stabiliser, proton donor
Asp114 Asp85A Asp 85, which forms a hydrogen bond with His 64 Ne2 neutralises the positive charge of His 64 created by the ester bond cleavage during catalysis. increase basicity, 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, bimolecular nucleophilic addition, overall reactant used, coordination to a metal ion, decoordination from a metal ion, overall product formed, unimolecular elimination by the conjugate base

References

  1. Sugiyama M et al. (2002), J Biol Chem, 277, 20051-20058. A Novel Prokaryotic Phospholipase A2. CHARACTERIZATION, GENE CLONING, AND SOLUTION STRUCTURE. DOI:10.1074/jbc.m200264200. PMID:11897786.
  2. Matoba Y et al. (2003), Proteins, 51, 453-469. Atomic resolution structure of prokaryotic phospholipase A2: Analysis of internal motion and implication for a catalytic mechanism. DOI:10.1002/prot.10360. PMID:12696056.
  3. Matoba Y et al. (2002), J Biol Chem, 277, 20059-20069. The crystal structure of prokaryotic phospholipase A2. DOI:10.1074/jbc.M200263200. PMID:11897785.

Step 1. The His93 and Asp114 dyad activates a water molecule which performs a nucleophilic attack on the ester carbonyl carbon. This results in an oxyanion intermediate forming which is stabilized by the calcium ion- the oxyanion becomes ligated to the calcium in the place of one of the water molecules.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His64A electrostatic stabiliser
Asp85A electrostatic stabiliser
Asp85A increase basicity
Asp43A metal ligand
Leu44A (main-N) metal ligand
Asp65A metal ligand
His64A proton acceptor

Chemical Components

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

Step 2. The tetrahedral intermediate collapses with His93 protonating the leaving group- generating the products.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp85A electrostatic stabiliser
Asp43A metal ligand
Leu44A (main-N) metal ligand
Asp65A metal ligand
His64A proton donor

Chemical Components

overall product formed, proton transfer, ingold: unimolecular elimination by the conjugate base
Download: Image, Marvin File

Step 1. The His93 and Asp114 dyad activates a water molecule which performs a nucleophilic attack on the ester carbonyl carbon. This results in an oxyanion intermediate forming which is stabilized by the calcium ion- the oxyanion becomes ligated to the calcium in the place of one of the water molecules.

Step 2. The tetrahedral intermediate collapses with His93 protonating the leaving group- generating the products.

Products.

Contributors

Emma Penn, Gemma L. Holliday, James Willey