Phospholipase D

 

Phospholipase D catalyses the hydrolysis of phospholipids (normally phosphatidyl choline) into phosphatidic acid (PA) and the polar head group (normally choline). The PA generated can be converted into second messengers such as diacylglycerol (DAG) and is also suggested to cause changes in lipid bilayer properties. Phospholipase D will additionally catalyse transphosphatidylation reactions in vitro when an alcohol is present as the nucleophilic donor. It has been shown to be part of a superfamily that includes cardiolipin synthases, phosphatidyl serine synthases, poxvirus envelope proteins and several endonucleases.

 

Reference Protein and Structure

Sequence
P84147 UniProt (3.1.4.4) IPR025202 (Sequence Homologues) (PDB Homologues)
Biological species
Streptomyces sp. PMF (Bacteria) Uniprot
PDB
1v0y - Phospholipase D from Streptomyces sp. strain PMF soaked with the substrate dibutyrylphosphatidylcholine. (1.71 Å) PDBe PDBsum 1v0y
Catalytic CATH Domains
3.30.870.10 CATHdb (see all for 1v0y)
Click To Show Structure

Enzyme Reaction (EC:3.1.4.4)

1,2-diacyl-sn-glycero-3-phosphocholine
CHEBI:57643ChEBI
+
water
CHEBI:15377ChEBI
→
choline
CHEBI:15354ChEBI
+
1,2-diacyl-sn-glycerol 3-phosphate(2-)
CHEBI:58608ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Choline phosphatase, Lecithinase D, Lipophosphodiesterase II,

Enzyme Mechanism

Introduction

The catalysed hydrolysis reaction occurs in two steps. First, nucleophilic attack by His 170 on the substrate phosphate leads to displacement of the alcohol head group (e.g. choline if the substrate is phosphatidyl choline) and formation of a His-phosphatidate intermediate with a covalent P-N bond to His 170 N-epsilon. The departing polar head group is protonated by His 448. In the second step, His 448 deprotonates a water molecule that attacks the His-phosphatidate intermediate to displace His 170 and produce phosphatidate. Asp 473 and Asp 202 interact with and modify the pKa values of His 170 and His 448 respectively.

Catalytic Residues Roles

UniProt PDB* (1v0y)
His167 His170(167)A Attacks phosphate of substrate to form a covalent His-Phosphatidate intermediate that is subsequently hydrolysed. covalently attached, nucleofuge, nucleophile
Asp199 Asp202(199)A Modifies pKa of His 448. modifies pKa, electrostatic stabiliser
His440 His448(440)A Protonates departing alcohol group (eg choline) during formation of the His-Phosphatidate intermediate. Later deprotonates the water molecule that attacks this intermediate. increase nucleophilicity, proton acceptor, proton donor
Asp465 Asp473(465)A Modifies pKa of His 170. increase nucleophilicity, 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

overall reactant used, intermediate formation, bimolecular nucleophilic addition, overall product formed, unimolecular elimination by the conjugate base, proton transfer, intermediate terminated, bimolecular nucleophilic substitution, native state of enzyme regenerated

References

  1. Leiros I et al. (2004), J Mol Biol, 339, 805-820. The Reaction Mechanism of Phospholipase D from Streptomyces sp. Strain PMF. Snapshots along the Reaction Pathway Reveal a Pentacoordinate Reaction Intermediate and an Unexpected Final Product. DOI:10.1016/j.jmb.2004.04.003. PMID:15165852.
  2. Mahankali M et al. (2015), Cell Signal, 27, 69-81. Mechanism of enzymatic reaction and protein-protein interactions of PLD from a 3D structural model. DOI:10.1016/j.cellsig.2014.09.008. PMID:25308783.
  3. Gottlin EB et al. (1998), Proc Natl Acad Sci U S A, 95, 9202-9207. Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate. DOI:10.1073/pnas.95.16.9202. PMID:9689058.

Step 1. His170 attacks the phosphate forming a penta-covalent intermediate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp473(465)A electrostatic stabiliser
His170(167)A covalently attached
Asp473(465)A increase nucleophilicity
His170(167)A nucleophile

Chemical Components

overall reactant used, intermediate formation, ingold: bimolecular nucleophilic addition

Step 2. The penta-covalent intermediate collapses and His448 donates a proton to the choline leaving group.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His170(167)A covalently attached
Asp202(199)A electrostatic stabiliser, modifies pKa
His448(440)A proton donor

Chemical Components

overall product formed, ingold: unimolecular elimination by the conjugate base, proton transfer

Step 3. His448 deprotonates a water molecule, activating it for nucleophilic attack on the phosphate. This causes His170 to be displaced.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp202(199)A electrostatic stabiliser
Asp473(465)A electrostatic stabiliser
Asp202(199)A modifies pKa
His448(440)A increase nucleophilicity
His170(167)A nucleofuge
His448(440)A proton acceptor

Chemical Components

overall product formed, intermediate terminated, ingold: bimolecular nucleophilic substitution, native state of enzyme regenerated, proton transfer
Download: Image, Marvin File

Step 1. His170 attacks the phosphate forming a penta-covalent intermediate.

Step 2. The penta-covalent intermediate collapses and His448 donates a proton to the choline leaving group.

Step 3. His448 deprotonates a water molecule, activating it for nucleophilic attack on the phosphate. This causes His170 to be displaced.

Products.

Contributors

Steven Smith, Gemma L. Holliday, James Willey