Phospholipase C

 

Phospholipase C (PLC) from Bacillus cereus is a monomeric protein with a degree of similarly to mammalian PLCs and can mimic the action of mammalian PLCs for example by stimulating prostaglandin synthesis.

The active site contains three Zn(II) ions which activate the cleavage of membrane phospholipids, liberating the polar head group and diacylglycerol. Although phosphatidylcholine is the preferred substrate, phosphatidylserine and phosphatidylethanolamine are also accepted.

 

Reference Protein and Structure

Sequence
P09598 UniProt (3.1.4.3) IPR008947 (Sequence Homologues) (PDB Homologues)
Biological species
Bacillus cereus (Bacteria) Uniprot
PDB
1ah7 - PHOSPHOLIPASE C FROM BACILLUS CEREUS (1.501 Å) PDBe PDBsum 1ah7
Catalytic CATH Domains
1.10.575.10 CATHdb (see all for 1ah7)
Cofactors
Zinc(2+) (3) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.1.4.3)

water
CHEBI:15377ChEBI
+
phosphatidylcholine(1+)
CHEBI:49183ChEBI
→
1,2-diacyl-sn-glycerol
CHEBI:17815ChEBI
+
phosphocholine
CHEBI:18132ChEBI
Alternative enzyme names: Alpha-toxin, Clostridium oedematiens beta- and gamma-toxins, Clostridium welchii alpha-toxin, Heat-labile hemolysin, Lecithinase C, Lipophosphodiesterase C, Lipophosphodiesterase I, Phosphatidase C,

Enzyme Mechanism

Introduction

Crystallographic data has shown the substrate phosphate to coordinate directly to the tri-metallic active site, as well as proximate amino acids.

The general base (Asp55) deprotonates water, which initiates a nucleophilic attack on the phosphorus atom of the phosphodiester linkage. This results in two products, phosphorylcholine and diacylglycerol. Diacylglycerol should be activated by a general acid, which could be one of the Zn(II) ions or could be another amino acid, but the identity of this is unknown.

Catalytic Residues Roles

UniProt PDB* (1ah7)
Asp93 Asp55A General base. hydrogen bond acceptor, hydrogen bond donor, metal ligand, proton acceptor, proton donor
His156, Asp160, His166, His180, Glu184, His52, His107 His118A, Asp122A, His128A, His142A, Glu146A, His14A, His69A Binds one of the Zn(II) ions. metal ligand
*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

bimolecular nucleophilic substitution, overall reactant used, overall product formed, intermediate formation, hydrolysis, proton transfer, rate-determining step, intermediate terminated, native state of enzyme regenerated, inferred reaction step

References

  1. Lyu Y et al. (2016), Biotechnol Lett, 38, 23-31. Recent research progress with phospholipase C from Bacillus cereus. DOI:10.1007/s10529-015-1962-6. PMID:26437973.
  2. Huang Q et al. (2016), Biochim Biophys Acta, 1864, 697-705. Recombinant broad-range phospholipase C from Listeria monocytogenes exhibits optimal activity at acidic pH. DOI:10.1016/j.bbapap.2016.03.008. PMID:26976751.
  3. Martin SF et al. (1999), Biochemistry, 38, 4403-4408. Catalytic Cycle of the Phosphatidylcholine-Preferring Phospholipase C fromBacillus cereus. Solvent Viscosity, Deuterium Isotope Effects, and Proton Inventory Studies†. DOI:10.1021/bi9821216. PMID:10194360.
  4. Martin SF et al. (1998), Biochemistry, 37, 5755-5760. General Base Catalysis by the Phosphatidylcholine-Preferring Phospholipase C fromBacillus cereus:  The Role of Glu4 and Asp55†. DOI:10.1021/bi972948k. PMID:9548962.
  5. Martin SF et al. (1996), Biochemistry, 35, 12970-12977. Expression and Site-Directed Mutagenesis of the Phosphatidylcholine-Preferring Phospholipase C ofBacillus cereus:  Probing the Role of the Active Site Glu146†. DOI:10.1021/bi961316f. PMID:8841144.
  6. Hansen S et al. (1993), J Mol Biol, 234, 179-187. Crystal Structure of Phospholipase C from Bacillus cereus Complexed with a Substrate Analog. DOI:10.1006/jmbi.1993.1572. PMID:8230197.
  7. Hough E et al. (1989), Nature, 338, 357-360. High-resolution (1.5 Å) crystal structure of phospholipase C from Bacillus cereus. DOI:10.1038/338357a0. PMID:2493587.

Step 1. Asp55 deprotonates water, which initiates nucleophilic attack on the phosphate in a substitution reaction, eliminating 1,2-diacylglycerol.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp55A hydrogen bond acceptor
Asp55A metal ligand
His118A metal ligand
Asp122A metal ligand
His69A metal ligand
Glu146A metal ligand
His142A metal ligand
His128A metal ligand
His14A metal ligand
Asp55A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, intermediate formation, hydrolysis, proton transfer, rate-determining step

Step 2. Diacylglycerol deprotonates Asp55 in an inferred return step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp55A hydrogen bond donor, metal ligand
His118A metal ligand
Asp122A metal ligand
His69A metal ligand
Glu146A metal ligand
His142A metal ligand
His128A metal ligand
His14A metal ligand
Asp55A proton donor

Chemical Components

proton transfer, overall product formed, intermediate terminated, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Asp55 deprotonates water, which initiates nucleophilic attack on the phosphate in a substitution reaction, eliminating 1,2-diacylglycerol.

Step 2. Diacylglycerol deprotonates Asp55 in an inferred return step.

Products.

Introduction

The phosphate group of the substrate binds to the trinuclear zinc cluster mainly through direct coordination to Zn1 and Zn2, and hydrogen bonding to the Zn2-bound water molecule. The hydroxide bridging Zn1 and Zn3 makes the nucleophilic attack on the phosphorus center, concertedly with the dissociation of the leaving alkoxide, which takes a proton from the Zn2-bound water molecule. In the subsequent step, the Zn2 bound hydroxide makes a reverse attack, resulting in the regeneration of the bridging hydroxide.

Catalytic Residues Roles

UniProt PDB* (1ah7)
Asp93 Asp55A Acts as a general acid/base, abstracting a proton from the zinc-activated water. It is returned to its initial protonation state by the leaving hydroxide ion. hydrogen bond acceptor, metal ligand, proton acceptor, proton donor
His156, Asp160, His166, His180, Glu184, His52, His107 His118A, Asp122A, His128A, His142A, Glu146A, His14A, His69A Binds one of the Zn(II) ions. metal ligand
*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 substitution, overall reactant used, overall product formed, intermediate formation, hydrolysis

References

  1. Lyu Y et al. (2016), Biotechnol Lett, 38, 23-31. Recent research progress with phospholipase C from Bacillus cereus. DOI:10.1007/s10529-015-1962-6. PMID:26437973.
  2. Liao RZ et al. (2010), J Phys Chem B, 114, 2533-2540. Reaction Mechanism of the Trinuclear Zinc Enzyme Phospholipase C: A Density Functional Theory Study. DOI:10.1021/jp910992f. PMID:20121060.

Step 1. Asp55 deprotonates zinc activated water, which initiates the nucleophilic attack. The DAG substrate is re-protonated from a second zinc-bound water molecule.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
His14A metal ligand
His128A metal ligand
His142A metal ligand
Glu146A metal ligand
Asp122A metal ligand
His69A metal ligand
His118A metal ligand
Asp55A metal ligand
Asp55A hydrogen bond acceptor
Asp55A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, intermediate formation, hydrolysis

Step 2. The second zinc-bound water molecule attacks the phosphate to re-generate the original zinc-activated water and release the product from the active site.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp55A metal ligand
His118A metal ligand
Asp122A metal ligand
His69A metal ligand
Glu146A metal ligand
His142A metal ligand
His128A metal ligand
His14A metal ligand
Asp55A proton donor

Chemical Components

proton transfer
Download: Image, Marvin File

Step 1. Asp55 deprotonates zinc activated water, which initiates the nucleophilic attack. The DAG substrate is re-protonated from a second zinc-bound water molecule.

Step 2. The second zinc-bound water molecule attacks the phosphate to re-generate the original zinc-activated water and release the product from the active site.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Sophie T. Williams, Stuart Lucas, Craig Porter, Katherine Ferris