Sphingomyelin phosphodiesterase D (Class I)

 

Sphingomyelinase D (SMase D), isolated from Loxosceles laeta, catalyses the hydrolysis of sphingomyelin to ceramide 1-phosphate (C1P) and choline, and the hydrolysis of lysophosphatidyl choline to lysophosphatidic acid (LPA) and choline. This enzyme belongs to class I of the SMase D enzymes. SMase D is a component of Loxosceles spider venom and and is the principle toxin causing dermonecrosis, acute renal failure, platelet aggregation and systemic intravascular haemolysis. SMase D activity is not found anywhere else in the animal kingdom but some pathogenic bacteria, such as Corynebacterium pseudotuberculosis, produce a similar enzyme as an exotoxin. This enzyme belongs to the arthropod phospholipase D family, Class I subfamily.

In addition to hydrolysing sphingomyelin, the enzyme can hydrolyse lysophosphatidyl choline to lysophosphatidic acid and choline.

 

Reference Protein and Structure

Sequence
Q8I914 UniProt (4.6.1.-) IPR017946 (Sequence Homologues) (PDB Homologues)
Biological species
Loxosceles laeta (South american recluse spider) Uniprot
PDB
2f9r - Crystal structure of the inactive state of the Smase I, a sphingomyelinase D from Loxosceles laeta venom (1.85 Å) PDBe PDBsum 2f9r
Catalytic CATH Domains
3.20.20.190 CATHdb (see all for 2f9r)
Cofactors
Magnesium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.1.4.41)

water
CHEBI:15377ChEBI
+
sphingomyelin d18:1
CHEBI:17636ChEBI
→
choline
CHEBI:15354ChEBI
+
N-acylsphingosine 1-phosphate(2-)
CHEBI:57674ChEBI
+
hydron
CHEBI:15378ChEBI
Alternative enzyme names: Sphingomyelinase D,

Enzyme Mechanism

Introduction

His47 is the nucleophile for attack on the phosphorous of the scissile phosphodiester. The resulting penta-coordinated intermediate is stabilised through interactions with Trp230 and Lys93, and water-mediated interactions to Mg(II). The intermediate collapses and the choline leaving group is protonated by His12. Water is deprotonated by His12 and then is the nucleophile for attack on the phosphorous of the intermediate. The resulting penta-coordinated intermediate is stabilised as before and collapses to eliminate His47 and form C1P.

Catalytic Residues Roles

UniProt PDB* (2f9r)
Glu58, Asp117 Glu32B, Asp91B These residues are involved in coordinating the magnesium ion. metal ligand
His38 His12B His12 protonates the choline leaving group and deprotonates water for hydrolysis of the intermediate. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Trp256 Trp230B Trp230 stabilises the penta-coordinated intermediates during the reaction. hydrogen bond donor, electrostatic stabiliser
His73 His47B His47 is the nucleophile for attack on the phosphorous of the scissile phosphodiester. It is the leaving group during the hydrolysis of the enzyme-substrate intermediate. covalently attached, hydrogen bond donor, nucleophile, nucleofuge
Gly74 (main-C) Gly48B (main-C) The backbone carbonyl of Gly48 forms a hydrogen bond to His47ND1. This causes His47 to be in a hyperpolarized state, aiding nucleophilic attack on the substrate. activator, hydrogen bond acceptor, hydrogen bond donor, electrostatic stabiliser
Asp60, Asp259, Asn278, Asp78 Asp34B, Asp233B, Asn252B, Asp52B Part of a hydrogen bonding network involving Asp52, Asp34, Asp233 and Asn252. It may be involved in modulating the pKa values of His12 and His47. attractive charge-charge interaction, hydrogen bond acceptor, metal ligand, electrostatic stabiliser
Lys119 Lys93B Lys93 stabilises the penta-coordinated intermediates during the reaction. attractive charge-charge interaction, hydrogen bond donor, 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

bimolecular nucleophilic addition, overall reactant used, intermediate formation, unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, overall product formed, hydrolysis, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate terminated

References

  1. Murakami MT et al. (2005), J Biol Chem, 280, 13658-13664. Structural Basis for Metal Ion Coordination and the Catalytic Mechanism of Sphingomyelinases D. DOI:10.1074/jbc.m412437200. PMID:15654080.
  2. Masood R et al. (2018), Int J Biol Macromol, 107, 1054-1065. Spider's venom phospholipases D: A structural review. DOI:10.1016/j.ijbiomac.2017.09.081. PMID:28951301.
  3. Ago H et al. (2006), J Biol Chem, 281, 16157-16167. Structural Basis of the Sphingomyelin Phosphodiesterase Activity in Neutral Sphingomyelinase from Bacillus cereus. DOI:10.1074/jbc.m601089200. PMID:16595670.
  4. Murakami MT et al. (2006), Biochem Biophys Res Commun, 342, 323-329. Structural insights into the catalytic mechanism of sphingomyelinases D and evolutionary relationship to glycerophosphodiester phosphodiesterases. DOI:10.1016/j.bbrc.2006.01.123. PMID:16480957.
  5. Obama T et al. (2003), Biol Pharm Bull, 26, 920-926. His151 and His296 Are the Acid-Base Catalytic Residues of Bacillus cereus Sphingomyelinase in Sphingomyelin Hydrolysis. DOI:10.1248/bpb.26.920. PMID:12843611.

Step 1. His47 is the nucleophile for attack on the phosphorous of the scissile phosphodiester. A penta-coordinated intermediate is formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp233B activator, hydrogen bond acceptor
Asn252B activator, hydrogen bond acceptor, hydrogen bond donor
Gly48B (main-C) activator, hydrogen bond acceptor, electrostatic stabiliser
Asp52B activator, hydrogen bond acceptor
His12B hydrogen bond donor
Lys93B electrostatic stabiliser, attractive charge-charge interaction, hydrogen bond donor
Asp34B attractive charge-charge interaction, hydrogen bond acceptor, metal ligand, electrostatic stabiliser
His47B hydrogen bond donor
Trp230B hydrogen bond donor, electrostatic stabiliser
Glu32B metal ligand
Asp91B metal ligand
His47B nucleophile

Chemical Components

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

Step 2. The intermediate collapses and eliminates choline, which is protonated by His12.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp233B activator, hydrogen bond acceptor
Asn252B activator, hydrogen bond acceptor, hydrogen bond donor
Gly48B (main-C) hydrogen bond donor
Asp52B activator, hydrogen bond acceptor
His12B hydrogen bond donor
Lys93B electrostatic stabiliser, hydrogen bond donor
Asp34B electrostatic stabiliser, hydrogen bond acceptor, attractive charge-charge interaction, metal ligand
His47B covalently attached, hydrogen bond donor
Trp230B electrostatic stabiliser, hydrogen bond donor
Glu32B metal ligand
Asp91B metal ligand
His12B proton donor

Chemical Components

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

Step 3. His12 deprotonates water, which then acts as the nucleophile for attack on the phosphorous centre of the intermediate. A penta-coordinated intermediate is formed.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp233B activator, hydrogen bond acceptor
Asn252B activator, hydrogen bond acceptor, hydrogen bond donor
Gly48B (main-C) hydrogen bond acceptor
Asp52B activator, hydrogen bond acceptor
His12B hydrogen bond donor, hydrogen bond acceptor
Lys93B attractive charge-charge interaction, hydrogen bond donor, electrostatic stabiliser
Asp34B electrostatic stabiliser, hydrogen bond acceptor, attractive charge-charge interaction, metal ligand
His47B covalently attached, hydrogen bond donor
Trp230B electrostatic stabiliser, hydrogen bond donor
Glu32B metal ligand
Asp91B metal ligand
His12B proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic addition, hydrolysis, intermediate formation, overall reactant used

Step 4. The intermediate collapses and eliminates His47. This forms ceramide 1-phosphate.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Asp233B activator, hydrogen bond acceptor
Asn252B activator, hydrogen bond acceptor, hydrogen bond donor
Gly48B (main-C) hydrogen bond acceptor, electrostatic stabiliser
Asp52B activator, hydrogen bond acceptor
His12B hydrogen bond donor
Lys93B electrostatic stabiliser, hydrogen bond donor
Asp34B electrostatic stabiliser, hydrogen bond acceptor, attractive charge-charge interaction, metal ligand
His47B hydrogen bond donor
Trp230B electrostatic stabiliser, hydrogen bond donor
Glu32B metal ligand
Asp91B metal ligand
His47B nucleofuge

Chemical Components

ingold: unimolecular elimination by the conjugate base, enzyme-substrate complex cleavage, native state of enzyme regenerated, intermediate collapse, intermediate terminated, overall product formed
Download: Image, Marvin File

Step 1. His47 is the nucleophile for attack on the phosphorous of the scissile phosphodiester. A penta-coordinated intermediate is formed.

Step 2. The intermediate collapses and eliminates choline, which is protonated by His12.

Step 3. His12 deprotonates water, which then acts as the nucleophile for attack on the phosphorous centre of the intermediate. A penta-coordinated intermediate is formed.

Step 4. The intermediate collapses and eliminates His47. This forms ceramide 1-phosphate.

Products.

Contributors

Judith A. Reeks, Gemma L. Holliday, James Willey