Sandbox Reserved 313
| This Sandbox is Reserved from January 10, 2010, through April 10, 2011 for use in BCMB 307-Proteins course taught by Andrea Gorrell at the University of Northern British Columbia, Prince George, BC, Canada. |
To get started:
More help: Help:Editing |
| |||||||||
| 2dds, resolution 1.80Å () | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Ligands: | |||||||||
| Activity: | Sphingomyelin phosphodiesterase, with EC number 3.1.4.12 | ||||||||
| Related: | 2ddr, 2ddt | ||||||||
| |||||||||
| |||||||||
| |||||||||
| Resources: | FirstGlance, OCA, PDBsum, RCSB, TOPSAN | ||||||||
| Coordinates: | save as pdb, mmCIF, xml | ||||||||
IntroductionIntroduction
Sphingomyelin phosphodiesterase (Sphingomyelinase): (SMase) is an enzyme which catalyzes the hydrolysis of sphingomyelin (SM) to ceramide and phosphocholine. This enzyme also has hemolytic activity where red blood cells are ruptured and hemoglobin is released into the blood plasma. [1]. SMase is a member of the DNA-I superfamily involved in hydrolytic cleavage during metabolism reactions. This enzyme has become the object of renewed interest since the discovery of the sphingomyelin signal transduction pathway which is involved in apoptosis. This pathway is initiated by a neutral sphingomyelinase hydrolysis of sphingomyelin in the plasma membrane to generate ceramide. Ceramide acts as a secondary messenger which causes the stimulation of the cascade effect of kinases and transcription factors which activate programmed cell death[2].
Types of SMase[3]:Types of SMase[3]:
Acid sphingomyelinase (aSMase) - aSMase is a soluble lysosomal hydrolase with an optical acivity at pH 5. Acid-SMase has two enzymatic forms: one is targeted to the endo-lysosomal compartment where it coordinates Zn, and the other can be realeased extracellularly through the golgi secretory pathway[4]. A deficiency in results in the lysosomal storage disorder Niemann-Pick disease. Acid-SMase normally metabolizes sphingomyelin which is found in every cell of the body. When aSMase is lacking in the cell, SM builds up and eventually results in cell death[5].
Secretory sphingomyelinase (S-SMase) - S-Smase is found in human vascular endothelial cells and arises from the acid-SMase gene through differential protein trafficking of a precurser. The precurser can be targeted to the Gogli secretory pathway or lysosomes. S-SMase hydrolyses (SM) found in plasma membranes and lipoprotein molecules but has also been found to play an important role in intracellular or paracrine ceramide second messenger signaling pathways. S-SMase is activated by normal levels of Zn2+ and operates generally at acidic pH levels, but can hydrolyze atherogenic lipoproteins at neutral pH[6].
Neutral Mg2+-dependent sphingomyelinases (nSMase) - The membrane bound dependent nSMase has an neatral pH optimum and is found predominantly in the brain. This neutral SMase enzyme relies on Magnesium and is activated by unsaturated fatty acids and phosphatidylserine. Magnesium dependent SMase operates in the plasmamembrane [7].
Neutral Mg2+-independent sphingomyelinases - The activities of magnesium independent nSMase are found predominantly in the cytosol and are not very well known[3].
Alkaline sphingomyelinase (bSMase) - Alkaline SMase is found in the intestine and hydrolyses sphingomyelin in both the lumen and the mucosal membrane and requires bile salts for activity. This enzyme shares some similarities with the nucleotide pyrophosphatase(NPP) family and is called NPP7. The enzyme has a hydrophobic domain at the N and C terminus, where the N terminus acts as a signal peptide (which is eventually cleaved) and the C terminus acts as a signal anchor which attaches the enzyme to membranes[3][8].
Bacterial sphingomyelinase - This enzyme is secreted from a bacterial cell and it has been shown that both bacterial SMase and mammalian DNase I catalyze the hydrolysis of the phosphodiester bonds of substrates which depends on the divalent metal ions present. both bacterial SMase and mammalian DNase I have a highly conserved pentapeptide sequence SDHYP which include the catalytically important residues Asp 251 and His 252[9].
Structure and FunctionStructure and Function
Recently, in the 1980's, the primary structure of sphingomyelinase was determined by cloning the first N-SMases from Bacillus cereus and Staphylococcus aureus and by the subsequent sequencing of their cDNAs [7]. The crystal structure of sphingomyelinase has been solved using the bacterium Listeria ivanovii and Bacillus cereus(Bc-SMase) to gain further insight into its catalytic activities [7]. The overall structure of Bc-SMase has been determined to consist of a β-sandwich with α/β motifs[1]. Through SMase structure identification, it has been determined to be a member of the DNA I-like superfamily having geometrically identical amino acid residues as the enzymes in this superfamily. The only different with SMase, is that it has a unique hydrophobic beta-hairpin structure. The crystal structure revealed that this unique beta-hairpin region has two solvent exposed aromatic amino acids, Trp-284 and Phe-285, on the top which bind to the cell surfaces to catalyze hemolysis. The hydrolysis and hemolytic activity of Bc-SMase occur in a metal ion dependent manner. Bc-Smase is found in complex with divalent metal ions, Co2+, Mg2+ or Ca2+ in the central cleft of this enzyme. The central cleft acts as an active site.
Active SiteActive Site
The of the Bc-SMase cotains the amino acid residues and where Glu-53, Asp-195 and residues are essential for the hydrolytic activity of the enzyme. In the central cleft, bound Ca2+ displays a hepta-coordination pattern which is different from the Co2+ and Mg2+ bound forms which are in a double-hexa-coordination forming a double octahedral bi-pyramid. Mg2+ binds to Glu-53 and is required for SM hydrolytic activity and Mg2+ with Ca2+ are required for hemolytic activity[1].
MechanismMechanism
It has been proposed by Ago, Hideo. et al 2006, that the mechanism of Bc-SMase is similar to that of bovine pancreatic DNase 1 because Bc-SMase and bovine DNase 1 are homologous proteins which both have conserved alleged catalytic amino acid residues and a similar molecular structure[1]. The proposed hydrolytic activity of Bc-SMAse cloned from Bacillus cereus is coordinated by essential water bridged divalent metal ions. Two metal ions which are bound to the Glu-53 and residues in the central cleft which orientate the substrate in the active site. The divalent cation which is linked to His-296 provides a general base water and a phosphate from sphingomelin binds to the central cleft at the site of the water bridged double metal ions. The divalent metal ion which is located at Glu-53 then binds to sphingomelin by directly interacting with the amide oxygen and the ester oxygen(O4). The water briged divalent metal ions, along with Asn-197 chains bind to the oxygens located on thephosphate of sphingomyelin which results in a negative charge on the phosphate group. This results in the phosphorous of SM becoming positively charged. The pKa value of the bound water molecule is lowered through the complex with divalent metal ions and this results in a activated H2O molecule which attacks the phosphorous of SM. Phosphocholine and ceramide are formed through the delocalization of the phosphorous by the divalent metal ionos, through the increased negative charge of the oxygens[1].
ImplicationsImplications
The crystal structure of Bc-SMase has some significant implications for unlocking the poorly characterized structure of neutral sphingomyelinase (nSMase) found in mammals. Bc-SMase is a homologue of nSMase, sharing similar metal ion dependence, amino acid sequence identity, and a similar hydrolytic mechanism[1].
ReferencesReferences
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 Ago H, Oda M, Takahashi M, Tsuge H, Ochi S, Katunuma N, Miyano M, Sakurai J. Structural basis of the sphingomyelin phosphodiesterase activity in neutral sphingomyelinase from Bacillus cereus. J Biol Chem. 2006 Jun 9;281(23):16157-67. Epub 2006 Apr 4. PMID:16595670 doi:10.1074/jbc.M601089200
- ↑ Kolesnick RN, Haimovitz-Friedman A, Fuks Z. The sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor, Fas, and ionizing radiation. Biochem Cell Biol. 1994 Nov-Dec;72(11-12):471-4. PMID:7544586
- ↑ 3.0 3.1 3.2 Goni FM, Alonso A. Sphingomyelinases: enzymology and membrane activity. FEBS Lett. 2002 Oct 30;531(1):38-46. PMID:12401200
- ↑ Jenkins RW, Idkowiak-Baldys J, Simbari F, Canals D, Roddy P, Riner CD, Clarke CJ, Hannun YA. A novel mechanism of lysosomal acid sphingomyelinase maturation: requirement for carboxyl-terminal proteolytic processing. J Biol Chem. 2011 Feb 4;286(5):3777-88. Epub 2010 Nov 22. PMID:21098024 doi:10.1074/jbc.M110.155234
- ↑ Smith EL, Schuchman EH. The unexpected role of acid sphingomyelinase in cell death and the pathophysiology of common diseases. FASEB J. 2008 Oct;22(10):3419-31. Epub 2008 Jun 20. PMID:18567738 doi:10.1096/fj.08-108043
- ↑ Tabas I. Secretory sphingomyelinase. Chem Phys Lipids. 1999 Nov;102(1-2):123-30. PMID:11001566
- ↑ 7.0 7.1 7.2 Bernardo K, Krut O, Wiegmann K, Kreder D, Micheli M, Schafer R, Sickman A, Schmidt WE, Schroder JM, Meyer HE, Sandhoff K, Kronke M. Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain. J Biol Chem. 2000 Mar 17;275(11):7641-7. PMID:10713073 Cite error: Invalid
<ref>tag; name "gp7" defined multiple times with different content - ↑ Duan RD. Alkaline sphingomyelinase: an old enzyme with novel implications. Biochim Biophys Acta. 2006 Mar;1761(3):281-91. Epub 2006 Mar 30. PMID:16631405 doi:10.1016/j.bbalip.2006.03.007
- ↑ Matsuo Y, Yamada A, Tsukamoto K, Tamura H, Ikezawa H, Nakamura H, Nishikawa K. A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I. Protein Sci. 1996 Dec;5(12):2459-67. PMID:8976554 doi:10.1002/pro.5560051208