Sphingomyelin phosphodiesterase
Encyclopedia
Sphingomyelin phosphodiesterase or simply Sphingomyelinase (SMase) is a hydrolase
enzyme that is involved in sphingolipid
metabolism reactions. SMase is a member of the DNase I superfamily of enzymes and is responsible for breaking sphingomyelin
(SM) down into phosphocholine
and ceramide
. The activation of SMase has been suggested as a major route for the production of ceramide in response to cellular stresses.
Of these, the lysosomal acidic SMase and the magnesium-dependent neutral SMase are considered major candidates for the production of ceramide in the cellular response to stress.
– a lysosomal storage disease characterized by deficiencies in acid SMase. Subsequent study found that this enzyme was the product of a distinct gene, had an optimum pH of 7.4, was dependent on Mg2+ ions for activity, and was particularly enriched in brain. However, a more recent study in bovine brain suggested the existence of multiple N-SMase isoforms with different biochemical and chromatographical properties.
A major breakthrough came in the mid 1980s with the cloning of the first N-SMases from Bacillus cereus
and Staphylococcus aureus
. Using the sequences of these bacterial sphingomyelinases in homology searches ultimately led to the identification of the yeast N-SMases ISC1 in the budding yeast Saccharomyces cerevisiae
and the mammalian N-SMase enzymes, nSMase1 and nSMase2. The identity between mammalian, yeast and bacterial SMases is very low - being approximately 20% between nSMase2 and the B. cereus SMase. However, an alignment of the sequences (see figure) indicate a number of conserved residues throughout the family, particularly in the catalytic region of the enzymes. This has led to the suggestion of a common catalytic mechanism for the N-SMase family.
Interestingly, a third N-SMase protein - termed nSMase3 - was recently cloned and characterized. Surprisingly, nSMase3 bears little sequence similarity to either nSMase1 or nSMase2. However, there appears to be a high degree of evolutionary conservation from lower to higher organisms suggesting that it may comprise a unique and distinct N-SMase. The high expression of nSMase3 in heart and skeletal muscle also suggests potential roles in heart function.
The solving of the crystal structure of the neutral sphingomyelinase from Listeria ivanovii and Bacillus cereus
have allowed a fuller understanding of their enzymatic site. The active site
of the B. cereus
SMase comprises the residues Asn
-16, Glu
-53, Asp
-195, Asn-197, and His
-296. Of these, the residues Glu-53, Asp-195, and His-296 are known to be essential for activity. The relative catalytic activities of SMase when metal ions are bound to the active site have been studied for divalent metal ions Co2+, Mn2+, Mg2+, Ca2+, and Sr2+. Of these five metal ions, Co2+, Mn2+, and Mg2+ bound to the active site result in high catalytic activity of SMase. Ca2+ and Sr2+ bound to the active site exhibit much lower catalytic activity of SMase. When one Mg2+ ion or two Co2+ ions bind to the active site, double hexa-coordinated
geometry results with two octahedral bi-pyramids for Co2+ and one octahedral bi-pyramid for Mg2+. When one Ca2+ ion binds to the active site, a hepta-coordinated geometry results. Therefore, the difference in catalytic activity for metal ions is predicted to be due to geometrical differences. Of Co2+ and Mg2+, SMase has better reactivity when two Co2+ ions are bound to SMase; when these Co2+ ions are bound, Glu-53 and His-296 each bind one divalent metal cation. These cations are surrounded by bridged water molecules and function as Lewis acids.
has also shed light on their catalytic mechanisms. The active site of SMase contains Glu and His residues that are each bound to one or two divalent metal cations, usually Co2+, Mg2+, or Ca2+ for optimum performance. These two cations assist in catalysis by recruiting SM to the active site of SMase. The divalent cation bound to the Glu residue interacts with the amido-oxygen and ester
-oxygen between C1 and the phosphate
group of SM; an Asn residue and the divalent metal cation bound to the His residue bind to the oxygen atoms of the phosphate group of SM. This stabilizes the phosphate group’s negative charge. The metal cation bound to the His residue and Asp and Asn side chains lower the pKa
value of one of the bridged water molecules, thus activating a water molecule. This water molecule then acts as a nucleophile
and attacks the phosphate group of SM, creating a pentavalent phosphorus atom whose negative charge is stabilized by the divalent metal cations. The phosphate then reforms its tetrahedral conformation and results in the products ceramide
and phosphocholine
. However, it is currently not clear if the mechanism of action of the acidic sphingomyelinase is the same, owing to the lack of a crystal structure.
Hydrolase
In biochemistry, a hydrolase is an enzyme that catalyzes the hydrolysis of a chemical bond. For example, an enzyme that catalyzed the following reaction is a hydrolase:-Nomenclature:...
enzyme that is involved in sphingolipid
Sphingolipid
Sphingolipids are a class of lipids containing a backbone of sphingoid bases, a set of aliphatic amino alcohols that includes sphingosine. They were discovered in brain extracts in the 1870s and were named for the mythological Sphinx because of their enigmatic nature. These compounds play...
metabolism reactions. SMase is a member of the DNase I superfamily of enzymes and is responsible for breaking sphingomyelin
Sphingomyelin
Sphingomyelin is a type of sphingolipid found in animal cell membranes, especially in the membranous myelin sheath that surrounds some nerve cell axons. It usually consists of phosphorylcholine and ceramide...
(SM) down into phosphocholine
Phosphocholine
Phosphocholine is an intermediate in the synthesis of phosphatidylcholine in tissues. Phosphocholine is made in a reaction, catalyzed by choline kinase, that converts ATP + Choline into Phosphocholine and ADP...
and ceramide
Ceramide
Ceramides are a family of lipid molecules. A ceramide is composed of sphingosine and a fatty acid. Ceramides are found in high concentrations within the cell membrane of cells. They are one of the component lipids that make up sphingomyelin, one of the major lipids in the lipid bilayer...
. The activation of SMase has been suggested as a major route for the production of ceramide in response to cellular stresses.
The Sphingomyelinase Family
Currently, five types of SMase have been identified. These are classified according to their cation dependence and pH optima of action and are:- LysosomalLysosomethumb|350px|Schematic of typical animal cell, showing subcellular components. [[Organelle]]s: [[nucleoli]] [[cell nucleus|nucleus]] [[ribosomes]] [[vesicle |vesicle]] rough [[endoplasmic reticulum]]...
Acid SMase - Secreted zinc-dependent Acid SMase
- Magnesium-dependent Neutral SMase
- Magnesium-independent Neutral SMase
- Alkaline SMase
Of these, the lysosomal acidic SMase and the magnesium-dependent neutral SMase are considered major candidates for the production of ceramide in the cellular response to stress.
Neutral Sphingomyelinase
Neutral sphingomyelinase (N-SMase) activity was first described in fibroblasts from patients with Niemann-Pick diseaseNiemann-Pick disease
Niemann–Pick disease refers to a group of fatal inherited metabolic disorders that are included in the larger family of lysosomal storage diseases .-Signs and symptoms:Symptoms are related to the organs in which they accumulate...
– a lysosomal storage disease characterized by deficiencies in acid SMase. Subsequent study found that this enzyme was the product of a distinct gene, had an optimum pH of 7.4, was dependent on Mg2+ ions for activity, and was particularly enriched in brain. However, a more recent study in bovine brain suggested the existence of multiple N-SMase isoforms with different biochemical and chromatographical properties.
A major breakthrough came in the mid 1980s with the cloning of the first N-SMases from Bacillus cereus
Bacillus cereus
Bacillus cereus is an endemic, soil-dwelling, Gram-positive, rod-shaped, beta hemolytic bacterium. Some strains are harmful to humans and cause foodborne illness, while other strains can be beneficial as probiotics for animals...
and Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus is a facultative anaerobic Gram-positive coccal bacterium. It is frequently found as part of the normal skin flora on the skin and nasal passages. It is estimated that 20% of the human population are long-term carriers of S. aureus. S. aureus is the most common species of...
. Using the sequences of these bacterial sphingomyelinases in homology searches ultimately led to the identification of the yeast N-SMases ISC1 in the budding yeast Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
and the mammalian N-SMase enzymes, nSMase1 and nSMase2. The identity between mammalian, yeast and bacterial SMases is very low - being approximately 20% between nSMase2 and the B. cereus SMase. However, an alignment of the sequences (see figure) indicate a number of conserved residues throughout the family, particularly in the catalytic region of the enzymes. This has led to the suggestion of a common catalytic mechanism for the N-SMase family.
Interestingly, a third N-SMase protein - termed nSMase3 - was recently cloned and characterized. Surprisingly, nSMase3 bears little sequence similarity to either nSMase1 or nSMase2. However, there appears to be a high degree of evolutionary conservation from lower to higher organisms suggesting that it may comprise a unique and distinct N-SMase. The high expression of nSMase3 in heart and skeletal muscle also suggests potential roles in heart function.
Active Site
Bacillus cereus
Bacillus cereus is an endemic, soil-dwelling, Gram-positive, rod-shaped, beta hemolytic bacterium. Some strains are harmful to humans and cause foodborne illness, while other strains can be beneficial as probiotics for animals...
have allowed a fuller understanding of their enzymatic site. The active site
Active site
In biology the active site is part of an enzyme where substrates bind and undergo a chemical reaction. The majority of enzymes are proteins but RNA enzymes called ribozymes also exist. The active site of an enzyme is usually found in a cleft or pocket that is lined by amino acid residues that...
of the B. cereus
Bacillus cereus
Bacillus cereus is an endemic, soil-dwelling, Gram-positive, rod-shaped, beta hemolytic bacterium. Some strains are harmful to humans and cause foodborne illness, while other strains can be beneficial as probiotics for animals...
SMase comprises the residues Asn
ASN
ASN may refer to:Organisations:* ASN Bank in the Netherlands* American Society of Nephrology* American Society for Neurochemistry* American Society of Neuroimaging* American Society for Nutrition...
-16, Glu
Glutamic acid
Glutamic acid is one of the 20 proteinogenic amino acids, and its codons are GAA and GAG. It is a non-essential amino acid. The carboxylate anions and salts of glutamic acid are known as glutamates...
-53, Asp
Aspartic acid
Aspartic acid is an α-amino acid with the chemical formula HOOCCHCH2COOH. The carboxylate anion, salt, or ester of aspartic acid is known as aspartate. The L-isomer of aspartate is one of the 20 proteinogenic amino acids, i.e., the building blocks of proteins...
-195, Asn-197, and His
Histidine
Histidine Histidine, an essential amino acid, has a positively charged imidazole functional group. It is one of the 22 proteinogenic amino acids. Its codons are CAU and CAC. Histidine was first isolated by German physician Albrecht Kossel in 1896. Histidine is an essential amino acid in humans...
-296. Of these, the residues Glu-53, Asp-195, and His-296 are known to be essential for activity. The relative catalytic activities of SMase when metal ions are bound to the active site have been studied for divalent metal ions Co2+, Mn2+, Mg2+, Ca2+, and Sr2+. Of these five metal ions, Co2+, Mn2+, and Mg2+ bound to the active site result in high catalytic activity of SMase. Ca2+ and Sr2+ bound to the active site exhibit much lower catalytic activity of SMase. When one Mg2+ ion or two Co2+ ions bind to the active site, double hexa-coordinated
Coordinate covalent bond
A dipolar bond, also known as dative covalent bond or coordinate bond is a kind of 2-centre, 2-electron covalent bond in which the two electrons derive from the same atom. Typically, a dipolar bond is formed when a Lewis base donates a pair of electrons to a Lewis acid. This description of bonding...
geometry results with two octahedral bi-pyramids for Co2+ and one octahedral bi-pyramid for Mg2+. When one Ca2+ ion binds to the active site, a hepta-coordinated geometry results. Therefore, the difference in catalytic activity for metal ions is predicted to be due to geometrical differences. Of Co2+ and Mg2+, SMase has better reactivity when two Co2+ ions are bound to SMase; when these Co2+ ions are bound, Glu-53 and His-296 each bind one divalent metal cation. These cations are surrounded by bridged water molecules and function as Lewis acids.
Mechanism
The solving of the crystal structure of the neutral sphingomyelinase from Listeria ivanovii and Bacillus cereusBacillus cereus
Bacillus cereus is an endemic, soil-dwelling, Gram-positive, rod-shaped, beta hemolytic bacterium. Some strains are harmful to humans and cause foodborne illness, while other strains can be beneficial as probiotics for animals...
has also shed light on their catalytic mechanisms. The active site of SMase contains Glu and His residues that are each bound to one or two divalent metal cations, usually Co2+, Mg2+, or Ca2+ for optimum performance. These two cations assist in catalysis by recruiting SM to the active site of SMase. The divalent cation bound to the Glu residue interacts with the amido-oxygen and ester
Ester
Esters are chemical compounds derived by reacting an oxoacid with a hydroxyl compound such as an alcohol or phenol. Esters are usually derived from an inorganic acid or organic acid in which at least one -OH group is replaced by an -O-alkyl group, and most commonly from carboxylic acids and...
-oxygen between C1 and the phosphate
Phosphate
A phosphate, an inorganic chemical, is a salt of phosphoric acid. In organic chemistry, a phosphate, or organophosphate, is an ester of phosphoric acid. Organic phosphates are important in biochemistry and biogeochemistry or ecology. Inorganic phosphates are mined to obtain phosphorus for use in...
group of SM; an Asn residue and the divalent metal cation bound to the His residue bind to the oxygen atoms of the phosphate group of SM. This stabilizes the phosphate group’s negative charge. The metal cation bound to the His residue and Asp and Asn side chains lower the pKa
PKA
PKA, pKa, or other similar variations may stand for:* pKa, the symbol for the acid dissociation constant at logarithmic scale* Protein kinase A, a class of cAMP-dependent enzymes* Pi Kappa Alpha, the North-American social fraternity...
value of one of the bridged water molecules, thus activating a water molecule. This water molecule then acts as a nucleophile
Nucleophile
A nucleophile is a species that donates an electron-pair to an electrophile to form a chemical bond in a reaction. All molecules or ions with a free pair of electrons can act as nucleophiles. Because nucleophiles donate electrons, they are by definition Lewis bases.Nucleophilic describes the...
and attacks the phosphate group of SM, creating a pentavalent phosphorus atom whose negative charge is stabilized by the divalent metal cations. The phosphate then reforms its tetrahedral conformation and results in the products ceramide
Ceramide
Ceramides are a family of lipid molecules. A ceramide is composed of sphingosine and a fatty acid. Ceramides are found in high concentrations within the cell membrane of cells. They are one of the component lipids that make up sphingomyelin, one of the major lipids in the lipid bilayer...
and phosphocholine
Phosphocholine
Phosphocholine is an intermediate in the synthesis of phosphatidylcholine in tissues. Phosphocholine is made in a reaction, catalyzed by choline kinase, that converts ATP + Choline into Phosphocholine and ADP...
. However, it is currently not clear if the mechanism of action of the acidic sphingomyelinase is the same, owing to the lack of a crystal structure.