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Molecular cloning and expression of Mn(2+)-dependent sphingomyelinase/hemolysin of an aquatic bacterium, Pseudomonas sp. strain TK4.
- Source :
-
Journal of bacteriology [J Bacteriol] 2002 Jan; Vol. 184 (2), pp. 540-6. - Publication Year :
- 2002
-
Abstract
- We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn(2+). The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.
- Subjects :
- Amino Acid Sequence
Animals
Base Sequence
Cloning, Molecular
DNA, Bacterial
Escherichia coli metabolism
Genetic Vectors metabolism
Hemolysin Proteins classification
Hemolysin Proteins isolation & purification
Hemolysin Proteins metabolism
Hydrolysis
Molecular Sequence Data
Mutagenesis
Pseudomonas genetics
Recombinant Fusion Proteins classification
Recombinant Fusion Proteins genetics
Recombinant Fusion Proteins isolation & purification
Recombinant Fusion Proteins metabolism
Sequence Analysis, DNA
Sequence Homology, Amino Acid
Sheep
Sphingomyelin Phosphodiesterase classification
Sphingomyelin Phosphodiesterase isolation & purification
Sphingomyelin Phosphodiesterase metabolism
Sphingomyelins metabolism
Substrate Specificity
Gene Expression
Hemolysin Proteins genetics
Manganese metabolism
Pseudomonas enzymology
Sphingomyelin Phosphodiesterase genetics
Subjects
Details
- Language :
- English
- ISSN :
- 0021-9193
- Volume :
- 184
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of bacteriology
- Publication Type :
- Academic Journal
- Accession number :
- 11751833
- Full Text :
- https://doi.org/10.1128/JB.184.2.540-546.2002