Molecular cloning and expression of Mn(2+)-dependent sphingomyelinase/hemolysin of an aquatic bacterium, Pseudomonas sp. strain TK4.
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.
Sueyoshi
Noriyuki
N
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Science, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Kita
Katsuhiro
K
Okino
Nozomu
N
Sakaguchi
Keishi
K
Nakamura
Takashi
T
Ito
Makoto
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000378:metabolism; D006460:Hemolysin Proteins / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006868:Hydrolysis; D008345:Manganese / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012756:Sheep; D013108:Sphingomyelin Phosphodiesterase / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism; D013379:Substrate Specificity
D004269:DNA, Bacterial; D006460:Hemolysin Proteins; D011993:Recombinant Fusion Proteins; D013109:Sphingomyelins; D008345:Manganese; C445307:TK4 sphingomyelinase; D013108:Sphingomyelin Phosphodiesterase