PMID:11827965
| Citation |
Furusato, M, Sueyoshi, N, Mitsutake, S, Sakaguchi, K, Kita, K, Okino, N, Ichinose, S, Omori, A and Ito, M (2002) Molecular cloning and characterization of sphingolipid ceramide N-deacylase from a marine bacterium, Shewanella alga G8. J. Biol. Chem. 277:17300-7 |
|---|---|
| Abstract |
Recently, lyso-sphingolipids have been identified as ligands for several orphan G protein-coupled receptors, although the molecular mechanism for their generation has yet to be clarified. Here, we report the molecular cloning of the enzyme, which catalyzes the generation of lyso-sphingolipids from various sphingolipids (sphingolipid ceramide N-deacylase). The 75-kDa enzyme was purified from the marine bacterium, Shewanella alga G8, and its gene was cloned from a G8 genomic library using sequences of the purified enzyme. The cloned enzyme was composed of 992 amino acids, including a signal sequence of 35 residues, and its molecular weight was estimated to be 109,843. Significant sequence similarities were found with an unknown protein of Streptomyces fradiae Y59 and a Lumbricus terrestris lectin but not other known functional proteins. The 106-kDa recombinant enzyme expressed in Escherichia coli hydrolyzed various glycosphingolipids and sphingomyelin, although it seems to be much less active than the native 75-kDa enzyme. In vitro translation using wheat germ extract revealed the activity of a 75-kDa deletion mutant lacking a C terminus to be much stronger than that of the full-length enzyme, suggesting that C-terminal processing is necessary for full activity. |
| Links |
PubMed Online version:10.1074/jbc.M110688200 |
| Keywords |
Amidohydrolases/chemistry; Amidohydrolases/genetics; Amidohydrolases/isolation & purification; Amino Acid Sequence; Base Sequence; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Gene Deletion; Glycosphingolipids/chemistry; Hydrogen-Ion Concentration; Molecular Sequence Data; Phylogeny; Plasmids/metabolism; Protein Binding; Protein Biosynthesis; Protein Structure, Tertiary; Shewanella/enzymology; Sphingomyelins/chemistry; Time Factors |
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