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J. Biol. Chem., Vol. 277, Issue 19, 17300-17307, May 10, 2002

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Molecular Cloning and Characterization of Sphingolipid Ceramide N-Deacylase from a Marine Bacterium, Shewanella alga G8^*

Masako Furusato, Noriyuki Sueyoshi, Susumu Mitsutake, Keishi Sakaguchi, Katsuhiro Kita, Nozomu Okino, Sachiyo Ichinose^§, Akira Omori^§, and Makoto Ito^¶

From the  Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 and the ^§ Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida 194-8511, Tokyo, Japan

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.

The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number AB079849.

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