Molecular cloning and characterization of sphingolipid ceramide N-deacylase from a marine bacterium, Shewanella alga G8.
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.
Furusato
Masako
M
Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Sueyoshi
Noriyuki
N
Mitsutake
Susumu
S
Sakaguchi
Keishi
K
Kita
Katsuhiro
K
Okino
Nozomu
N
Ichinose
Sachiyo
S
Omori
Akira
A
Ito
Makoto
M
D000581:Amidohydrolases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D006028:Glycosphingolipids / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D020592:Shewanella / Q000201:enzymology*; D013109:Sphingomyelins / Q000737:chemistry; D013997:Time Factors
D006028:Glycosphingolipids; D013109:Sphingomyelins; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase