Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa.

We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides, i.e. it hydrolyzed ceramides containing C12:0-C18:0 fatty acids and 7-nitrobenz-2-oxa-1, 3-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K. (1995) J. Biol. Chem. 270, 24370-24374). This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent Km of 139 microM, and a Vmax of 5.3 micromol/min/mg using N-palmitoylsphingosine as the substrate. The enzyme appears to require Ca2+ for expression of the activity. Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. Our study demonstrated that ceramidase is widely distributed from bacteria to mammals.
AuthorsN Okino, M Tani, S Imayama, M Ito
JournalThe Journal of biological chemistry (J Biol Chem) Vol. 273 Issue 23 Pg. 14368-73 (Jun 5 1998) ISSN: 0021-9258 [Print] UNITED STATES
PMID9603946 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Bacterial Proteins
  • Ceramides
  • Amidohydrolases
  • Ceramidases
  • Calcium
  • Adult
  • Amidohydrolases (chemistry)
  • Bacterial Proteins (chemistry)
  • Calcium (pharmacology)
  • Ceramidases
  • Ceramides (chemistry)
  • Dermatitis, Atopic (microbiology)
  • Female
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Weight
  • Pseudomonas aeruginosa (enzymology, pathogenicity)
  • Substrate Specificity

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