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.
Okino
N
N
Laboratory of Marine Biochemistry, Faculty of Agriculture, Kyushu University, Fukuoka 812-8581, Japan.
Tani
M
M
Imayama
S
S
Ito
M
M
D000328:Adult; D000581:Amidohydrolases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D002118:Calcium / Q000494:pharmacology; D055572:Ceramidases; D002518:Ceramides / Q000737:chemistry; D003876:Dermatitis, Atopic / Q000382:microbiology; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000472:pathogenicity; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002518:Ceramides; D000581:Amidohydrolases; D055572:Ceramidases; D002118:Calcium