Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa.
Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aeruginosa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 273 (1998) 14368-14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hydrolysis reaction of the CDase was clearly distinguishable from the reaction of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDase catalyzed the condensation of a free fatty acid to sphingosine (Sph) without cofactors but did not catalyze the transfer of a fatty acid from acyl-CoA to Sph. The reverse hydrolysis reaction proceeded most efficiently in the presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reaction tended to be favored with an increase in the concentration of the detergent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently condensed to Sph. In contrast, the stereo-specificity of the reverse reaction for the sphingoid bases is very strict; the D-erythro form of Sph, not the L-erythro or D/L-threo one, was only acceptable for the reverse reaction. Chemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that the two reactions are catalyzed at the same catalytic domain.
Kita
K
K
Department of Bioscience and Biotechnology, Graduate School Kyushu University, Fukuoka, Japan.
Okino
N
N
Ito
M
M
D000581:Amidohydrolases / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D002413:Cations, Divalent; D055572:Ceramidases; D003902:Detergents; D004734:Energy Metabolism; D005227:Fatty Acids / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008345:Manganese / Q000378:metabolism; D017830:Octoxynol; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D002413:Cations, Divalent; D003902:Detergents; D005227:Fatty Acids; D011993:Recombinant Fusion Proteins; D008345:Manganese; D017830:Octoxynol; D000581:Amidohydrolases; D055572:Ceramidases; D002118:Calcium