Reverse hydrolysis reaction of a recombinant alkaline ceramidase of Pseudomonas aeruginosa

Recently, we purified an alkaline ceramidase (CDase) of Pseudomonas aerugin osa and found that the enzyme catalyzed a reversible reaction in which the N-acyl linkage of ceramide was hydrolyzed or synthesized [J. Biol. Chem. 27 3 (1998) 14368-14373]. Here, we report the characterization of the reverse hydrolysis reaction of the CDase using a recombinant enzyme. The reverse hy drolysis reaction of the CDase was clearly distinguishable from the reactio n of an acyl-coenzyme A (CoA) dependent N-acyltransferase, because the CDas e catalyzed the condensation of a free fatty acid to sphingosine (Sph) with out cofactors but did not catalyze the transfer of a fatty acid from acyr-C oA to Sph. The reverse hydrolysis reaction proceeded most efficiently in th e presence of 0.05% Triton X-100 at neutral pH, while the hydrolysis reacti on tended to be favored with an increase in the concentration of the deterg ent at alkaline pH. The specificity of the reverse reaction for fatty acids is quite broad; saturated and unsaturated fatty acids were efficiently con densed 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. C hemical modification of the enzyme protein affected or did not affect both the hydrolysis and reverse reactions to the same extent, suggesting that th e two reactions are catalyzed at the same catalytic domain. (C) 2000 Elsevi er Science B.V. All rights reserved.