Synthesis of a novel fluorescent ceramide analogue and its use in the characterization of recombinant ceramidase from Pseudomonas aeruginosa PA01

Ceramidase (CDase) hydrolyses the N-acyl linkage of the sphingolipid ceramide. We synthesized the non-fluorescent ceramide analogue (4E,2S,3R)-2-N-(10-pyrenedecanoyl)-1,3,17-trihydroxy-17- (3,5-dinitrobenzoyl)-4-heptadecene (10) that becomes fluorescent upon hydrolysis of its N-acyl bond. This novel substrate was used to study several kinetic aspects of the recombinant CDase from the pathogenic bacterium Pseudomonas aeruginosa PA01. Maximum CDase activity was observed above 1.5 μM substrate, with an apparent K_m of 0.5±0.1 μM and a turnover of 5.5 min^-1. CDase activity depends on divalent cations without a strong specificity. CDase is inhibited by sphingosine and by several sphingosine analogues. The lack of inhibition by several mammalian CDase inhibitors such as D-erythro-MAPP, L-erythro-MAPP or N-oleoylethanolamine points to a novel active site and/or substrate binding region. The CDase assay described here offers the opportunity to develop and screen for specific bacterial CDase inhibitors of pharmaceutical interest.