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 microM substrate, with an apparent K(m) of 0.5+/-0.1 microM 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.
Nieuwenhuizen
Willem F
WF
Microbial Genetics, University of Tübingen, Waldhäuser Strasse 70/8, 72076 Tübingen, Germany. nieuwenhuizen@voeding.tno.nl
van Leeuwen
Sander
S
Götz
Friedrich
F
Egmond
Maarten R
MR
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D055572:Ceramidases; D002518:Ceramides / Q000138:chemical synthesis* / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D010713:Phosphatidylcholines / Q000737:chemistry* / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000201:enzymology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism
C460280:13-(PLPC-OOH); D002518:Ceramides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D000581:Amidohydrolases; D055572:Ceramidases