Title
Synthesis of a novel fluorescent ceramide analogue and its use in the characterization of recombinant ceramidase from Pseudomonas aeruginosa PA01
Author
Nieuwenhuizen, W.F.
van Leeuwen, S.
Götz, F.
Egmond, M.R.
Centraal Instituut voor Voedingsonderzoek TNO
Publication year
2002
Abstract
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 Km 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. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Chemicals/CAS: 13-(PLPC-OOH); Amidohydrolases, EC 3.5.-; ceramidase, EC 3.5.1.23; Ceramides; Enzyme Inhibitors; Fluorescent Dyes; Phosphatidylcholines; Recombinant Proteins
Subject
Nutrition
Amidohydrolases
Binding Sites
Ceramides
Enzyme Inhibitors
Fluorescent Dyes
Hydrogen-Ion Concentration
Kinetics
Magnetic Resonance Spectroscopy
Molecular Structure
Phosphatidylcholines
Pseudomonas aeruginosa
Recombinant Proteins
Bacteria (microorganisms)
Mammalia
Pseudomonas
Pseudomonas aeruginosa
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http://resolver.tudelft.nl/uuid:e37b5521-7788-4244-b064-868b62abefaa
DOI
https://doi.org/10.1016/s0009-3084(01)00206-7
TNO identifier
57470
ISSN
0009-3084
Source
Chemistry and Physics of Lipids, 114 (2), 181-191
Document type
article