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

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Author: Nieuwenhuizen, W.F. · Leeuwen, S. van · Götz, F. · Egmond, M.R.
Type:article
Date:2002
Institution: Centraal Instituut voor Voedingsonderzoek TNO
Source:Chemistry and Physics of Lipids, 2, 114, 181-191
Identifier: 57470
doi: doi:10.1016/S0009-3084(01)00206-7
Keywords: 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

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