Inhibition of yeast inositol phosphorylceramide synthase by aureobasidin A measured by a fluorometric assay.
Inositol phosphorylceramide synthase (IPC synthase) is an essential and unique enzyme in fungal sphingolipid biosynthesis and is the target of the cyclic nonadepsipeptide antibiotic aureobasidin A. As a first step towards understanding the mechanism of aureobasidin A inhibition, we developed a fluorometric HPLC assay for IPC synthase using the Saccharomyces cerevisiae enzyme and the fluorescent substrate analog 6-[N-(7-nitro-2,1, 3-benzoxadiazol-4-yl)amino]-hexanoyl ceramide (C(6)-NBD-cer). The kinetic parameters for C(6)-NBD-cer were comparable to those for the synthetic substrate N-acetylsphinganine used previously. Aureobasidin A acted as a tight-binding, non-competitive inhibitor with respect to C(6)-NBD-cer and had a K(i) of 0.55 nM.
Zhong
W
W
Department of Antimicrobial Research, DuPont Pharmaceuticals Company, Experimental Station, Bldg. E400, Wilmington, DE, USA.
Murphy
D J
DJ
Georgopapadakou
N H
NH
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives; D000935:Antifungal Agents / Q000494:pharmacology*; D002518:Ceramides; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D047630:Depsipeptides; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D005470:Fluorometry; D006602:Hexosyltransferases / Q000037:antagonists & inhibitors*; D007700:Kinetics; D010456:Peptides, Cyclic / Q000494:pharmacology; D012441:Saccharomyces cerevisiae
D000935:Antifungal Agents; D002518:Ceramides; D047630:Depsipeptides; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010456:Peptides, Cyclic; C071398:aureobasidin A; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D006602:Hexosyltransferases; C088714:phosphatidylinositol-ceramide phosphoinositol transferase; D009327:4-Chloro-7-nitrobenzofurazan