A substrate mimic allows high-throughput assay of the FabA protein and consequently the identification of a novel inhibitor of Pseudomonas aeruginosa FabA
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Eukaryotes and prokaryotes possess fatty acid synthase (FAS) biosynthetic pathway(s) that comprise iterative chain elongation, reduction, and dehydration reactions. The bacterial FASII pathway differs significantly from human FAS pathways and is a long-standing target for antibiotic development against Gram-negative bacteria due to differences from the human FAS, and several existing antibacterial agents are known to inhibit FASII enzymes. N-acetylcysteamine (NAC) fatty acid thioesters have been used as mimics of the natural acyl carrier protein (ACP) pathway intermediates to assay FASII enzymes, and we now report an assay of FabV from Pseudomonas aeruginosa using (E)-2-decenoyl-NAC. In addition, we have converted an existing UV absorbance assay for FabA, the bifunctional dehydration/epimerization enzyme and key target in the FAS II pathway, into a high throughput enzyme coupled fluorescence assay that has been employed to screen a library of diverse small molecules. With this approach, N-(4-chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (N42FTA) was found to competitively inhibit (pIC50 = 5.7 ± 0.2) the processing of 3-hydroxydecanoyl-NAC by P. aeruginosa FabA. N42FTA was shown to be potent in blocking crosslinking of E. coli ACP and FabA, a direct mimic of the biological process. The co-complex structure of N42FTA with P. aeruginosa FabA protein rationalizes affinity and suggests future design opportunities. Employing NAC fatty acid mimics to developing further high throughput assays for individual enzymes in the FASII pathway should aid in the discovery of new antimicrobials.
Moynie , L , Hope , A G , Finzel , K , Schmidberger , J , Leckie , S M , Schneider , G , Burkart , M D , Smith , A D , Gray , D W & Naismith , J 2016 , ' A substrate mimic allows high-throughput assay of the FabA protein and consequently the identification of a novel inhibitor of Pseudomonas aeruginosa FabA ' Journal of Molecular Biology , vol 428 , no. 1 , pp. 108-120 . DOI: 10.1016/j.jmb.2015.10.027
Journal of Molecular Biology
© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
The research leading to these results has received funding from the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° 223461, Senior Investigator Award WT100209MA (JHN), Swedish Science Council (GS), Wellcome Trust Strategic grant 100476/Z/12/Z (DWG) and National Institutes of Health R01GM095970 (MB). JHN & ADS are Royal Society Wolfson Merit Award holders.