Projects per year
Abstract
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.
Original language | English |
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Pages (from-to) | 108-120 |
Number of pages | 13 |
Journal | Journal of Molecular Biology |
Volume | 428 |
Issue number | 1 |
Early online date | 10 Nov 2015 |
DOIs | |
Publication status | Published - 16 Jan 2016 |
Keywords
- HTS
- Pathogen
- Drug discovery
- Crystal structure
- Co-complex
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Dive into the research topics of 'A substrate mimic allows high-throughput assay of the FabA protein and consequently the identification of a novel inhibitor of Pseudomonas aeruginosa FabA'. Together they form a unique fingerprint.Projects
- 2 Finished
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The Human SPT Complex: The Human Serine Palmitoyltransferase (SPT) Complex; Specificity, Structure, Regulation and Inhibition
Naismith, J. (PI)
31/12/14 → 29/01/18
Project: Standard
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Transport and Polymerisation INV Award: Transport and polymerisation of bacterial polysaccharides
Naismith, J. (PI)
1/07/13 → 30/06/20
Project: Standard
Datasets
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Crystal Structure of 3-hydroxydecanoyl-Acyl Carrier Protein Dehydratase (FabA) from Pseudomonas aeruginosa in complex with N-(4- Chlorobenzyl)-3-(2-furyl)-1H-1,2,4-triazol-5-amine (PDB entry 4CL6)
Moynie, L. (Creator), Hope, A. G. (Creator), Finzel, K. (Creator), Schmidberger, J. (Creator), Leckie, S. M. (Creator), Schneider, G. (Creator), Burkart, M. D. (Creator), Smith, A. D. (Creator), Gray, D. W. (Creator) & Naismith, J. H. (Creator), Protein Data Bank (PDB), 21 Jan 2015
DOI: 10.2210/pdb4CL6/pdb, https://www.rcsb.org/structure/4CL6
Dataset