This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by He, X.
Right arrow Articles by Reynolds, K. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by He, X.
Right arrow Articles by Reynolds, K. A.

 Previous Article  |  Next Article 

Antimicrobial Agents and Chemotherapy, May 2002, p. 1310-1318, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1310-1318.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Purification, Characterization, and Identification of Novel Inhibitors of the ß-Ketoacyl-Acyl Carrier Protein Synthase III (FabH) from Staphylococcus aureus

Xin He and Kevin A. Reynolds*

Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, Virginia 23219

Received 30 July 2001/ Returned for modification 23 December 2001/ Accepted 1 February 2002

Staphylococcus aureus is a versatile and dangerous pathogen and one of the major causes of community-acquired and hospital-acquired infections. The rise of multidrug-resistant strains of S. aureus requires the development of new antibiotics with previously unexploited mechanisms of action, such as inhibition of the ß-ketoacyl-acyl carrier protein (ACP) synthase III (FabH). This enzyme initiates fatty acid biosynthesis in a bacterial type II fatty acid synthase, catalyzing a decarboxylative condensation between malonyl-ACP and an acyl coenzyme A (CoA) substrate and is essential for viability. We have identified only one fabH in the genome of S. aureus and have shown that it encodes a protein with 57, 40, and 34% amino acid sequence identity with the FabH proteins of Bacillus subtilis (bFabH1), Escherichia coli (ecFabH), and Mycobacterium tuberculosis (mtFabH). Additional genomic sequence analysis revealed that this S. aureus FabH (saFabH) is not mutated in certain methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA) strains. saFabH was expressed in E. coli with an N-terminal polyhistidine tag and subsequently purified by metal chelate and size exclusion chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a molecular mass of 37 kDa, while gel filtration demonstrated a mass of 66.7 kDa, suggesting a noncovalent homodimeric structure for saFabH. The apparent Km for malonyl-ACP was 1.76 ± 0.40 µM, and the enzyme was active with acetyl-CoA (kcat, 16.18 min-1; Km, 6.18 ± 0.9 µM), butyryl-CoA (kcat, 42.90 min-1; Km, 2.32 ± 0.12 µM), and isobutyryl-CoA (kcat, 98.0 min-1; Km, 0.32 ± 0.04 µM). saFabH was weakly inhibited by thiolactomycin (50% inhibitory concentration [IC50], >100 µM) yet was efficiently inhibited by two new FabH inhibitors, 5-chloro-4-phenyl-[1,2]-dithiol-3-one (IC50, 1.87 ± 0.10 µM) and 4-phenyl-5-phenylimino-[1,2,4]dithiazolidin-3-one (IC50, 0.775 ± 0.08 µM).

* Corresponding author. Mailing address: Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219. Phone: (804) 828-7575. Fax: (804) 827-3664. E-mail: kareynol{at}hsc.vcu.edu.

Antimicrobial Agents and Chemotherapy, May 2002, p. 1310-1318, Vol. 46, No. 5
0066-4804/02/$04.00+0     DOI: 10.1128/AAC.46.5.1310-1318.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Zhang, Y.-M., White, S. W., Rock, C. O. (2006). Inhibiting Bacterial Fatty Acid Synthesis. J. Biol. Chem. 281: 17541-17544 [Abstract] [Full Text]  
  • Brown, A. K., Sridharan, S., Kremer, L., Lindenberg, S., Dover, L. G., Sacchettini, J. C., Besra, G. S. (2005). Probing the Mechanism of the Mycobacterium tuberculosis {beta}-Ketoacyl-Acyl Carrier Protein Synthase III mtFabH: FACTORS INFLUENCING CATALYSIS AND SUBSTRATE SPECIFICITY. J. Biol. Chem. 280: 32539-32547 [Abstract] [Full Text]  
  • Li, Y., Florova, G., Reynolds, K. A. (2005). Alteration of the Fatty Acid Profile of Streptomyces coelicolor by Replacement of the Initiation Enzyme 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH). J. Bacteriol. 187: 3795-3799 [Abstract] [Full Text]  
  • Kodali, S., Galgoci, A., Young, K., Painter, R., Silver, L. L., Herath, K. B., Singh, S. B., Cully, D., Barrett, J. F., Schmatz, D., Wang, J. (2005). Determination of Selectivity and Efficacy of Fatty Acid Synthesis Inhibitors. J. Biol. Chem. 280: 1669-1677 [Abstract] [Full Text]  
  • He, X., Reeve, A. M., Desai, U. R., Kellogg, G. E., Reynolds, K. A. (2004). 1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH). Antimicrob. Agents Chemother. 48: 3093-3102 [Abstract] [Full Text]  
  • Freiberg, C., Brunner, N. A., Schiffer, G., Lampe, T., Pohlmann, J., Brands, M., Raabe, M., Habich, D., Ziegelbauer, K. (2004). Identification and Characterization of the First Class of Potent Bacterial Acetyl-CoA Carboxylase Inhibitors with Antibacterial Activity. J. Biol. Chem. 279: 26066-26073 [Abstract] [Full Text]  
  • Nowak-Thompson, B., Hammer, P. E., Hill, D. S., Stafford, J., Torkewitz, N., Gaffney, T. D., Lam, S. T., Molnar, I., Ligon, J. M. (2003). 2,5-Dialkylresorcinol Biosynthesis in Pseudomonas aurantiaca: Novel Head-to-Head Condensation of Two Fatty Acid-Derived Precursors. J. Bacteriol. 185: 860-869 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»