Summary
The emergence of bacterial pathogens resistant to current antibiotics has caused an urgent demand for new treatments. Peptide deformylase (PDF) has become an exciting target for designing novel antibiotics. To facilitate the screening of PDF inhibitors, three robust, coupled assays have been developed. The first method couples the PDF reaction with that of formate dehydrogenase. Formate dehydrogenase oxidizes formate into CO2 with a concomitant reduction of NAD+ to NADH, which can be monitored spectrophotometrically. The second method involves Aeromonas aminopeptidase (AAP) as the coupling enzyme and an artificial substrate, f-Met-Leu-p-nitroanilide. The sequential action of PDF and AAP releases p-nitroanilide as a highly chromogenic product. In the third method, f-Met-Lys-7-amino-4-methylcoumarin is used as the substrate. Deformylation by PDF gives an excellent substrate for dipeptidyl peptidase I, which releases the dipeptide Met-Lys and fluorogenic 7-amino-4-methylcoumarin. The combination of these assay methods should meet the needs of most laboratories.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Bibliography
Giglione, C., Pierre, M., and Meinnel, T.(2000)Peptide deformylaseas a target for new generation, broad spectrum antimicrobial agents. Mol. Microbiol. 36, 1197–1205.
Pei, D. (2001) Peptide deformylase: A target for novel antibiotics? Emerging Ther. Targets 5, 23–40.
Yuan, Z, Trias, J., and White, R. J.(2001)Deformylase as a novelantibacterial target. Drug Discovery Today 6, 954–961.
Clements, J. M.,Beckett, R. P., Brown, A.,Catlin, G., Lobell, M ,Palan, S., Thomas, W., Whittaker, M., Wood, S., Salama, S.,Baker, P. J., Rodgers,H.F., Barynin, V., Rice, D. W., and Hunter, M. G. (2001) Antibiotic activity and characterization of BB-3497, a novel peptide deformylase inhibitor. Antimicrob. Agents Chemother. 45, 563–570.
Meinnel, T.,Mechulam, Y., and Blanquet, S. (1993) Methionine as translation start signal: A review of the enzymes of the pathway in Escherichia coli. Biochimie 75, 1061–1075.
Ben-Bassart, A., Baur, K., Chang, S. Y., Myambo, K., Boosman, A., and Chang, S. (1987) Processing of the initiation methionine from proteins: Properties of the Escherichia coli methionine aminopeptidase and its gene structure. J. Bacteriol. 169, 751–757.
Mazel, D., Pochet, S., and Marliere, P. (1994) Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation. EMBO J. 13, 914–923.
Meinnel, T., and Blanquet, S. (1994) Characterization of the Thermus thermophilus locus encoding peptide deformylase and methionyl-tRNA(fMet) formyltransferase. J. Bacteriol. 176, 7387–7390.
Margolis, P. Hackbarth, C. J., Young, D. C., Wang, W., Chen, D., Yuan, Z., White, R., Trias, J. (2001) Peptide deformylase in S. aureus: Resistance to inhibition is mediated by mutations in the formyltransferase gene. Antimicrob. Agents Chemother. 44, 825–1831.
Rajagopalan, P. T. R., Yu, X. C., and Pei, D. (1997) Peptide deformylase: A new type of mononuclear iron protein. J. Am. Chem. Soc. 119, 12418–12419.
Groche, D., Becker, A., Schlichting, I., Kabsch, W., Schultz, S., and Wagner, A. F. V. (1998) Isolation and crystallization of functionally competent Escherichia coli peptide deformylase forms containing either iron or nickel in the active site. Biochem. Biophys. Res. Comm. 246, 342–346.
Nguyen, K. T., Hu, X., Colton, C., Chakratarti, R., Zhu, M. X., and Pei, D. (2003) Characterization of a human peptide deformylase: Implications for antibacterial drug design. Biochemistry 42, 9952–9958.
Lazennec, C., and Meinnel, T. (1997) Formate dehydrogenase-coupled spectrophotometric assay of peptide deformylase. Anal. Biochem. 244, 180–182.
Rajagopalan, P. T. R., Datta, A., and Pei, D. (1997) Purification, characterization, and inhibition of peptide deformylase from Escherichia coli. Biochemistry 36, 13910–13918.
Wei, Y., and Pei, D. (1997) Continuous spectrophotometric assay of peptide deformylase. Anal. Biochem. 250, 29–34.
Nguyen, K. T., Hu, X., and Pei, D. (2004) Slow-binding inhibition of peptide deformylase by cyclic peptidomimetics as revealed by a new spectrophotometric assay. Bioorg. Chem. 32, 178–191.
Rajagopalan, P. T. R., and Pei, D. (1998) Oxygen mediated inactivation of peptide deformylase. J. Biol. Chem. 273, 22305–22310.
Rajagopalan, P. T. R., Grimme, S., and Pei, D. (2000) Characterization of Cobalt(II)-substituted peptide deformylase: Function of the metal ion and the catalytic residue Glu-133. Biochemistry 39, 779–790.
Hu, X., Nguyen, K. T., Jiang, V. C., Lofland, D., Moser, H. E., and Pei, D. (2004) Macrocyclic inhibitors for peptide deformylase: A structure-activity relationship study of the ring size. J. Med. Chem. 47, 4941–4949.
Prescott, J. M., and Wilkes, S. H. (1976) Aeromonas aminopeptidase. Methods Enzylmol. 45, 530–543.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press Inc.
About this protocol
Cite this protocol
Nguyen, K.T., Pei, D. (2008). High-Throughput Screening of Peptide Deformylase Inhibitors. In: Champney, W.S. (eds) New Antibiotic Targets. Methods In Molecular Medicine™, vol 142. Humana Press. https://doi.org/10.1007/978-1-59745-246-5_10
Download citation
DOI: https://doi.org/10.1007/978-1-59745-246-5_10
Publisher Name: Humana Press
Print ISBN: 978-1-58829-915-4
Online ISBN: 978-1-59745-246-5
eBook Packages: Springer Protocols