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Peptide aldehyde inhibitors of bacterial peptide deformylases
D J Durand1, B Gordon Green, J F O'Connell
1Department of Enzymology, Merck Research Laboratories, Rahway, New Jersey, 07065, USA.
Abstract:
Bacterial peptide deformylases (PDF, EC 3.5.1.27) are metalloenzymes that cleave the N-formyl groups from N-blocked methionine polypeptides. Peptide aldehydes containing a methional or norleucinal inhibited recombinant peptide deformylase from gram-negative Escherichia coli and gram-positive Bacillus subtilis. The most potent inhibitor was calpeptin, N-CBZ-Leu-norleucinal, which was a competitive inhibitor of the zinc-containing metalloenzymes, E. coli and B. subtilis PDF with Ki values of 26.0 and 55.6 microM, respectively. Cobalt-substituted E. coli and B. subtilis deformylases were also inhibited by these aldehydes with Ki values for calpeptin of 9.5 and 12.4 microM, respectively. Distinct spectral changes were observed upon binding of calpeptin to the Co(II)-deformylases, consistent with the noncovalent binding of the inhibitor rather than the formation of a covalent complex. In contrast, the chelator 1,10-phenanthroline caused the time-dependent inhibition of B. subtilis Co(II)-PDF activity with the loss of the active site metal. The fact that calpeptin was nearly equipotent against deformylases from both gram-negative and gram-positive bacterial sources lends further support to the idea that a single deformylase inhibitor might have broad-spectrum antibacterial activity.
Insights
Calpeptin effectively inhibits bacterial peptide deformylases (PDF) from both gram-negative and gram-positive bacteria. This potent inhibitor shows potential for broad-spectrum antibacterial activity by targeting essential metalloenzymes.
Area of Science:
- Biochemistry
- Enzymology
- Microbiology
Background:
- Bacterial peptide deformylases (PDF) are essential metalloenzymes responsible for removing N-formyl groups from nascent bacterial proteins.
- Inhibition of PDF can disrupt bacterial protein synthesis, presenting a potential target for novel antibiotics.
Purpose of the Study:
- To investigate the inhibitory potential of peptide aldehydes against bacterial peptide deformylases.
- To characterize the inhibition mechanism and spectrum of activity of potent PDF inhibitors.
Main Methods:
- Recombinant peptide deformylases from Escherichia coli (gram-negative) and Bacillus subtilis (gram-positive) were used.
- Enzyme inhibition assays were performed using peptide aldehydes, including calpeptin.
- Kinetic analysis (Ki values) and spectroscopic methods were employed to study inhibitor binding.
Main Results:
- Calpeptin (N-CBZ-Leu-norleucinal) demonstrated potent, competitive inhibition against both E. coli and B. subtilis PDF (Ki values in the microM range).
- Cobalt-substituted deformylases showed similar inhibition by calpeptin, with spectral changes indicating noncovalent binding.
- 1,10-phenanthroline caused time-dependent inhibition and metal loss, distinct from calpeptin's mechanism.
Conclusions:
- Calpeptin is a highly effective inhibitor of bacterial peptide deformylases from diverse bacterial species.
- The noncovalent binding mechanism and broad-spectrum activity suggest calpeptin's potential as a lead compound for developing new antibacterial agents.