Mechanism of Time-Dependent Inhibition of Polypeptide Deformylase by Actinonin

Abstract

Polypeptide deformylase (PDF) is an essential bacterial metalloenzyme responsible for the removal of the N-formyl group from the N-terminal methionine of nascent polypeptides. Inhibition of bacterial PDF enzymes by actinonin, a naturally occurring antibacterial agent, has been characterized using steady-state and transient kinetic methods. Slow binding of actinonin to these enzymes is observed under steady-state conditions. Progress curve analysis is consistent with a two-step binding mechanism, in which tightening of the initial encounter complex (EI) results in a final complex (EI*) with an extremely slow, but observable, off-rate (t(1/2) for inhibitor dissociation >or=0.77 days). Stopped-flow measurement of PDF fluorescence confirms formation of EI and provides a direct measurement of the association rate. Rapid dilution studies establish that the potency of actinonin is enhanced by more than 2000-fold upon tightening of EI to form EI*, from K(i) = 530 nM (EI) to Ki*<or= 0.23 nM (EI*). In sharp contrast, the previously reported small molecule PDF inhibitor, SB-543668, is a competitive, readily reversible inhibitor (t(1/2) for dissociation = 2.8 s). In addition, we demonstrate that BB-3497 is also a time-dependent inhibitor of PDF with an extremely slow off-rate. The two-step inhibition model detailed herein for the inhibition of Staphylococcus aureus PDF by actinonin and BB-3497 is consistent with a recent report on the time-dependent inhibition of Escherichia coli PDF by a macrocyclic peptidomimetic inhibitor [Ngugen, K. T., et al. (2004) Bioorg. Chem. 32, 178-191]. This study substantially extends our understanding of PDF inhibition and may facilitate the development of novel antibiotics.

Phylogenomic and Biochemical Characterization of ThreeLegionella pneumophilaPolypeptide Deformylases

“…Here, we have provided strong evidence that PDF is the molecular target of PDF inhibitors, particularly BB-3497, with anti-L. pneumophila activity. As described for other bacterial species (35), both actinonin and BB-3497 exhibit time-dependent inhibition of all three L. pneumophila PDFs, with slow rates of dissociation from the enzymes. While the structural basis for time dependency is undefined at present, it is clear that the net effect of this time-dependent process is a significant potency increase ranging from 20-to 160-fold, comparing K i to K i *.…”

“…Time-dependent inhibition of PDF has been reported previously (15,26,35) and is best described by a two-step mechanism in which the initial encounter complex, EI, tightens to form EI* with a slow off rate (k 6 ).…”

“…We found that both compounds are time-dependent inhibitors (see Materials and Methods) of these isozymes (Table 3). Initial binding constants (K i ) and the overall inhibition constants, K i *, representing the affinity of the final EI* complex, were determined as described previously (35). As shown in Table 3, both inhibitors exhibited time-dependent inhibition, with their initial binding K i values in the range of hundreds of nM to 12 M in the case of actinonin against Ni 2ϩ PdfA.…”

“…PDF is essential for bacterial-cell viability in Escherichia coli, Streptococcus pneumoniae, and Staphylococcus aureus (5,6,22). Moreover, actinonin, a naturally occurring antibiotic that is a potent time-dependent inhibitor of PDF (35), inhibits bacterial growth by targeting the enzyme (6). Several newly derived PDF inhibitors have been shown to possess in vitro activity and to be efficacious in animal models against several gram-positive pathogens (17,19,21).…”

Frequency of Spontaneous Resistance to Peptide Deformylase Inhibitor GSK1322322 in Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae

“…Q57 is part of a conserved region present only in class II enzymes, and V71 is located within the highly conserved motif 1, which is involved in the structural stability and catalytic mechanism of PDF. It has been shown that PDF inhibitors such as GSK1322322 display strong time-dependent inhibition, which contributes to their antibacterial potency (32). Recent kinetic studies support the hypothesis that the time-dependent nature of PDF inhibition is due to both the binding of the compounds to the active site metal of PDF and the hydrogen-bonding network between the inhibitor and certain key residues of the protein, including V71 (33).…”