Peptide Deformylase in Staphylococcus aureus : Resistance to Inhibition Is Mediated by Mutations in the Formyltransferase Gene (original) (raw)
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Resistance of Streptococcus pneumoniae to Deformylase Inhibitors Is Due to Mutations in defB
Antimicrobial Agents and Chemotherapy, 2001
Resistance to peptide deformylase inhibitors in Escherichia coli or Staphylococcus aureus is due to inactivation of transformylase activity. Knockout experiments in Streptococcus pneumoniae R6x indicate that the transformylase (fmt) and deformylase (defB) genes are essential and that a def paralog (defA) is not. Actinoninresistant mutants of S. pneumoniae ATCC 49619 harbor mutations in defB but not in fmt. Reintroduction of the mutated defB gene into wild-type S. pneumoniae R6x recreates the resistance phenotype. The altered enzyme displays decreased sensitivity to actinonin.
Journal of Antimicrobial Chemotherapy, 2012
Objectives: To determine how the fitness cost of deformylase inhibitor resistance conferred by fmt mutations can be genetically compensated. Methods: Resistant mutants were isolated and characterized with regard to their growth rates in vitro and in neutropenic mice, MIC and DNA sequence. Faster-growing compensated mutants were isolated by serial passage in culture medium, and for a subset of the resistant and compensated mutants whole-genome sequencing was performed. Results: Staphylococcus aureus mutants resistant to the peptide deformylase inhibitor actinonin had mutations in the fmt gene that conferred high-level actinonin resistance and reduced bacterial growth rate. Compensated mutants that remained fully resistant to actinonin and showed increased growth rates appeared within 30-60 generations of growth. Whole-genome sequencing and localized DNA sequencing of mutated candidate genes showed that alterations in the gene agrC were present in the majority of compensated strains. Resistant and compensated mutants grew at similar rates as the wild-type in a mouse thigh infection model. Conclusions: Resistance to deformylase inhibitors due to fmt mutations reduces bacterial growth rates, but these costs can be reduced by mutations in the agrC gene. Mutants defective in fmt (with or without compensatory agrC mutations) grew well in an animal model, implying that they can also cause infection in a host.
Deformylase as a novel antibacterial target
Drug Discovery Today, 2001
Bacterial genomics has revealed a plethora of previously unknown targets of potential use in the discovery of novel antibacterial drugs. However, so far little has emerged from this approach. Peptide deformylase is an interesting target that was discovered more than 30 years ago, but was not exploited until recently. The reawakening of interest in this target resulted from an improved understanding of the enzyme, making it a more tractable and attractive target. Information on the properties of the enzyme, such as its three-dimensional structure, the activity of inhibitors, its resistance and suitability as a target are discussed. deformylase, a distinctive enzyme of eubacterial translation. EMBO J.
Characterization of peptide deformylase homologues from Staphylococcus epidermidis
Microbiology, 2010
The emergence of multi-drug-resistant strains of Staphylococcus epidermidis emphasizes the need to develop new antibiotics. The unique and essential role of the peptide deformylase (PDF) in catalysing the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria makes it an attractive antibacterial drug target. In the present study, both deformylase homologues from S. epidermidis (SePDF-1 and SePDF-2) were cloned and expressed, and their enzymic activities were characterized. Co2+-substituted SePDF-1 exhibited much higher enzymic activity (k cat/K m 6.3×104 M−1 s−1) than those of Ni2+- and Zn2+-substituted SePDF-1, and SePDF-1 showed much weaker binding ability towards Ni2+ than towards Co2+ and Zn2+, which is different from PDF in Staphylococcus aureus (SaPDF), although they share 80 % amino-acid sequence identity. The determined crystal structure of SePDF-1 was similar to that of (SaPDF), except for differences in the metal-binding sites. The other d...
Crystal Structure of Type II Peptide Deformylase from Staphylococcus aureus
Journal of Biological Chemistry, 2002
The first crystal structure of Class II peptide deformylase has been determined. The enzyme from Staphylococcus aureus has been over-expressed and purified in Escherichia coli and the structure determined by X-ray crystallography to 1.9 Å resolution. The purified iron-enriched form of S. aureus peptide deformylase enzyme retained high activity over many months. In contrast, the iron-enriched form of the E. coli enzyme is very labile. Comparison of the two structures details many differences; however, there is no structural explanation for the dramatic activity differences we observed. The protein structure of the S. aureus enzyme reveals a fold similar, but not identical to the well-characterized Escherichia coli enzyme. The most striking deviation of the S. aureus from E. coli structure is the unique conformation of the C-terminal amino acids. The distinctive C-terminal helix of the latter is replaced by a strand in S. aureus that wraps around the enzyme terminating near the active site. While there are no differences at the amino acid level near the active site metal ion, significant changes are noted in the peptide binding cleft that may play a role in the design of general peptide deformylase inhibitors.
Characterization of a Human Peptide Deformylase: Implications for Antibacterial Drug Design †
Biochemistry, 2003
Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA i , resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes, making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencing has revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding Homo sapiens PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-aminoacid targeting sequence was overexpressed in Escherichia coli. The recombinant, Co 2+ -substituted protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhanced green fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion. However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation for the apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is at least partially due to mutation of a highly conserved residue (Leu-91 in E. coli PDF) in mammalian PDF. PDF inhibitors had no detectable effect on two different human cell lines. These results suggest that HsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylation and validates PDF as an excellent target for antibacterial drug design.
Antimicrobial Agents and Chemotherapy, 2001
An assay was developed to determine the activity of peptide deformylase (PDF) inhibitors under conditions as close as possible to the physiological situation. The assay principle is the detection of N-terminal [ 35 S]methionine labeling of a protein that contains no internal methionine. If PDF is active, the deformylation of the methionine renders the peptide a substrate for methionine aminopeptidase, resulting in the removal of the N-terminal methionine label. In the presence of a PDF inhibitor, the deformylation is blocked so that the N-formylated peptide is not processed and the label is detected. Using this assay, it is possible to determine the PDF activity under near-physiological conditions in a cell-free transcription-translation system as well as in intact bacterial cells.
Biochemical Pharmacology, 2006
Although peptide deformylase (PDF, EC 3.5.1.27) was first described in 1968, the instability of enzyme preparations prevented it from being seriously considered as a target until this problem was finally solved in 1998. PDFs essentiality was first demonstrated in Escherichia coli in 1994. Genomic analyses have shown this enzyme to be present in all eubacteria. PDF homologs have also been found in eukaryotes including Homo sapiens. The function and relevance of the human chromosomal homolog to the safety of PDF inhibitors as therapeutic agents is not clear at this stage. Although there is considerable sequence variation between the different bacterial PDFs, there are three strongly conserved motifs that together constitute a critical metal binding site. The observation that PDF is a metalloenzyme has led to the design of inhibitors containing metal chelating pharmacophores. The most potent of these synthetic inhibitors are active against a range of clinically relevant respiratory tract pathogens in vitro and in vivo, including those resistant to current antibiotics. Mutants resistant to PDF inhibitors have been obtained in the laboratory; these resulted from mutations in the genes for transformylase (EC 2.1.2.9) or PDF. The mechanism involved and its frequency were pathogen-dependent. The two most advanced PDF inhibitor leads, which are both reverse hydroxamates, have progressed to phase 1 clinical trials and were well tolerated.
Journal of bacteriology, 1993
Overexpression of the fms gene, the first translation unit of a dicistronic operon that also encodes methionyl-tRNA(fMet) formyltransferase in Escherichia coli, sustains the overproduction of peptide deformylase activity in crude extracts. This suggests that the fms gene encodes the peptide deformylase. Moreover, the fms gene product has a motif characteristic of metalloproteases, an activity compatible with deformylase. The corresponding protein could be purified to homogeneity. However, its enzymatic activity could not be retained during the purification procedure. As could be expected from the occurrence in its amino acid sequence of a zinc-binding motif characteristic of metallopeptidases, the purified fms product displayed one tightly bound zinc atom.