Expression of N-formylated proteins in Escherichia coli

In bacteria, protein expression initiates with a formyl-methionine group. Addition of the antibiotic actinonin, a known peptide deformylase inhibitor, at the time of induction of protein expression results in the retention of the formyl group by the overexpressed protein. In addition, because deformylation is a prerequisite for removal of the initiating methionine, this post-translational processing step is also prevented by actinonin, and the N-formyl methionine residue is retained by proteins from which it is normally removed. We have demonstrated the applicability of this system for obtaining N-modified forms of several different proteins and use one of these modified molecules to show that the N-terminal amino group is not required for ClpXP degradation of proteins bearing an N-terminal recognition signal.     

Structural basis for the design of antibiotics targeting peptide deformylase

While protein synthesis in bacteria begins with a formylated methionine, the formyl group of the nascent polypeptide is removed by peptide deformylase. Since eukaryotic protein synthesis does not involve formylation and deformylation at the N-terminus, there has been increasing interest in peptide deformylase as a potential target for antibacterial chemotherapy. Toward this end and to aid in the design of effective antibiotics targeting peptide deformylase, the structures of the protein-inhibitor complexes of both the cobalt and the zinc containing Escherichia coli peptide deformylase bound to the transition-state analogue, (S)-2-O-(H-phosphonoxy)-L-caproyl-L-leucyl-p-nitroanilide (PCLNA), have been determined. The proteins for both deformylase-inhibitor complexes show basically the same fold as for the native enzyme. The PCLNA inhibitor adopts an extended conformation and fits nicely into a hydrophobic cavity located near the metal site. On the basis of these structures, guidelines for the design of high-affinity deformylase inhibitors are suggested. As our results show that the protein residues which interact with the PCLNA inhibitor are conserved over a wide variety of species, we suggest that antibiotics targeting deformylase could have wide applicability.     

Increased production of peptide deformylase eliminates retention of formylmethionine in bovine somatotropin overproduced in Escherichia coli

In Escherichia coli and most other microorganisms, peptide synthesis is started at methionine start codons which are read only by N-formyl-methionine-tRNA. The formyl group is normally removed from the N-terminal Met residue of the peptide by peptide deformylase (PDF). However, it has been observed that overproduction of proteins in recombinant bacteria often yields protein products which are incompletely deformylated. Certain proteins could be poor substrates for PDF and exhibit incomplete deformylation, particularly when they are overproduced. Strains of E. coli which overproduce bovine somatotropin (BST) have a significant fraction of the BST with the formyl group retained. The PDF gene was isolated and positioned into a BST production vector in such a way that the BST and PDF genes were coexpressed. In strains containing this coexpression vector, the levels of PDF were increased and formylated BST was undetectable.     

Peptide deformylase inhibitors with non-peptide scaffold: synthesis and structure-activity relationships

Peptide deformylase (PDF), which removes the formyl group at the N-terminal methionine residue of nascent protein, has been recognized as a potent target for antibacterial therapy. We report herein the synthesis and structure-activity relationship studies of non-peptide PDF inhibitors.     

Formyl peptide receptor-mediated proinflammatory consequences of peptide deformylase inhibition in Staphylococcus aureus

The biosynthesis of proteins with N-terminal formylated methionine residues and subsequent protein deformylation are unique and invariant bacterial processes. They are exploited by the capacity of the human innate immune system to sense formylated peptides (FPs) and targeted by the deformylation-blocking antibiotic actinonin. We show that human polymorphonuclear leukocytes respond via the formyl peptide receptor (FPR) with increased calcium ion fluxes, chemotactic migration, IL-8 release, and CD11b upregulation to the human pathogen Staphylococcus aureus upon actinonin treatment. These data underscore the crucial role of bacterial FPs in innate immunity and indicate that deformylase inhibition may have considerable proinflammatory consequences.     

Genetic characterization of polypeptide deformylase, a distinctive enzyme of eubacterial translation.

Deformylase performs an essential step in the maturation of proteins in eubacteria, by removing the formyl group from the N-terminal methionine residue of ribosome-synthesized polypeptides. In spite of this important role in translation, the enzyme had so far eluded characterization because of its instability. We report the isolation of the deformylase gene of Escherichia coli, def, by overexpression of a genomic library from a high-copy-number plasmid and selection for utilization of the substrate analogue formyl-leucyl-methionine as a source of methionine. The def gene encodes a 169 amino acid polypeptide that bears no obvious resemblance to other known proteins. It forms an operon with the fmt gene, that encodes the initiator methionyl-tRNA(i) transformylase, which was recently characterized (Guillon et al., J. Bacteriol., 174, 4294-4301, 1992). This operon was mapped at min 72 of the E. coli chromosome. The def gene could be inactivated if the fmt gene was also inactivated, or if biosynthesis of N10-formyl-tetrahydrofolate, the formyl donor in methionyl-tRNA(i) transformylation, was blocked by trimethoprim. These findings designate deformylase as a target for antibacterial chemotherapy.     

Activation of antibacterial prodrugs by peptide deformylase

5'-Dipeptidyl derivatives of 5-fluorodeoxyuridine (FdU) (1a-d) were synthesized. These compounds are biologically inactive but can be activated by peptide deformylase, which removes the N-terminal formyl group of the dipeptide, to release the active drug FdU via an intramolecular cyclization reaction. Because the deformylase is ubiquitous among bacteria but absent in mammalian cells, 1a-d provide a novel class of potential antibacterial agents.     

1H, 13C and 15N NMR assignments of the E. coli peptide deformylase in complex with a natural inhibitor called actinonin

In eubacteria, the formyl group of nascent polypeptides is removed by peptide deformylase protein (PDF). This is the reason why PDF has received special attention in the course of the search for new antibacterial agents. We observed by NMR that actinonin, a natural inhibitor, induced drastic changes in the HSQC spectrum of E. coli PDF. We report here the complete NMR chemical shift assignments of PDF resonances bound to actinonin.     

A direct spectrophotometric assay for peptide deformylase.

A direct UV-VIS spectrophotometric assay has been developed for peptide deformylase. This assay employs a novel class of peptide mimetics as deformylase substrates which, upon enzymatic removal of the N-terminal formyl group, rapidly release free thiols. The released thiols are quantitated using Ellman's reagent. A variety of peptide analogues that contain beta-thiaphenylalanine or beta-thiamethionine as the N-terminal residue were synthesized and found to be excellent substrates of the peptide deformylase from Escherichia coli (k(cat)/K(M) = 6.9 x 10(5) M(-1) s(-1) for the most reactive substrate). The deformylase reaction is conveniently monitored on a UV-VIS spectrophotometer in a continuous fashion. The versatility of the assay has been demonstrated by its application to kinetic characterization of the deformylase, pH profile studies, and enzyme inhibition assays. The assay can also be performed in an end-point fashion. The results demonstrate that this assay is a simple, highly sensitive, and rapid method to study kinetic properties of deformylases without the use of any coupling enzymes.     

The role of peptide deformylase in protein biosynthesis: a proteomic study

Recently we investigated the influence of classical and emerging antibiotics on the proteome of Bacillus subtilis including in our studies actinonin, a potent novel inhibitor of peptide deformylase. The protein synthesis pattern under actinonin treatment changed so dramatically that a direct comparison to the control pattern was impossible. Dual channel imaging revealed that actinonin treatment caused the majority of newly synthesised proteins to accumulate in spots different from the ones usually observed, indicating a more acidic isoelectric point. Two strategies were used to investigate the nature of the charge shift. In the first place, protein patterns of a conditional peptide deformylase mutant under nonrepressing and repressing conditions were compared. Secondly, several protein pairs excised from two-dimensional (2-D) gels of the peptide deformylase mutant, exponentially growing untreated wild-type and the actinonin treated wild-type were investigated with matrix-assisted laser desorption/ionization and electrospray ionization (ESI) time of flight mass spectrometry (TOF MS) for the existence of N-terminal formylation. Under nonrepressing conditions the mutant protein pattern resembled that of the wild-type. The loss of peptide deformylase activity under repressing conditions led to the same pI shift observed for actinonin treatment in the wild-type. Quadrupole TOF-MS on 11 protein pairs proved that the remaining N-terminal formyl residue was indeed responsible for the charge shift. Eight of these protein pairs were also present on 2-D gels of exponentially growing B. subtilis, where the more acidic, still formylated protein species represented the smaller parts.     

Synthesis and deformylation of Staphylococcus aureus delta-toxin are linked to tricarboxylic acid cycle activity

In bacteria, translation initiates with formyl-methionine; however, the N-terminal formyl group is usually removed by peptide deformylase, an enzymatic activity requiring iron. Staphylococcus aureus delta-toxin is a 26-amino-acid polypeptide secreted predominantly with a formylated N-terminal methionine, which led us to investigate regulation of delta-toxin deformylation. We observed that during exponential and early postexponential growth, delta-toxin accumulated in the culture medium in formylated and deformylated forms. In contrast, only formylated delta-toxin accumulated after the early postexponential phase. The transition from producing both species of delta-toxin to producing only formyl-methionine-containing delta-toxin coincided with increased tricarboxylic acid (TCA) cycle activity. The TCA cycle contains several iron-requiring enzymes, which led us to hypothesize that TCA cycle induction depletes the iron in the culture medium, thereby inhibiting peptide deformylase activity. As expected, S. aureus depletes the iron in the culture medium between the postexponential and stationary phases of growth. Inhibition of delta-toxin deformylation was relieved by TCA cycle inactivation or by addition of supplemental iron to the culture medium. Of interest, peptides containing formyl-methionine are potent chemoattractants for neutrophils, suggesting that delta-toxin deformylation may have functional consequences. We found neutrophil chemotactic activity only with formylated delta-toxin. The S. aureus TCA cycle is derepressed upon depletion of rapidly catabolizable carbon sources; this coincides with the transition to producing only formylated delta-toxin and results in an increased inflammatory response. The proinflammatory response should increase host cell damage and result in the release of nutrients. Taken together, these results establish that there is an important linkage between bacterial metabolism and pathogenesis.     

Expression of Escherichia coli methionyl-tRNA formyltransferase in Saccharomyces cerevisiae leads to formylation of the cytoplasmic initiator tRNA and possibly to initiation of protein synthesis with formylmethionine

Protein synthesis in eukaryotic cytoplasm and in archaebacteria is initiated with methionine, whereas, that in eubacteria and in eukaryotic organelles, such as mitochondria and chloroplasts, is initiated with formylmethionine. In view of this clear distinction, we have investigated whether protein synthesis in the eukaryotic cytoplasm can be initiated with formylmethionine, and, if so, what the consequences are to the cell. For this purpose, we have expressed in an inducible manner the Escherichia coli methionyl-tRNA formyltransferase (MTF) in the cytoplasm of the yeast Saccharomyces cerevisiae. Expression of active MTF, but not of an inactive mutant, leads to formylation of methionine attached to the yeast cytoplasmic initiator tRNA to the extent of about 70%. As a consequence, the yeast strain grows slowly. Coexpression of the E. coli polypeptide deformylase (DEF), which removes the formyl group from the N-terminal formylmethionine in a polypeptide, rescues the slow-growth phenotype, whereas, coexpression of an inactive mutant of DEF does not. These results suggest that the cytoplasmic protein-synthesizing system of yeast, like that of eubacteria, can at least to some extent utilize formylated initiator Met-tRNA to initiate protein synthesis and that initiation of proteins with formylmethionine leads to the slow-growth phenotype. Removal of the formyl group in these proteins by DEF would explain the rescue of the slow-growth phenotype.     

Energetic rationale for an unexpected and abrupt reversal of guanidinium chloride-induced unfolding of peptide deformylase

Peptide deformylase (PDF) catalyzes the removal of formyl group from the N-terminal methionine residues of nascent proteins in prokaryotes, and this enzyme is a high priority target for antibiotic design. In pursuit of delineating the structural-functional features of Escherichia coli PDF (EcPDF), we investigated the mechanistic pathway for the guanidinium chloride (GdmCl)-induced unfolding of the enzyme by monitoring the secondary structural changes via CD spectroscopy. The experimental data revealed that EcPDF is a highly stable enzyme, and it undergoes slow denaturation in the presence of varying concentrations of GdmCl. The most interesting aspect of these studies has been the abrupt reversal of the unfolding pathway at low to moderate concentrations of the denaturant, but not at high concentration. An energetic rationale for such an unprecedented feature in protein chemistry is offered.     

Peptide deformylase inhibitors of Mycobacterium tuberculosis: Synthesis,structural investigations,and biological results

Bacterial peptide deformylase (PDF) belongs to a subfamily of metalloproteases catalyzing the removal of the N-terminal formyl group from newly synthesized proteins. We report the synthesis and biological activity of highly potent inhibitors of Mycobacterium tuberculosis (Mtb) PDF enzyme as well as the first X-ray crystal structure of Mtb PDF. Structure–activity relationship and crystallographic data clarified the structural requirements for high enzyme potency and cell based potency. Activities against single and multi-drug-resistant Mtb strains are also reported.     

Inhibition of bacterial peptide deformylase by biaryl acid analogs

Peptide deformylase is an essential eubacterial metalloenzyme involved in the maturation of proteins by cleaving the N-formyl group from N-blocked methionine polypeptides. Biaryl acid analogs containing tetrazole, acyl sulfonamide, or carboxylate pharmacophores were found to be potent inhibitors of recombinant Escherichia coli peptide deformylase. Two of these compounds, a biphenyl tetrazole, compound 1, and a biphenyl acyl sulfonamide, compound 4, were competitive inhibitors with K(i) values of 1.2 and 6.0 microM, respectively. By analogy to the binding of related compounds to other metalloenzymes such as Bacteroides fragilis metallo-beta-lactamase CcrA and human carbonic anhydrase, a mechanism of inhibition is proposed for these peptide deformylase inhibitors where the acidic moieties form direct ionic interactions with the active site metal cation.     

Inhibition and structure-activity studies of methionine hydroxamic acid derivatives with bacterial peptide deformylase

The posttranslational deformylation of N-formyl-Met-polypeptides by the metalloenzyme, peptide deformylase, is essential for bacterial growth. Methionine hydroxamic acid derivatives were found to inhibit recombinant Escherichia coli peptide deformylase activity containing either zinc or cobalt. The binding of methionine hydroxamate and hydrazide inhibitors to cobalt-substituted deformylase caused spectral changes consistent with the formation of a pentacoordinate metal complex similar to that of actinonin, a psuedopeptide hydroxamate inhibitor. The spectral and kinetic data support the binding of these N-substituted L-methionine derivatives in a reverse orientation with respect to N-formyl-Met-peptide substrates within the active site. Based on this hypothesis a second generation of N-substituted methionyl hydroxamic acids were evaluated and found to possess greater inhibitory potency. These results may provide the basis for the design of more potent and selective deformylase inhibitors as potential antibacterial agents.     

Rice peptide deformylase PDF1B is crucial for development of chloroplasts

Because protein synthesis begins with N-formylmethionine in plant endosymbiotic organelles, removal of the formyl group by peptide deformylase (PDF) is essential to allowing the excision of the first methionine. Rice contains three copies (OsPDF1A, OsPDF1B and OsPDF1B2) of the PDF genes. Unlike OsPDF1A and OsPDF1B, OsPDF1B2 is apparently non-functional, with several deleterious substitutions and deletions. OsPDF1A is more strongly expressed in the roots, while OsPDF1B is expressed at higher levels in mature leaves. Transient expression of PDF-green fluorescent protein (GFP) fusion proteins in the protoplasts demonstrates that, unlike OsPDF1A, OsPDF1B is localized in both the chloroplasts and the mitochondria. We used T-DNA insertional alleles to elucidate functional roles associated with OsPDF1B. Homozygous plants of pdf1b/pdf1b exhibited the phenotypes of chlorina and growth retardation. Histochemical analysis showed that the length of their mesophyll cells was increased 4- to 5-fold, resulting in a reduction in the total number of cells. Transmission electron microscopy analyses revealed that chloroplasts were severely damaged and mitochondria appeared to be mildly altered in the pdf1b mutants. Expression of genes encoded in the chloroplasts and mitochondria was altered in the mutants. Based on these results, we conclude that OsPDF1B is essential for the development of chloroplast and perhaps mitochondria.     

Structure analysis of peptide deformylases from Streptococcus pneumoniae,Staphylococcus aureus,Thermotoga maritima and Pseudomonas aeruginosa: snapshots of the oxygen sensitivity of peptide deformylase

Peptide deformylase (PDF) has received considerable attention during the last few years as a potential target for a new type of antibiotics. It is an essential enzyme in eubacteria for the removal of the formyl group from the N terminus of the nascent polypeptide chain. We have solved the X-ray structures of four members of this enzyme family, two from the Gram-positive pathogens Streptococcus pneumoniae and Staphylococcus aureus, and two from the Gram-negative bacteria Thermotoga maritima and Pseudomonas aeruginosa. Combined with the known structures from the Escherichia coli enzyme and the recently solved structure of the eukaryotic deformylase from Plasmodium falciparum, a complete picture of the peptide deformylase structure and function relationship is emerging. This understanding could help guide a more rational design of inhibitors. A structure-based comparison between PDFs reveals some conserved differences between type I and type II enzymes. Moreover, our structures provide insights into the known instability of PDF caused by oxidation of the metal-ligating cysteine residue.     

Enzymatic properties of Escherichia coli peptide deformylase.

Since its discovery in crude extracts in the late sixties, Escherichia coli peptide deformylase activity could not be further characterized because of an apparent extreme instability. We show that this behavior was caused by an inadequate activity assay, involving substrate concentration inhibition and substrate precipitation in crude extracts. The homogeneous protein, as it was previously purified (T. Meinnel and S. Blanquet J. Bacteriol. 175:7737-7740, 1993), had actually retained its initial activity. The influence on the deformylation reaction of several factors was studied and used to improve the activity assay. Pure peptide deformylase proves to act only on peptide substrates with an N-formylmethionyl moiety. In agreement with the occurrence of zinc in the enzyme, peptide deformylase activity is inhibited by 1,10-phenanthroline.     

Synthesis and antibacterial activity of peptide deformylase inhibitors

Peptide deformylase catalyzes the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria. Its essential character in bacterial cells makes it an attractive target for antibacterial drug design. In this work, we have rationally designed and synthesized a series of peptide thiols that act as potent, reversible inhibitors of purified recombinant peptide deformylase from Escherichia coli and Bacillus subtilis. The most potent inhibitor has a K(I) value of 11 nM toward the B. subtilis enzyme. These inhibitors showed antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations (MIC) as low as 5 microM ( approximately 2 microg/mL). The PDF inhibitors induce bacterial cell lysis and are bactericidal toward all four bacterial strains that have been tested, B. subtilis, Staphylococcus epidermidis, Enterococcus faecalis, and E. coli. Resistance evaluation of one of the inhibitors (1b) against B. subtilis showed that no resistant clone could be found from >1 x 10(9) cells. Quantitative analysis using a set of inhibitors designed to possess varying potencies against the deformylase enzyme revealed a linear correlation between the MIC values and the K(I) values. These results suggest that peptide deformylase is the likely molecular target responsible for the antibacterial activity of these inhibitors and is therefore a viable target for antibacterial drug design.