Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis

Arabidopsis (ecotype Columbia-0) genes, AtDEF1 and AtDEF2, represent eukaryotic homologs of the essential prokaryotic gene encoding peptide deformylase. Both deduced proteins contain three conserved protein motifs found in the active site of all eubacterial peptide deformylases, and N-terminal extensions identifiable as chloroplast-targeting sequences. Radiolabeled full-length AtDEF1 was imported and processed by isolated pea (Pisum sativum L. Laxton's Progress No. 9) chloroplasts and AtDEF1 and 2 were immunologically detected in Arabidopsis leaf and chloroplast stromal protein extracts. The partial cDNAs encoding the processed forms of Arabidopsis peptide deformylase 1 and 2 (pAtDEF1 and 2, respectively) were expressed in Escherichia coli and purified using C-terminal hexahistidyl tags. Both recombinant Arabidopsis peptide deformylases had peptide deformylase activity with unique kinetic parameters that differed from those reported for the E. coli enzyme. Actinonin, a specific peptide deformylase inhibitor, was effective in vitro against Arabidopsis peptide deformylase 1 and 2 activity, respectively. Exposure of several plant species including Arabidopsis to actinonin resulted in chlorosis and severe reductions in plant growth and development. The results suggest an essential role for peptide deformylase in protein processing in all plant plastids.     

Characterization of the L399P and R447G mutants of hsc70: the decrease in refolding activity is correlated with an increase in the rate of substrate dissociation

Peptide deformylase (DEF; EC 3.5.1.88) removes the N-formyl group from nascent polypeptides. Two nuclear-encoded DEFs in Arabidopsis thaliana (At) are localized to chloroplasts, and thus, the N-termini of chloroplast-translated proteins may be a consequence of AtDEFs' substrate specificity. Using peptide analogs of select chloroplast-translated proteins, AtDEF1 activity was as much as 100-fold lower than AtDEF2 activity and showed little variance with peptide sequence. However, AtDEF2 activity was significantly influenced by peptide sequence, with the most efficiently processed substrate mimicking the N-terminus of the nascent D1 polypeptide, a core protein of photosystem II. Though AtDEF2's specificity was predictive of N-formyl retention for some chloroplast proteins, exceptions suggests that additional factors in vivo aid in determining the retention of an N-formyl group.     

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.     

Over-expression of peptide deformylase in chloroplasts confers actinonin resistance,but is not a suitable selective marker system for plastid transformation

Arabidopsis thaliana peptide deformylase PDF1B was expressed in tobacco chloroplasts using spectinomycin as the selective agent. The foreign protein accumulated in chloroplasts (6% of the total soluble protein) and was enzymatically active. Transplastomic plants were evaluated for resistance to the peptide deformylase inhibitor actinonin. In vitro seed germination in the presence of actinonin and in planta application of the inhibitor demonstrated the resistance of the transformed plants. In addition, transgenic leaf explants were able to develop shoots via organogenesis in the presence of actinonin. However, when the combination of the PDF1B gene and actinonin was used as the primary selective marker system for chloroplast transformation of tobacco, all developed shoots were escapes. Therefore, under the experimental conditions tested, the use of this system for plastid transformation would be limited to function as a secondary selective marker.     

Inhibition of peptide deformylase in Nicotiana tabacum leads to decreased D1 protein accumulation, ultimately resulting in a reduction of photosystem II complexes

Eukaryotic homologs of bacterial peptide deformylases were recently found in several vascular plants and may be essential in chloroplast protein processing. Treating tobacco seedlings with the peptide deformylase inhibitor actinonin resulted in leaf chlorosis and reduced growth and development, indicative of a systemic movement of the inhibitor. Photosystem II (PSII) activity was reduced, manifested as a significant decrease in the maximum quantum efficiency of photosystem II. Accumulation and assembly of nascent D1 protein into PSII monomers was also reduced, eventually leading to PSII disassembly and leaf necrosis. Processing and assembly of D1 protein in tobacco was a major and potentially critical target of peptide deformylase inhibition. These results confirm that N-terminal deformylation is an essential step in the accumulation and assembly of PSII subunit polypeptides in the chloroplasts of vascular plants.     

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.     

The crystal structures of four peptide deformylases bound to the antibiotic actinonin reveal two distinct types: a platform for the structure-based design of antibacterial agents

Bacterial peptide deformylase (PDF) belongs to a sub-family of metalloproteases that catalyse the removal of the N-terminal formyl group from newly synthesised proteins. PDF is essential in prokaryotes and conserved throughout the eubacteria. It is therefore considered an attractive target for developing new antibacterial agents. Here, we report the crystal structures of four bacterial deformylases, free or bound to the naturally occurring antibiotic actinonin, including two from the major bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus. The overall tertiary structure is essentially conserved but shows significant differences, namely at the C terminus, which are directly related to the deformylase type (i.e. I or II) they belong to. The geometry around the catalytic metal ion exhibits a high level of similarity within the different enzymes, as does the binding mode of actinonin to the various deformylases. However, some significant structural differences are found in the vicinity of the active site, highlighting the structural and molecular requirements for the design of a deformylase inhibitor active against a broad spectrum of bacterial strains.     

Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts

Unexpected peptide deformylase (PDF) genes were recently retrieved in numerous marine phage genomes. While various hypotheses dealing with the occurrence of these intriguing sequences have been made, no further characterization and functional studies have been described thus far. In this study, we characterize the bacteriophage Vp16 PDF enzyme, as representative member of the newly identified C-terminally truncated viral PDFs. We show here that conditions classically used for bacterial PDFs lead to an enzyme exhibiting weak activity. Nonetheless, our integrated biophysical and biochemical approaches reveal specific effects of pH and metals on Vp16 PDF stability and activity. A novel purification protocol taking in account these data allowed strong improvement of Vp16 PDF specific activity to values similar to those of bacterial PDFs. We next show that Vp16 PDF is as sensitive to the natural inhibitor compound of PDFs, actinonin, as bacterial PDFs. Comparison of the 3D structures of Vp16 and E. coli PDFs bound to actinonin also reveals that both PDFs display identical substrate binding mode. We conclude that bacteriophage Vp16 PDF protein has functional peptide deformylase activity and we suggest that encoded phage PDFs might be important for viral fitness.     

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.     

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.     

Actinonin, a naturally occurring antibacterial agent, is a potent deformylase inhibitor

Peptide deformylase (PDF) is essential in prokaryotes and absent in mammalian cells, thus making it an attractive target for the discovery of novel antibiotics. We have identified actinonin, a naturally occurring antibacterial agent, as a potent PDF inhibitor. The dissociation constant for this compound was 0.3 x 10(-)(9) M against Ni-PDF from Escherichia coli; the PDF from Staphylococcus aureus gave a similar value. Microbiological evaluation revealed that actinonin is a bacteriostatic agent with activity against Gram-positive and fastidious Gram-negative microorganisms. The PDF gene, def, was placed under control of P(BAD) in E. coli tolC, permitting regulation of PDF expression levels in the cell by varying the external arabinose concentration. The susceptibility of this strain to actinonin increases with decreased levels of PDF expression, indicating that actinonin inhibits bacterial growth by targeting this enzyme. Actinonin provides an excellent starting point from which to derive a more potent PDF inhibitor that has a broader spectrum of antibacterial activity.     

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.     

A new human peptide deformylase inhibitable by actinonin

Peptide deformylases (PDFs) have been investigated as potential specific targets for antibiotics, but the possible existence of a functional human PDF (HsPDF) presents a potential hurdle to the design of specific drugs. We have expression cloned a HsPDF that has deformylase activity, although it is a slower and catalytically less active enzyme than bacterial or plant PDFs. A cobalt-substituted form of HsPDF (but not nickel or zinc) is active, and the enzyme appears to be active at a pH between 6.0 and 7.2, a temperature range of 25-50 degrees C, and in a low KCl ionic strength buffer. Actinonin inhibits HsPDF activity with an IC50 of 43 nM and kills Daudi and HL60 human cancer cell lines with an LC50 of 5.3 and 8.8 microM, respectively. The inhibition of HsPDF may provide an explanation for the mechanism by which actinonin is cytotoxic against various human tumor cell lines.     

Identification of eukaryotic peptide deformylases reveals universality of N-terminal protein processing mechanisms

The N-terminal protein processing pathway is an essential mechanism found in all organisms. However, it is widely believed that deformylase, a key enzyme involved in this process in bacteria, does not exist in eukaryotes, thus making it a target for antibacterial agents such as actinonin. In an attempt to define this process in higher eukaryotes we have used Arabidopsis thaliana as a model organism. Two deformylase cDNAs, the first identified in any eukaryotic system, and six distinct methionine aminopeptidase cDNAs were cloned. The corresponding proteins were characterized in vivo and in vitro. Methionine aminopeptidases were found in the cytoplasm and in the organelles, while deformylases were localized in the organelles only. Our work shows that higher plants have a much more complex machinery for methionine removal than previously suspected. We were also able to identify deformylase homologues from several animals and clone the corresponding cDNA from human cells. Our data provide the first evidence that lower and higher eukaryotes, as well as bacteria, share a similar N-terminal protein processing machinery, indicating universality of this system.     

Structure and Activity of Human Mitochondrial Peptide Deformylase, a Novel Cancer Target

Peptide deformylase proteins (PDFs) participate in the N-terminal methionine excision pathway of newly synthesized peptides. We show that the human PDF (HsPDF) can deformylate its putative substrates derived from mitochondrial DNA-encoded proteins. The first structural model of a mammalian PDF (1.7 Å), HsPDF, shows a dimer with conserved topology of the catalytic residues and fold as non-mammalian PDFs. The HsPDF C-terminus topology and the presence of a helical loop (H2 and H3), however, shape a characteristic active site entrance. The structure of HsPDF bound to the peptidomimetic inhibitor actinonin (1.7 Å) identified the substrate-binding site. A defined S1′ pocket, but no S2′ or S3′ substrate-binding pockets, exists. A conservation of PDF-actinonin interaction across PDFs was observed. Despite the lack of true S2′ and S3′ binding pockets, confirmed through peptide binding modeling, enzyme kinetics suggest a combined contribution from P2′and P3′ positions of a formylated peptide substrate to turnover.     

The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes.

The Mi locus of tomato confers resistance to root knot nematodes. Tomato DNA spanning the locus was isolated as bacterial artificial chromosome clones, and 52 kb of contiguous DNA was sequenced. Three open reading frames were identified with similarity to cloned plant disease resistance genes. Two of them, Mi-1.1 and Mi-1.2, appear to be intact genes; the third is a pseudogene. A 4-kb mRNA hybridizing with these genes is present in tomato roots. Complementation studies using cloned copies of Mi-1.1 and Mi-1.2 indicated that Mi-1.2, but not Mi-1.1, is sufficient to confer resistance to a susceptible tomato line with the progeny of transformants segregating for resistance. The cloned gene most similar to Mi-1.2 is Prf, a tomato gene required for resistance to Pseudomonas syringae. Prf and Mi-1.2 share several structural motifs, including a nucleotide binding site and a leucine-rich repeat region, that are characteristic of a family of plant proteins, including several that are required for resistance against viruses, bacteria, fungi, and now, nematodes.     

Metalloprotease inhibitors GM6001 and TAPI-0 inhibit the obligate intracellular human pathogen Chlamydia trachomatis by targeting peptide deformylase of the bacterium

Chlamydia trachomatis is an obligate intracellular bacterium responsible for a number of human diseases. The mechanism underlying the intracellular parasitology of Chlamydiae remains poorly understood. In searching for host factors required for chlamydial infection, we discovered that C. trachomatis growth was effectively inhibited with GM6001 and TAPI-0, two compounds known as specific inhibitors of matrix metalloproteases. The inhibition was independent of chlamydial entry of the cell, suggesting that the loss of extracellular metalloprotease activities of the host cell is unlikely to be the mechanism for the growth suppression. Nucleotide sequences of candidate metalloprotease genes remained unchanged in a chlamydial variant designated GR10, which had been selected for resistance to the inhibitors. Nevertheless, GR10 displayed a single base mutation in the presumable promoter region of the gene for peptide deformylase (PDF), a metal-dependent enzyme that removes the N-formyl group from newly synthesized bacterial proteins. The mutation correlated with an increased PDF expression level and resistance to actinonin, a known PDF inhibitor with antibacterial activity, as compared with the parental strain. Recombinant chlamydial PDF was covalently labeled with a hydroxamate-based molecular probe designated AspR1, which was developed for the detection of metalloproteases. The AspR1 labeling of the chlamydial PDF became significantly less efficient in the presence of excessive amounts of GM6001 and TAPI-0. Finally, the PDF enzyme activity was efficiently inhibited with GM6001 and TAPI-0. Taken together, our results suggest that the metalloprotease inhibitors suppress chlamydial growth by targeting the bacterial PDF. These findings have important biochemical and medical implications.     

Characterization of peptide deformylase2 from B. cereus

Peptide deformylase (PDF) is a metalloenzyme that removes the N-terminal formyl groups from newly synthesized proteins. It is essential for bacterial survival, and is therefore-considered as a potential target for antimicrobial chemotherapy. However, some bacteria including medically relevant pathogens possess two or more def-like genes. Here we have examined two PDFs from Bacillus cereus. The two share only 32% sequence identity and the crystal structures show overall similarity with PDF2 having a longer C-terminus. However, there are differences at the two active sites, and these differences appear to contribute to the activity difference seen between the two. BcPDF2 is found as a dimer in the crystal form with two additional actinonin bound at that interface.     

Peptide deformylase inhibitors with retro-amide scaffold: Synthesis and structure–activity relationships

Peptide deformylase (PDF) is a metalloprotease catalyzing the removal of a formyl group from newly synthesized proteins. Thus inhibition of PDF activity is considered to be one of the most effective antibiotic strategies. Reported herein are the synthesis and structure–activity relationship studies of retro-amide inhibitors based on actinonin, a naturally occurring PDF inhibitor. Analysis of the structure–activity relationships led to the discovery of 7a, which exhibits potent enzyme inhibition and antibacterial activity against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis.