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1.
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. 相似文献
2.
Peptide aldehyde inhibitors of bacterial peptide deformylases. 总被引:3,自引:0,他引:3
D J Durand B Gordon Green J F O'Connell S K Grant 《Archives of biochemistry and biophysics》1999,367(2):297-302
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. 相似文献
3.
Eukaryotic peptide deformylases. Nuclear-encoded and chloroplast-targeted enzymes in Arabidopsis
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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. 相似文献
4.
Characterization of the Thermus thermophilus locus encoding peptide deformylase and methionyl-tRNA(fMet) formyltransferase. 总被引:2,自引:1,他引:1
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An Escherichia coli strain with thermosensitive expression of the gene encoding peptide deformylase (fms) has been constructed. At nonpermissive temperatures, this strain fails to grow. The essential character of the fms gene was further used to clone by heterologous complementation the locus corresponding to Thermus thermophilus peptide deformylase. The cloned fragment also carries the methionyl-tRNA(fMet) formyltransferase gene (fmt). It is located immediately downstream from the fms gene, as in E. coli. Further sequence analysis of the region surrounding the E. coli fms-fmt locus indicates that the genes bordering the fms-fmt region are not conserved in T. thermophilus. 相似文献
5.
Peptide deformylase (PDF) catalyses the removal of the N-formyl group from the nascent polypeptide during protein maturation. The PDF of Mycobacterium tuberculosis H37Rv (MtbPDF), overexpressed and purified from Escherichia coli, was characterized as an iron-containing enzyme with stability towards H(2) O(2) and moderate thermostability. Substitution of two conserved residues (G49 and L107) from MtbPDF with the corresponding residues found in human PDF affected its deformylase activity. Among characterized PDFs, glycine (G151) in motif III instead of conserved aspartate is characteristic of M. tuberculosis. Although the G151D mutation in MtbPDF increased its deformylase activity and thermostability, it also affected enzyme stability towards H(2) O(2) . Molecular dynamics and docking results confirmed improved substrate binding and catalysis for the G151D mutant and the study provides another possible molecular basis for the stability of MtbPDF against oxidizing agents. 相似文献
6.
The peptide deformylase in bacteria is involved in removal of N-formyl group from newly synthesized proteins. The gene encoding this enzyme from Mycobacterium tuberculosis was cloned and expressed in Escherichia coli. The enzyme activity of the recombinant protein (mPDF) was insensitive to modulation by common monovalent/divalent cations. Kinetic analysis, using N-formylmethionine-alanine as the substrate, yielded K(cat)/K(m) of approximately 1220 M(-1)s(-1). Actinonin, a naturally occurring antibiotic, and 1,10-ortho-phenanthroline strongly inhibited the enzyme activity. The mPDF was very stable at 30 degrees C with a half-life of approximately 4h and exhibited resistance to oxidizing agents, like H(2)O(2). Thus, the mPDF achieved distinction in its behavior among any reported iron-containing peptide deformylases. Furthermore, amino acid sequence analysis of mPDF revealed the presence of an unusually long carboxy-terminal end (residues 182-197), which is atypical for any gram-positive bacteria. Our results, through deletion analysis, for the first time established the role of this region in mPDF enzyme activity. 相似文献
7.
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. 相似文献
8.
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. 相似文献
9.
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. 相似文献
10.
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. 相似文献
11.
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. 相似文献
12.
Baldwin ET Harris MS Yem AW Wolfe CL Vosters AF Curry KA Murray RW Bock JH Marshall VP Cialdella JI Merchant MH Choi G Deibel MR 《The Journal of biological chemistry》2002,277(34):31163-31171
The first crystal structure of Class II peptide deformylase has been determined. The enzyme from Staphylococcus aureus has been overexpressed and purified in Escherichia coli and the structure determined by x-ray crystallography to 1.9 A 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 E. coli enzyme. The most striking deviation of the S. aureus from the 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 which wraps around the enzyme, terminating near the active site. Although there are no differences at the amino acid level near the active site metal ion, significant changes are noted in the peptide binding cleft which may play a role in the design of general peptide deformylase inhibitors. 相似文献
13.
Evidence that peptide deformylase and methionyl-tRNA(fMet) formyltransferase are encoded within the same operon in Escherichia coli. 总被引:4,自引:2,他引:2
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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. 相似文献
14.
Pratt LM Beckett RP Davies SJ Launchbury SB Miller A Spavold ZM Todd RS Whittaker M 《Bioorganic & medicinal chemistry letters》2001,11(19):2585-2588
By screening a library of metalloenzyme inhibitors, the N-formyl-hydroxylamine derivative BB-3497 was identified as a potent inhibitor of Escherichia coli peptide deformylase with antibacterial activity both in vitro and in vivo. The homochiral synthesis of BB-3497, involving a novel asymmetric Michael addition reaction is described. 相似文献
15.
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. 相似文献
16.
Balakrishnan A Wang L Li X Ohman-Strickland P Malatesta P Fan H 《Microbiological research》2009,164(3):338-346
Chlamydia trachomatis is an obligate intracellular bacterium responsible for a number of health problems, including sexually transmitted infection in humans. We recently discovered that C. trachomatis infection in cell culture is highly susceptible to inhibitors of peptide deformylase, an enzyme that removes the N-formyl group from newly synthesized polypeptides. In this study, one of the deformylase inhibitors, GM6001, was tested for potential antichlamydial activity using a murine genital C. muridarum infection model. Topical application of GM6001 significantly reduced C. muridarum loading in BALB/c mice that were vaginally infected with the pathogen. In striking contrast, growth of the probiotic Lactobacillus plantarum is strongly resistant to the PDF inhibitor. GM6001 demonstrated no detectable toxicity against host cells. On the basis of these data and our previous observations, we conclude that further evaluation of PDF inhibitors for prevention and treatment of sexually transmitted chlamydial infection is warranted. 相似文献
17.
Yoo JS Zheng CJ Lee S Kwak JH Kim WG 《Bioorganic & medicinal chemistry letters》2006,16(18):4889-4892
A new 24-membered ring lactone, macrolactin N, was isolated from a culture broth of Bacillus subtilis and its structure was established by various spectral analysis. Macrolactin N inhibited Staphylococcus aureus peptide deformylase with an IC50 value of 7.5 microM and also showed antibacterial activity against Escherichia coli and S. aureus. 相似文献
18.
Byerly DW McElroy CA Foster MP 《Protein science : a publication of the Protein Society》2002,11(7):1850-1853
Identifying potential ligand binding sites on a protein surface is an important first step for targeted structure-based drug discovery. While performing control experiments with Escherichia coli peptide deformylase (PDF), we noted that the organic solvents used to solubilize some ligands perturbed many of the same resonances in PDF as the small molecule inhibitors. To further explore this observation, we recorded (15)N HSQC spectra of E. coli peptide deformylase (PDF) in the presence of trace quantities of several simple organic solvents (acetone, DMSO, ethanol, isopropanol) and identified their sites of interaction from local perturbation of amide chemical shifts. Analysis of the protein surface structure revealed that the ligand-induced shift perturbations map to the active site and one additional surface pocket. The correlation between sites of solvent and inhibitor binding highlights the utility of organic solvents to rapidly and effectively validate and characterize binding sites on proteins prior to designing a drug discovery screen. Further, the solvent-induced perturbations have implications for the use of organic solvents to dissolve candidate ligands in NMR-based screens. 相似文献
19.
Davies SJ Ayscough AP Beckett RP Bragg RA Clements JM Doel S Grew C Launchbury SB Perkins GM Pratt LM Smith HK Spavold ZM Thomas SW Todd RS Whittaker M 《Bioorganic & medicinal chemistry letters》2003,13(16):2709-2713
Structural modifications to the peptide deformylase inhibitor BB-3497 are described. In this paper, we describe the initial SAR around this lead for modifications to the methylene spacer and the P1' side chain. Enzyme inhibition and antibacterial activity data revealed that the optimum distance between the N-formyl hydroxylamine metal binding group and the P1' side chain is one unsubstituted methylene unit. Additionally, lipophilic P1' side chains that closely mimic the methionine residue in the substrate provided compounds with the best microbiological profile. 相似文献
20.
Kreusch A Spraggon G Lee CC Klock H McMullan D Ng K Shin T Vincent J Warner I Ericson C Lesley SA 《Journal of molecular biology》2003,330(2):309-321
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. 相似文献