Isolation and Crystallization of Functionally CompetentEscherichia coliPeptide Deformylase Forms Containing either Iron or Nickel in the Active Site

doi:10.1006/bbrc.1998.8616

Biochemical and Biophysical Research Communications

Volume 246, Issue 2, 19 May 1998, Pages 342-346

https://doi.org/10.1006/bbrc.1998.8616 Get rights and content

Abstract

Three metallo forms of peptide deformylase (PDF, EC 3.5.1.31) ofEscherichia coliwere prepared and crystallized (space group C2, diffraction limit 1.9 Å) for initiating the X-ray structure determination of the metal center in correlation with the catalytic functionality of this enzyme. The native Fe²⁺containing enzyme species was directly isolated from overproducing bacteria by using catalase as a buffer additive, which stabilizes the catalytic activity against oxidative destruction. The Ni²⁺containing form, which is oxygen-insensitive, was obtained by metal exchange with free Ni²⁺and found to be catalytically equally effective (k_cat/K_M= 10⁵M⁻¹s⁻¹for N-formyl-Met-Ala). The Zn²⁺form, prepared from the apoenzyme or by displacement of bound Ni²⁺by free Zn²⁺, proved virtually inactive.

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Cited by (114)

Peptide deformylases from Vibrio parahaemolyticus phage and bacteria display similar deformylase activity and inhibitor binding clefts
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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.
Biphenyl tetrazole-thiazolidinediones as novel bacterial peptide deformylase inhibitors: Synthesis, biological evaluations and molecular docking study
2016, Biomedicine and Pharmacotherapy
Herein, we report the synthesis and screening of biphenyl tetrazole-thiazolidinediones 14(a-j) as bacterial Peptide deformylase (PDF) enzyme inhibitors. The compounds 14b (IC₅₀ value = 16.25 μM), 14c (IC₅₀ value = 18.00 μM) and 14h (IC₅₀ value = 17.25 μM) had shown good PDF inhibition activity. The compounds 14b (MIC range = 20.75–35.41 μg/mL), 14c (MIC range = 19.41–26.00 μg/mL) and 14d (MIC range = 8.41–8.58 μg/mL) had also shown potent antibacterial activity when compared with standard ciprofloxacin (MIC range = 25–50 μg/mL). Thus, the active derivatives were not only potent PDF inhibitors but also efficient antibacterial agents. In order to gain more insight on the binding mode of the compounds with PDF enzyme, the synthesized compounds 14(a-j) were docked against PDF enzyme of E. coli and compounds exhibited good binding properties. The results suggest that this class of compounds have been potential for development and use in a future as antibacterial drugs.
Bacterial Peptide deformylase inhibition of cyano substituted biaryl analogs: Synthesis, in vitro biological evaluation, molecular docking study and in silico ADME prediction
2016, Bioorganic and Medicinal Chemistry
Herein, we report the synthesis and screening of cyano substituted biaryl analogs 5(a–m) as Peptide deformylase (PDF) enzyme inhibitors. The compounds 5a (IC₅₀ value = 13.16 μM), 5d (IC₅₀ value = 15.66 μM) and 5j (IC₅₀ value = 19.16 μM) had shown good PDF inhibition activity. The compounds 5a (MIC range = 11.00–15.83 μg/mL), 5b (MIC range = 23.75–28.50 μg/mL) and 5j (MIC range = 7.66–16.91 μg/mL) had also shown potent antibacterial activity when compared with ciprofloxacin (MIC range = 25–50 μg/mL). Thus, the active derivatives were not only potent PDF inhibitors but also efficient antibacterial agents. In order to gain more insight on the binding mode of the compounds with PDF, the synthesized compounds 5(a–m) were docked against PDF enzyme of Escherichia coli and compounds exhibited good binding properties. In silico ADME properties of synthesized compounds were also analyzed and showed potential to develop as good oral drug candidates.
Purification and use of E. coli peptide deformylase for peptide deprotection in chemoenzymatic peptide synthesis
2013, Protein Expression and Purification
Peptide deformylases (PDFs) catalyze the removal of the formyl group from the N-terminal methionine residue in nascent polypeptide chains in prokaryotes. Its deformylation activity makes PDF an attractive candidate for the biocatalytic deprotection of formylated peptides that are used in chemoenzymatic peptide synthesis. For this application it is essential to use PDF preparations that are free of contamination by peptidases that can cleave internal peptide bonds. Therefore, different purification methods were attempted and an industrially applicable purification procedure was developed based on a single anion-exchange chromatography step of an engineered PDF variant that was equipped with an anionic octaglutamate tag. The deformylation activity and stability of the engineered enzyme were similar to those of the wild-type PDF. This purification method furnished a PDF preparation with a 1500-fold decreased level of contamination by amidases and peptidases as compared to cell-free extract. It was shown that the enzyme could be used for deprotection of a formylated dipeptide that was prepared by thermolysin-mediated coupling.
Kinetic analyses and inhibition studies reveal novel features in peptide deformylase 1 from Trypanosoma cruzi
2012, Molecular and Biochemical Parasitology
In eubacteria and eukaryotic organelles N-terminal methionine excision requires the sequential action of two activities, a peptide deformylase (PDF), which systematically removes the N-formyl group present on all nascent polypeptides and methionine aminopeptidase (MAP), which exscinds methionine specifically and depends on the previous removal of the N-formyl group. In Trypanosoma cruzi two genes encoding bacterial PDF homologues have been identified and referred to as TcPDF-1 and TcPDF-2. Here we report the biochemical characterization of a truncated soluble version of TcPDF-1 lacking the hydrophobic N-terminal domain that is active with the bacterial PDF substrate formyl-methionyl-alanyl-serine but, in contrast to other PDFs, is not inhibited by actinonin. The enzyme is strongly activated by Cu²⁺ and inhibited by Ni²⁺. Our results show that T. cruzi PDF exhibits unique features thus providing a new avenue for the design of potential inhibitors for use in the treatment of diseases caused by trypanosomatid parasites.
The activity and cofactor preferences of N-acetyl-1-D-myoinosityl-2-amino- 2-deoxy-α-D-glucopyranoside deacetylase (MshB) change depending on environmental conditions
2011, Journal of Biological Chemistry
Actinomycetes, such as Mycobacterium species, are Gram-positive bacteria that utilize the small molecule mycothiol (MSH) as their primary reducing agent. Consequently, the enzymes involved in MSH biosynthesis are targets for drug development. The metal-dependent enzyme N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside deacetylase (MshB) catalyzes the hydrolysis of N-acetyl-1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside to form 1-d-myo-inosityl-2-amino-2-deoxy-α-d-glucopyranoside and acetate, the fourth overall step in MSH biosynthesis. Inhibitors of metalloenzymes typically contain a group that binds to the active site metal ion; thus, a comprehensive understanding of the native cofactor(s) of metalloenzymes is critical for the development of biologically effective inhibitors. Herein, we examined the effect of metal ions on the overall activity of MshB and probed the identity of the native cofactor. We found that the activity of MshB follows the trend Fe²⁺ > Co²⁺ > Zn²⁺ > Mn²⁺ and Ni²⁺. Additionally, our results show that the identity of the cofactor bound to purified MshB is highly dependent on the purification conditions used (aerobic versus anaerobic), as well as the metal ion content of the medium during protein expression. MshB prefers Fe²⁺ under anaerobic conditions regardless of the metal ion content of the medium and switches between Fe²⁺ and Zn²⁺ under aerobic conditions as the metal content of the medium is altered. These results indicate that the cofactor bound to MshB under biological conditions is dependent on environmental conditions, suggesting that MshB may be a cambialistic metallohydrolase that contains a dynamic cofactor. Consequently, biologically effective inhibitors will likely need to dually target Fe²⁺-MshB and Zn²⁺-MshB.

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Regular ArticleIsolation and Crystallization of Functionally CompetentEscherichia coliPeptide Deformylase Forms Containing either Iron or Nickel in the Active Site

Abstract

J. Mol. Biol.

J. Mol. Biol.

Methods Enzymol.

Clin. Chim. Acta

J. Biol. Chem.

J. Biol. Chem.

Anal. Biochem.

J. Inorg. Biochem.

EMBO J.

J. Bacteriol.

Regular Article
Isolation and Crystallization of Functionally CompetentEscherichia coliPeptide Deformylase Forms Containing either Iron or Nickel in the Active Site