The C‐terminal domain of peptide deformylase is disordered and dispensable for activity

Meinnel, Thierry; Lazennec, Christine; Dardel, Frédéric; Schmitter, Jean‐Marie; Blanquet, Sylvain

doi:10.1016/0014-5793(96)00357-2

Cited by 43 publications

(46 citation statements)

References 19 publications

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“…As residue 62 can be aligned with residue 1 of EcPDF, the catalytic domain of HsPDF was predicted to start immediately after this residue. In EcPDF, N-terminal deletions of more than 3 residues result in incorrect folding and inactivation of the enzyme (27). Consistent with this, the ⌬N78 form proved to be unstable and did not accumulate significantly in the soluble fraction (Fig.…”

Section: Identification In Animal Cdna Libraries and Cloning Of Homolsupporting

confidence: 69%

An Unusual Peptide Deformylase Features in the Human Mitochondrial N-terminal Methionine Excision Pathway

Serero

Giglione

Sardini

et al. 2003

Journal of Biological Chemistry

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126

137

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Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated.

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Section: Identification In Animal Cdna Libraries and Cloning Of Homolsupporting

confidence: 69%

An Unusual Peptide Deformylase Features in the Human Mitochondrial N-terminal Methionine Excision Pathway

Serero

Giglione

Sardini

et al. 2003

Journal of Biological Chemistry

Self Cite

126

137

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“…The structure of E. coli PDF has already been extensively characterized and will not be elaborated on further here (6,10,14,40). When the PDF structure with bound VRC4307 is examined, the hydroxamic acid is observed to coordinate the active-site Ni 2ϩ , as expected; the cyclopentyl ring system of VRC4307 lies in the S 1 Ј pocket of the enzyme (Fig.…”

Section: Resultsmentioning

confidence: 68%

N-Alkyl Urea Hydroxamic Acids as a New Class of Peptide Deformylase Inhibitors with Antibacterial Activity

Hackbarth

Chen

Lewis

et al. 2002

Antimicrob Agents Chemother

103

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Peptide deformylase (PDF) is a prokaryotic metalloenzyme that is essential for bacterial growth and is a new target for the development of antibacterial agents. All previously reported PDF inhibitors with sufficient antibacterial activity share the structural feature of a 2-substituted alkanoyl at the P 1 site. Using a combination of iterative parallel synthesis and traditional medicinal chemistry, we have identified a new class of PDF inhibitors with N-alkyl urea at the P 1 site. Compounds with MICs of <4 g/ml against gram-positive and gram-negative pathogens, including Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae, have been identified. The concentrations needed to inhibit 50% of enzyme activity (IC 50 s) for Escherichia coli Ni-PDF were <0.1 M, demonstrating the specificity of the inhibitors. In addition, these compounds were very selective for PDF, with IC 50 s of consistently >200 M for matrilysin and other mammalian metalloproteases. Structure-activity relationship analysis identified preferred substitutions resulting in improved potency and decreased cytotoxity. One of the compounds (VRC4307) was cocrystallized with PDF, and the enzymeinhibitor structure was determined at a resolution of 1.7 Å. This structural information indicated that the urea compounds adopt a binding position similar to that previously determined for succinate hydroxamates. Two compounds, VRC4232 and VRC4307, displayed in vivo efficacy in a mouse protection assay, with 50% protective doses of 30.8 and 17.9 mg/kg of body weight, respectively. These N-alkyl urea hydroxamic acids provide a starting point for identifying new PDF inhibitors that can serve as antimicrobial agents.

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“…We recently reported the importance of an insertion sequence and a carboxyl-terminal sequence in mPDF (relative to other PDFs) in determining its activity (15,16), this feature being distinct from the PDFs of other bacteria (7,37). In this study, we carried out structurefunction analyses of mPDF to confirm the identities of crucial amino acids in the insertion region that modulate the enzymatic function of this protein.…”

Section: Discussionmentioning

confidence: 98%

Three Consecutive Arginines Are Important for the Mycobacterial Peptide Deformylase Enzyme Activity

Saxena¹,

Kanudia

Datt

et al. 2008

Journal of Biological Chemistry

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Genes encoding the peptide deformylase enzyme (def) are present in all eubacteria and are involved in the deformylation of the N-formyl group of newly synthesized polypeptides during protein synthesis. We compared the amino acid sequences of this enzyme in different mycobacterial species and found that they are highly conserved (76% homology with 62% identity); however, when this comparison was extended to other eubacterial homologs, it emerged that the mycobacterial proteins have an insertion region containing three consecutive arginine residues (residues 77-79 in Mycobacterium tuberculosis peptide deformylase (mPDF)). Here, we demonstrate that these three arginines are important for the activity of mPDF. Circular dichroism studies of wild-type mPDF and of mPDF containing individual conservative substitutions (R77K, R78K, or R79K) or combined substitutions incorporated into a triple mutant (R77K/R78K/R79K) indicate that such mutations cause mPDF to undergo structural alterations. Molecular modeling of mPDF suggests that the three arginines are distal to the active site. Molecular dynamics simulations of wild-type and mutant mPDF structures indicate that the arginines may be involved in the stabilization of substrate binding pocket residues for their proper interaction with peptide(s). Treatment with 5-phosphothiorate-modified antisense oligodeoxyribonucleotides directed against different regions of def from M. tuberculosis inhibits growth of Mycobacterium smegmatis in culture. Taken together, these results hold out the possibility of future design of novel mycobacteria-specific PDF inhibitors.The first amino acid incorporated during polypeptide biosynthesis in eukaryotes is methionine. On the other hand, in prokaryotes and in eukaryotic organelles (mitochondria and chloroplasts), ribosomal protein biosynthesis is initiated with N-formyl-methionyl-tRNA. Thus, all nascent polypeptides in prokaryotes are formylated at their amino termini (1-3). Since peptidases acting at NH 2 termini are unable to utilize formylated NH 2 termini as substrates, the removal of the formyl group is mandatory for polypeptide maturation in all cases in which the removal of the NH 2 -terminal methionine is essential for protein folding or function. The enzyme, peptide deformylase (PDF), 3 catalyzes the deformylation of N-formylmethionine in nascent polypeptide chains in prokaryotes (3-5). PDF is a metalloprotease containing iron (as Fe 2ϩ ) or Zn 2ϩ (6 -8). The gene encoding peptide deformylase (def) is present throughout the eubacterial lineage (9). In several bacteria, it has been reported to be an essential gene (10 -12), currently considered to be an appropriate target for the development of antibacterials (13,14).We have earlier cloned, expressed, and characterized the peptide deformylase enzyme from Mycobacterium tuberculosis. We found that M. tuberculosis peptide deformylase (mPDF) is an iron-containing protein that is enzymatically active as a multimer (15, 16). Although conversion of Fe 2ϩ to Fe 3ϩ by environmental oxygen result...

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The C‐terminal domain of peptide deformylase is disordered and dispensable for activity

Cited by 43 publications

References 19 publications

An Unusual Peptide Deformylase Features in the Human Mitochondrial N-terminal Methionine Excision Pathway

An Unusual Peptide Deformylase Features in the Human Mitochondrial N-terminal Methionine Excision Pathway

N-Alkyl Urea Hydroxamic Acids as a New Class of Peptide Deformylase Inhibitors with Antibacterial Activity

Three Consecutive Arginines Are Important for the Mycobacterial Peptide Deformylase Enzyme Activity

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