Characterization of a Human Peptide Deformylase:  Implications for Antibacterial Drug Design

Abstract

Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an N-formylmethionyl-tRNA(i), resulting in N-terminal formylation of all nascent polypeptides. Peptide deformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majority of bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes, making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencing has revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding Homo sapiens PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-amino-acid targeting sequence was overexpressed in Escherichia coli. The recombinant, Co(2+)-substituted protein is catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterial PDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhanced green fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion. However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation for the apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is at least partially due to mutation of a highly conserved residue (Leu-91 in E. coli PDF) in mammalian PDF. PDF inhibitors had no detectable effect on two different human cell lines. These results suggest that HsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylation and validates PDF as an excellent target for antibacterial drug design.

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

“…HsPDF were released (44). Seven of the thirteen ORFs of the human mitochondrial genome are predicted to be substrates of HsMAP1D, according to the known rules of MAP substrate selectivity (reviewed in Ref.…”

“…1 and Refs. 7 and 9), the possible existence of an active peptide deformylase in animals has been a controversial issue (6,7,13,44). One objection raised was that the sequences of mitochondrially encoded proteins from mammals indicate that their Fo groups are retained, whereas this is not the case for the corresponding proteins of plant mitochondrial genomes (see data compiled in Ref.…”

Interplay Between N-Terminal Methionine Excision and FtsH Protease Is Essential for Normal Chloroplast Development and Function in Arabidopsis

“…Two PDFs have been identified in plants: mitochondrion-targeted PDF1A and PDF1B, which are targeted to both mitochondria and plastids (Giglione et al, 2000;Dirk et al, 2001). Only PDF1A has been found in humans and other animals (Giglione et al, 2000;Nguyen et al, 2003;Serero et al, 2003;Lee et al, 2004), and a PDF3 was identified in trypanosomatids (Bouzaidi-Tiali et al, 2007). Note that the many proteins encoded by the nuclear genome, which are further imported in the plastids, are not processed by the chloroplastic NME (cNME) but rather by a dedicated transit stromal peptidase.…”

Proteomics-Based Analysis of <i>Phalaenopsis amabilis</i> in Response toward <i>Cymbidium</i> Mosaic Virus and/or <i>Odontoglossum</i> Ringspot Virus Infection

“…Peptide deformylase is a metalloprotease involved in the posttranslational deformylation of N-terminal methionine residue of newly synthesized proteins [29]. The expression of chloroplast peptide deformylase 1A was significantly reduced in all virus infected P. Amabilis leaves, especially in ORSV-infected leaves, suggesting that viral infection has significant impact on protein modification in P. amabilis, and the symptom of chlorotic stripes in infected leaves probably resulted from the dramatically reduced expression of peptide deformylase 1A in chloroplasts.…”