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

Abstract

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.

The Crystal Structure of Mitochondrial (Type 1A) Peptide Deformylase Provides Clear Guidelines for the Design of Inhibitors Specific for the Bacterial Forms

“…The mature protein (i.e. the protein accumulating in the mitochondrion) lacks about 60 residues of the N-terminal targeting presequence (31,32). We overproduced this protein (AtPDF1A⌬H in Ref.…”

“…Modeling of the Three-dimensional Structure of the Human PDFHumans also have a PDF1A (30,31). HsPDF1A is very similar to AtPDF1A but exhibits an extended N terminus, a shorter C terminus, and lower identity levels in the CD-loop (Fig.…”

“…There is a long insertion between ␤-strands ␤ 2 and ␤ 3 and systematic amino acid changes in the vicinity of the active site (see an alignment in Ref. 31). PDF1 molecules therefore form two classes: PDF1A and PDF1B (TABLE ONE).…”

“…Unlike PDF1B, which is specific to plants and Apicomplexa, PDF1A is found in almost all eukaryotes. Both classes of PDF1 are functional in deformylation and potently inhibited by actinonin (31,32). Deformylation has been shown to be an essential process in eukaryotes (33,34).…”

“…Enzymatic studies have also shown that PDF1As from plants and animals differ from bacterial PDFs in a number of ways. We investigated these differences, with a view to developing guidelines for the design of PDF-In devoid of anti-PDF1A activity (31,32). As actinonin derivatives inhibit cell proliferation by inhibiting human PDF1A (HsPDF1A), HsPDF1A has been proposed as a target for new anticancer agents (34).…”