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 ∼ 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 ° C with a half-life of ∼ 4 h and exhibited resistance to oxidizing agents, like H2O2. 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 all 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. (C) 2005 Elsevier Inc. All rights reserved.