The carboxy-terminal end of the peptide deformylase from Mycobacterium tuberculosis is indispensable for its enzymatic activity.
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 approximately 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 degrees C with a half-life of approximately 4h and exhibited resistance to oxidizing agents, like H(2)O(2). 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 an 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.
Saxena
Rahul
R
Institute of Microbial Technology, Sector 39A, Chandigarh 160 036, India.
Chakraborti
Pradip K
PK
D000581:Amidohydrolases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution; D004789:Enzyme Activation; D004795:Enzyme Stability; D006877:Hydroxamic Acids / Q000737:chemistry*; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
D006877:Hydroxamic Acids; D011994:Recombinant Proteins; D000581:Amidohydrolases; C084507:peptide deformylase; C010531:actinonin