title: Biochemical characterization of recombinant methionine aminopeptidases (MAPs) from Mycobacterium tuberculosis H37Rv.
abstract: Methionine aminopeptidase (MAP) performs the essential post-translational N-terminal methionine excision (NME) of nascent polypeptides during protein synthesis. To characterize MAP from Mycobacterium tuberculosis, two homolgues, mapA (Rv0734) and mapB (Rv2861c), were over expressed and purified as recombinant proteins in E. coli. In vitro activity assay of apo-MtbMAPs using L-Met-p-nitro anilide as substrate revealed MtbMAP A to be catalytically more efficient compared to MtbMAP B. Ni(2+) was the best activator of apo-MtbMAP A, whereas Ni(2+) and Co(2+) activated apo-MtbMAP B equally. MtbMAP B showed higher thermo-stability, but was feedback inhibited by higher concentrations of L-methionine. Aminopeptidase inhibitors like actinonin and bestatin inhibited both MtbMAPs, more prominently MtbMAP B. Among the site-directed mutants of MtbMAP B, substitution of metal-binding residue D142 completely abolished enzyme activity, whereas substitution of residues forming S1' pocket, C105S and T94C, had only moderate effects on substrate hydrolysis. Present study identified a specific insertion region in MtbMAP A sequence which differentiates it from other bacterial and eukaryotic MAPs. A deletion mutant lacking amino acids from this insertion region (MtbMAP A-∆164-176) was constructed to probe into their structural and functional role in activity and stability of MtbMAP A. The limited success in soluble expression of this deletion mutant suggests further optimizations of expression conditions or alternative bioinformatics approaches for further characterization of this deletion mutant of MtbMAP A.
authors.affiliation: Biotechnology Division, National Institute for Interdisciplinary Science and Technology, CSIR, Trivandrum, 695019 Kerala, India.
authors.forename: Sai Shyam
authors.initials: SS
authors.lastname: Narayanan
authors.forename: Kesavan Madhavan
authors.initials: KM
authors.lastname: Nampoothiri
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000626:Aminopeptidases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D003035:Cobalt / Q000737:chemistry; D017124:Conserved Sequence; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D063208:Methionyl Aminopeptidases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009532:Nickel / Q000737:chemistry; D011480:Protease Inhibitors / Q000737:chemistry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D003035:Cobalt; D009532:Nickel; D000626:Aminopeptidases; D063208:Methionyl Aminopeptidases