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-Metp-nitro anilide as substrate revealed MtbMAP A to be catalytically more efficient compared to MtbMAP B. Ni2? was the best activator of apo-MtbMAP A, whereas Ni2? and Co2? activated apo-MtbMAPB equally. MtbMAPB showed higher thermo-stability, but was feedback inhibited by higher concentrations of L-methionine. Amino peptidase 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 S10 pocket, C105S and T94C, had only moderate effects on substrate hydrolysis.Present study identified a specific insertion region in Mtb-MAP A sequence which differentiates it from other bacterial and eukaryotic MAPs. A deletion mutant lacking amino acids from this insertion region (MtbMAP A-D164-176) was constructed to probe into their structural and functional role in activity and stability of MtbMAPA. 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