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Updated: May 23, 2026
X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050
Published on: May 13, 2020
Biochemical characterization of recombinant methionine aminopeptidases (MAPs) from Mycobacterium tuberculosis H37Rv
Sai Shyam Narayanan1, Kesavan Madhavan Nampoothiri
1Biotechnology Division, National Institute for Interdisciplinary Science and Technology, CSIR, Trivandrum, 695019 Kerala, India.
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.
Insights
Mycobacterium tuberculosis methionine aminopeptidase (MAP) homologs, MtbMAP A and MtbMAP B, were characterized. MtbMAP A showed higher catalytic efficiency, while MtbMAP B exhibited greater thermostability and sensitivity to inhibitors.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Methionine aminopeptidase (MAP) is crucial for post-translational N-terminal methionine excision (NME) in protein synthesis.
- Two MAP homologs, mapA (Rv0734) and mapB (Rv2861c), were identified in Mycobacterium tuberculosis.
Purpose of the Study:
- To characterize the biochemical and functional properties of MtbMAP A and MtbMAP B.
- To investigate the role of specific residues and sequence features in MtbMAP activity and stability.
Main Methods:
- Recombinant expression and purification of MtbMAP A and MtbMAP B in E. coli.
- In vitro activity assays using L-Met-p-nitro anilide substrate.
- Metal ion activation studies and inhibition assays with specific inhibitors (actinonin, bestatin).
- Site-directed mutagenesis and construction of a deletion mutant (MtbMAP A-∆164-176).
Main Results:
- MtbMAP A demonstrated higher catalytic efficiency than MtbMAP B.
- Ni(2+) optimally activated MtbMAP A, while Ni(2+) and Co(2+) equally activated MtbMAP B.
- MtbMAP B displayed superior thermostability but was susceptible to feedback inhibition by L-methionine.
- Both MtbMAPs were inhibited by actinonin and bestatin, with MtbMAP B being more sensitive.
- Substitution of metal-binding residue D142 in MtbMAP B abolished activity; S1' pocket mutations had moderate effects.
- A unique insertion region in MtbMAP A was identified, and a deletion mutant showed limited soluble expression.
Conclusions:
- MtbMAP A and MtbMAP B possess distinct biochemical properties relevant to their function in M. tuberculosis.
- Metal ion cofactors and specific active site residues play critical roles in MtbMAP activity.
- The identified insertion region in MtbMAP A may contribute to its unique characteristics, warranting further investigation.
