Amino-terminal extension present in the methionine aminopeptidase type 1c of Mycobacterium tuberculosis is indispensible for its activity

Abstract

BackgroundMethionine aminopeptidase (MetAP) is a ubiquitous enzyme in both prokaryotes and eukaryotes, which catalyzes co-translational removal of N-terminal methionine from elongating polypeptide chains during protein synthesis. It specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. The MetAP action for exclusion of N-terminal methionine is mandatory in 50-70% of nascent proteins. Such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins.ResultsWe cloned genes encoding two such metalloproteases (MtMetAP1a and MtMetAP1c) present in Mycobacterium tuberculosis and expressed them as histidine-tagged proteins in Escherichia coli. Although they have different substrate preferences, for Met-Ala-Ser, we found, MtMetAP1c had significantly high enzyme turnover rate as opposed to MtMetAP1a. Circular dichroism spectroscopic studies as well as monitoring of enzyme activity indicated high temperature stability (up to 50°C) of MtMetAP1a compared to that of the MtMetAP1c. Modelling of MtMetAP1a based on MtMetAP1c crystal structure revealed the distinct spatial arrangements of identical active site amino acid residues and their mutations affected the enzymatic activities of both the proteins. Strikingly, we observed that 40 amino acid long N-terminal extension of MtMetAP1c, compared to its other family members, contributes towards the activity and stability of this enzyme, which has never been reported for any methionine aminopeptidase. Furthermore, mutational analysis revealed that Val-18 and Pro-19 of MtMetAP1c are crucial for its enzymatic activity. Consistent with this observation, molecular dynamic simulation studies of wild-type and these variants strongly suggest their involvement in maintaining active site conformation of MtMetAP1c.ConclusionOur findings unequivocally emphasized that N-terminal extension of MtMetAP1c contributes towards the functionality of the enzyme presumably by regulating active site residues through "action-at-a-distance" mechanism and we for the first time are reporting this unique function of the enzyme.

MapB Protein is the Essential Methionine Aminopeptidase in Mycobacterium tuberculosis

“…As we confirmed mapB is essential for M. bovis growth, we wanted to examine whether knockout mutants are rescued by expression of MAP with modified activity. It was previously reported that MapB V18G and MapB W255L were highly defective in their methionine aminopeptidase activity as the substitution W255L affects the catalytic pocket, whereas V18G affects the N-terminus extension of the protein [17]. We first designed these two mutation sequences separately and also a mapB sequence containing both point mutations: mapB V18G , mapB W255L and mapB V18G;W255L .…”

Purification of native HBHA from Mycobacterium avium subsp. paratuberculosis

“…Similar to the M. tuberculosis HBHA [23], native map-HBHA contains no initiation methionine at its N-terminus and that the first amino acid is thus an alanine residue. The cleavage of the initiation methionine is a post-translational common process in Mycobacteria [24,25]. The HR-MS/MS analyses also indicated that the amino-terminal alanine is acetylated.…”

Natural products against key Mycobacterium tuberculosis enzymatic targets: Emerging opportunities for drug discovery

“…However, many of them suffered from a poor activity against the pathogens. The bacterial secondary metabolite pyridomycin (43), produced by Streptomyces pyridomyceticus or Dactylosporangium fulvum [71], was found to be a direct inhibitor of InhA (Ki = 6.5 μM) and exhibited significant in vitro antimycobacterial activity against several Mtb strains, including H37Rv (MIC = 0.56 µg/mL), and INH-resistant clinical isolates [72].…”

“…3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7Ps), shikimate kinase (SK), and acetohydroxyacid synthase (AHAS); hence, their fundamental role makes them attractive targets for the development of new antibiotics. MetAP removes the N-terminal methionine residue from newly synthesized proteins[43]. employed a pharmacophore-based virtual screening using synthetic and NP databases to identify new DAH7Ps inhibitors[46].…”