Dataset Information

MapB Protein is the Essential Methionine Aminopeptidase in Mycobacterium tuberculosis.

ABSTRACT: M. tuberculosis (Mtb), which causes tuberculosis disease, continues to be a major global health threat. Correct identification of valid drug targets is important for the development of novel therapeutics that would shorten the current 6-9 month treatment regimen and target resistant bacteria. Methionine aminopeptidases (MetAP), which remove the N-terminal methionine from newly synthesized proteins, are essential in all life forms (eukaryotes and prokaryotes). The MetAPs contribute to the cotranslational control of proteins as they determine their half life (N-terminal end rule) and facilitate further modifications such as acetylation and others. Mtb (and M. bovis) possess two MetAP isoforms, MetAP1a and MetAP1c, encoded by the mapA and mapB genes, respectively. Conflicting evidence was reported in the literature on which of the two variants is essential. To resolve this question, we performed a targeted genetic deletion of each of these two genes. We show that a deletion mutant of mapA is viable with only a weak growth defect. In contrast, we provide two lines of genetic evidence that mapB is indispensable. Furthermore, construction of double-deletion mutants as well as the introduction of point mutations into mapB resulting in proteins with partial activity showed partial, but not full, redundancy between mapB and mapA. We propose that it is MetAP1c (mapB) that is essentially required for mycobacteria and discuss potential reasons for its vitality.

SUBMITTER: Vanunu M

PROVIDER: S-EPMC6562599 | biostudies-literature | 2019 Apr

REPOSITORIES: biostudies-literature

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MapB Protein is the Essential Methionine Aminopeptidase in <i>Mycobacterium tuberculosis</i>.

Vanunu Miriam M Schall Patrick P Reingewertz Tali-Haviv TH Chakraborti Pradip K PK Grimm Bernhard B Barkan Daniel D

Cells 20190428 5

<i>M. tuberculosis (Mtb)</i>, which causes tuberculosis disease, continues to be a major global health threat. Correct identification of valid drug targets is important for the development of novel therapeutics that would shorten the current 6-9 month treatment regimen and target resistant bacteria. Methionine aminopeptidases (MetAP), which remove the N-terminal methionine from newly synthesized proteins, are essential in all life forms (eukaryotes and prokaryotes). The MetAPs contribute to the ...[more]

PMID: 31035386

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Project description: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.

Project description:BACKGROUND: Tuberculosis remains a serious world-wide health threat which requires the characterisation of novel drug targets for the development of future antimycobacterials. One of the key obstacles in the definition of new targets is the large variety of metabolic alterations that occur between cells in the active growth and chronic/dormant phases of tuberculosis. The ideal biochemical target should be active in both growth phases. Methionine adenosyltransferase, which catalyses the formation of S-adenosylmethionine from methionine and ATP, is involved in polyamine biosynthesis during active growth and is also required for the methylation and cyclopropylation of mycolipids necessary for survival in the chronic phase. RESULTS: The gene encoding methionine adenosyltransferase has been cloned from Mycobacterium tuberculosis and the model organism M. smegmatis. Both enzymes retained all amino acids known to be involved in catalysing the reaction. While the M. smegmatis enzyme could be functionally expressed, the M. tuberculosis homologue was insoluble and inactive under a large variety of expression conditions. For the M. smegmatis enzyme, the Vmax for S-adenosylmethionine formation was 1.30 micromol/min/mg protein and the Km for methionine and ATP was 288 microM and 76 microM respectively. In addition, the enzyme was competitively inhibited by 8-azaguanine and azathioprine with a Ki of 4.7 mM and 3.7 mM respectively. Azathioprine inhibited the in vitro growth of M. smegmatis with a minimal inhibitory concentration (MIC) of 500 microM, while the MIC for 8-azaguanine was >1.0 mM. CONCLUSION: The methionine adenosyltransferase from both organisms had a primary structure very similar those previously characterised in other prokaryotic and eukaryotic organisms. The kinetic properties of the M. smegmatis enzyme were also similar to known prokaryotic methionine adenosyltransferases. Inhibition of the enzyme by 8-azaguanine and azathioprine provides a starting point for the synthesis of higher affinity purine-based inhibitors.

Dataset Information

MapB Protein is the Essential Methionine Aminopeptidase in Mycobacterium tuberculosis.

Publications

MapB Protein is the Essential Methionine Aminopeptidase in <i>Mycobacterium tuberculosis</i>.

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