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

Pavitra Kanudia,Pradip K Chakraborti,Monica Mittal,Sangaralingam Kumaran

doi:10.1186/1471-2091-12-35

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.

Highlights

Methionine 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
The purified proteins, on resolving in SDSPAGE when visualized in Coomassie Brilliant Blue stained gels, exhibited bands at 37.3 ± 1.7 kDa (n = 4) and 39.1 ± 1.2 kDa (n = 4) for MtMetAP1a and MtMetAP1c, respectively
We further addressed the adaptability of MtMetAP1a and MtMetAP1c enzymes to different temperatures

Summary

Introduction

Methionine 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. The N-terminal methionine excision (NME) is an essential co-translational proteolytic process responsible for the diversity of amino-termini of proteins in both prokaryotes and eukaryotes [1]. It is an irreversible reaction, which occurs soon after N-terminal residues of the nascent polypeptide chain emerge from the ribosome exit tunnel. The type 2 enzymes, in contrast to type 1, have an approximately 60 amino acid long a helical domain inserted within the catalytic region of the enzyme [6,7] This helical sub-domain shares neither sequence nor structural homology with any other known protein. Two new subclasses, type 1c and 2c, have been introduced where ~40 amino acid long N-terminal extension is present but zinc finger motif is absent [9,10]

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