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

Abstract

Background: 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. 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.

Results: We 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.

Conclusion: Our 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.

Figure 1

MtMetAP1a and MtMetAP1c are enzymatically active. (A) Methionine aminopeptidase activity with different substrates. Enzyme activity of the two methionine aminopeptidases was determined using different substrates (4 mM) as indicated and MtMetAP1a (1.7 nM) or MtMetAP1c (0.038 nM) of purified protein as mentioned in the methods. Notations used: MG, Met-Gly; MAS, Met-Ala-Ser; MGMM, Met-Gly-Met-Met; GGA, Gly-Gly-Ala. (B) Kinetic analysis of methionine aminopeptidase activity of MtMetAP1s. Kinetic analysis of methionine removal ability of MtMetAP1a (left) was carried out by using 1.7 nM of purified protein with increasing concentration of Met-Gly-Met-Met as substrate. The reaction was monitored for 15 min. 0.038 nM of purified protein was incubated for 5 min with increasing concentration of Met-Ala-Ser as substrate to determine the kinetic parameters of MtMetAP1c (Right). Insets, effect of increasing amount of protein on the enzyme activity. (C) Effect of different inhibitors and chelator on MtMetAPs activity. MetAP1s were preincubated for 15 min at room temperature with the indicated amount of the inhibitors/EDTA and then the activity assay was performed. (D) Effect of temperature on MtMetAP1s. The thermal unfolding was performed at the rate of 1°C/min and the change in the CD signal was monitored at 208 nm. Left and right panels represent the thermal denaturation graphs for MtMetAP1a and MtMetAP1c respectively. Upper insets show the absorption spectra of MtMetAP1a and MtMetAP1c monitored at 25°C and 50°C. Lower insets show the effect of preincubation for 10 min at indicated temperatures on the activity of two MtMetAP1s. MtMetAP1a and MtMetAP1c are depicted at (left) and (right) respectively. The assay was carried out at 30°C after preincubation.

Figure 2

Mutations of active site residues affect enzymatic activity of MtMetAPs. (A) Sequence alignment of the two mycobacterial methionine aminopeptidases was performed using Clustal X. Gaps in the sequences were introduced for optimum alignment. Asterisk and dots denote, identical and similar amino acids, respectively. Residues highlighted with black represent the 40 amino acid long N-terminal extension present in MtMetAP1c and those with gray are the mutated amino acids in the two MtMetAPs. (B) Structural alignment of MtMetAP1a (green) with respect to MtMetAP1c (pink). Residues in green (sticks) represent MtMetAP1a active site residues (His-88, His-193, Glu-219) and residues in pink (sticks) depict MtMetAP1c active site residues (His212, His-114, Glu-238). Residue in blue is Tyr-183 (MtMetAP1a) and in hot pink is Phe-202 (MtMetAP1c). (C) Methionine aminopeptidase activity of different point mutants. Enzyme activity for the indicated amount of wild-type and purified variants of two MtMetAP1s was monitored using 4 mM of substrate, Met-Gly-Met-Met (MtMetAP1a) and Met-Ala-Ser (MtMetAP1c). Insets, Western blot of mutant proteins using anti-His antibody, far-UV and near-UV CD spectra.

Figure 3

N-terminal deletion of MtMetAP1c affects its enzyme activity. (A) Deletion scheme. Notations used: WT, wild-type; Δ, deleted residues. (B) Assessment of methionine aminopeptidase activity of deletion variants. Methionine aminopeptidase activity assay was performed using indicated amount of wild-type and mutant proteins with 4 mM of Met-Ala-Ser as the substrate. (C) Coomassie stained SDS-PAGE gel (upper panel) and Western blot (lower panel) using anti- His antibody of the mutant proteins as shown in Figure 3B. (D) Far-UV CD spectra of the WT, Δ2-10 and Δ2-15 proteins. (E) Near-UV CD spectra of the WT, Δ2-10 and Δ2-15 proteins.

Figure 4

Val-18 and Pro-19 at N-terminal extension are crucial for MtMetAP1c enzyme activity. (A) Sequence alignment of MetAP1c from different gram positive bacteria. Notations used with accession number in parantheses: M tb-Mycobacterium tuberculosis (P0A5J2); M bovis-Mycobacterium bovis (P0A5J3); M avium-Mycobacterium avium (A0QJ09); M marinum-Mycobacterium marinum (B2HJQ5); M paratuberculosis-Mycobacterium paratuberculosis (Q73VS7); M leprae-Mycobacterium leprae (Q9CBU7); S coelicolor-Streptomyces coelicolor (Q9RKR2); S scabies-Streptomyces scabies (C9ZHA6); R erythropolis-Rhodococcus erythropolis (C0ZY62); N farcinica-Nocardia farcinica (Q5YSA3); G bronchialis-Gordonia bronchialis (D0LBG0); C diphtheriae-Corynebacterium diphtheriae (Q6NGL5); C glutamicum-Corynebacterium glutamicum (Q6M437); B mcbrellneri-Brevibacterium mcbrellneri (D4YNZ0); L xyli-Leifsonia xyli subsp. xyli (Q6AFH6). (B) Effect of mutations on the MtMetAP1c enzyme activity. The indicated amounts of wild-type and mutant proteins were used to monitor the enzyme activity with 4 mM Met-Ala-Ser as the substrate. (C) Far-UV CD spectra of the different mutant proteins.

Figure 5

Molecular dynamic simulations indicate Val-18 and Pro-19 maintain active site conformation of MtMetAP1c. (A) C_α RMSD plot as a function of time at 300K over a period of 20 ns simulations done for wild-type and different mutant proteins. (B) MD simulations. The snapshots superimposed for wild-type (left) and V18GP19G (right) for 15 ns run. (Inset), depicts the highlighted regions of snapshots at 5ns, 7.5ns, 10ns, 15ns. (C) Snapshots of single mutant (left, V18G, blue-gray) superimposed with double mutant (pink). Both overlap each other to maximum probability compared to the wild-type protein. The loop proximal to the site of mutation is out of plane of the active site. MD simulations snapshots of single mutant (right, P19A, yellow ribbon blue sticks) superimposed with wild-type (blue-gray). Both overlap each other to maximum probability compared to the double mutant. The loop proximal to the site of mutation shows configurations similar to wild-type in 3-D space i.e. in plane to the active site.