BACKGROUND
the n-terminal methionine excision  is an essential co-translational proteolytic process responsible for the diversity of amino-termini of proteins in both prokaryotes and eukaryotes  <cit> .
it is an irreversible reaction, which occurs soon after n-terminal residues of the nascent polypeptide chain emerge from the ribosome exit tunnel before the commencement of protein folding  <cit> .
the enzyme involved in the process is known as methionine aminopeptidase , which specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged  <cit> .
in prokaryotes and eukaryotic organelles where the initiator methionine is formylated, nme requires the action of another metalloprotease, peptide deformylase in addition to metap, which removes the n-formyl group present on all synthesized nascent polypeptides.
in fact, removal of n-formyl group is a prerequisite for the subsequent action of metap  <cit> .
nevertheless, 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  <cit> .
metaps are ubiquitous in distribution and are highly conserved throughout the phylogeny.
they were initially grouped in two classes, metap <dig> and metap <dig> on the basis of sequence comparison.
the type  <dig> enzymes, in contrast to type  <dig>  have an approximately  <dig> amino acid long α helical domain inserted within the catalytic region of the enzyme  <cit> .
this helical sub-domain shares neither sequence nor structural homology with any other known protein.
the metaps are further divided in two subclasses on the basis of absence  or presence  of n-terminal extension  <cit> .
the n-terminal extension present in type 1b contains two zinc finger motifs while that of type 2b has alternate stretches of polyacidic and polybasic residues.
recently, two new subclasses, type 1c and 2c, have been introduced where ~ <dig> amino acid long n-terminal extension is present but zinc finger motif is absent  <cit> .
apart from several types of metaps, presence of more than one copy of functional genes encoding this enzyme has been reported in both prokaryotes and eukaryotes.
in human, there are two such well characterized enzymes, which have been shown to be involved in cell proliferation  <cit> .
among four isoforms of metaps in plasmodium falciparum, three exhibited enzymatic activities  <cit> .
genetic studies further established the essentiality of this enzyme in both prokaryotes and eukaryotes  <cit> .
these findings led to the assumption that metap would be an ideal drug target and screening/designing of inhibitors against this enzyme might help in planning to combat the problem of rapid resurgence of diseases due to emergence of drug resistant strains of different pathogens.
in this context, we focussed on tuberculosis, which is caused by mycobacterium tuberculosis, and responsible for considerable human mortality throughout the world in recent years.
although bacteria generally have single metap <dig>  mycobacterial genome revealed the presence of 2- <dig> putative genes for metap1; for instance m. tuberculosis has two genes mapa  and mapb  encoding mtmetap1a and mtmetap1c respectively.
although, crystal structure of mtmetap1c was solved as an apoenzyme and in complex form  <cit> , biochemical characterization of the two mtmetaps has not been reported until very recently  <cit> .
however, detailed structure-function analysis of these enzymes has not yet been carried out.
in this scenario, we concentrated on both these mtmetap1s and in this article we report their distinct characteristics.
furthermore, we provide here evidence that catalytic domain of mtmetap1c alone is not sufficient for the enzyme activity.
RESULTS
mtmetap1a and mtmetap1c exhibit subtle difference in their behaviour
the mapa and mapb genes from m. tuberculosis were pcr amplified, cloned in pet-28c and expressed in e. coli strain bl <dig>  the recombinant n-terminal his-tagged proteins were purified to near homogeneity by immobilized metal affinity chromatography using ni-nta resin.
the purified proteins, on resolving in sds-page when visualized in coomassie brilliant blue stained gels, exhibited bands at  <dig>  ±  <dig>  kda  and  <dig>  ±  <dig>  kda  for mtmetap1a and mtmetap1c, respectively.
the authenticity of the expressed proteins was further confirmed by western blotting using anti-his antibody .
mtmetap1a and mtmetap1c displayed maximum activity with met-gly-met-met and met-ala-ser respectively.
the enzymatic specificity for the terminal methionine residue was confirmed by using gly-gly-ala as the substrate and as expected both the enzymes were unable to hydrolyze it .
furthermore, using different peptides as substrates revealed that at least a tripeptide is the prerequisite for their functionality, since both the enzymes showed almost negligible activity with the dipeptide met-gly.
activities of purified enzymes  as a function of increasing concentrations  of substrate  yielded typical michaelis-menten curves .
interestingly, kinetic parameters determined by fitting the data to michaelis-menten equation for both the enzymes for their preferred substrate indicated that they were active .
interestingly, using met-ala-ser as the substrate for both the enzymes the catalytic efficiency of mtmetap1c was strikingly high  compared to that of the mtmetap1a .
thus, our result is in consonance with a recent report that mtmetap1c is catalytically more efficient compared to mtmetap1a  <cit> .
expectedly, both the enzymes exhibited a linear increase in activity as a function of increasing protein concentrations .
enzyme assays were carried out as described under 'methods' following the incubation of purified mtmetap1a  or mtmetap1c  proteins with different concentrations  of met-ala-ser  or met-gly-met-met  as substrate.
km and vmax values were calculated from non-linear regression analysis by fitting the data to michaelis-menten equation, and the results are expressed as mean ± sd.
for calculating kcat values, the molecular mass of recombinant enzyme was considered as  <dig>  kda  or  <dig>  kda .
mean ± sd for kcat/km was determined after calculating it for individual experiment.
since metaps are known as dinuclear metalloproteases, we also monitored the mycobacterial enzyme activities in response to edta, a metal ion chelator.
as expected, metal ion chelator affected the activities of both the enzymes.
however, compared to that used for mtmetap1c enzyme activity of mtmetap1a was inhibited at a lower concentrations of edta .
leucine aminopeptidase inhibitors like amastatin and bestatin, have already been shown to inhibit metap activity by binding to its metal centre  <cit> .
as shown in figure 1c, amastatin affected both mtmetap1a and mtmetap1c enzyme activities; magnitude of inhibition, however, varied considerably between them .
bestatin, on the other hand, had no effect on the enzymatic activities of both the proteins within the concentration range tested in our experimental conditions.
we further addressed the adaptability of mtmetap1a and mtmetap1c enzymes to different temperatures.
for this purpose, both the enzymes following incubation at different temperatures  were assayed at 30°c for their activities.
the maximum activity for both the enzymes was noticed between 30°-37°c.
interestingly, mtmetap1a retained ~85% of its activity at 50°c as opposed to ~25% by the mtmetap1c compared to that observed at 30°c .
this observation led to the postulation that alteration in the enzyme activity might be occurring as a result of heating and this would very likely cause structural changes in both the proteins.
this possibility was explored by incubating both the proteins to 50°c and comparing their far-uv cd spectra between 190- <dig> nm.
mtmetap1a retained  of its secondary structure on heating at 50°c while mtmetap1c was unfolded .
in fact, determination of tm of both the proteins by cd spectroscopy yielded values of  <dig> °c for mtmetap1a and  <dig> °c for mtmetap1c .
these findings, therefore, established the difference in the adaptive ability of mtmetap1a to high temperature compared to that of the mtmetap1c and bring an end to the argument about the temperature tolerance of the mtmetap1s  <cit> .
thus, all these lines of evidence implicated the existence of subtle differences in the behaviour of two mycobacterial metaps.
contribution of active site residues on mtmetap <dig> enzyme activity
nucleotide derived amino acid sequence alignment of mtmetap1a with mtmetap1c revealed ~41% identity  and the crystal structure of mtmetap1c  has already been resolved.
since mtmetap1a did not show any significant identity with any other protein in the pdb database, we used mtmetap1c as the template for modelling the structure of this metalloprotease  <cit> .
the modelled structure of mtmetap1a was validated by procheck and it shows that 98% residues are in the most favoured regions of ramachandran plot.
the monomer of each protein consists of two pairs of α-helices  packed on the either side of the central antiparallel β-sheet .
interestingly, ~55% of the structure comprised of loops connecting the secondary structural elements.
however, the  <dig> amino acid long n-terminal extension of mtmetap1c, which is devoid of any secondary structural element, lies across the surface of its catalytic domain .
structural analysis predicted the presence of his- <dig>  his- <dig> and glu- <dig> in the active site of mtmetap1a, while residues like asp- <dig>  asp- <dig>  glu- <dig> and trp- <dig> are lining the perimeter of the active site.
the presence of homologous residues in mtmetap1c implies that the active site of both the proteins is essentially conserved .
to gain an insight on their contribution towards the enzyme activity, we generated several point mutants .
interestingly, the variants do not show any appreciable enzymatic activity even on using 10-fold excess protein compared to that of the wild-type .
in fact, mutating w236l in mtmetap1a and w255l in mtmetap1c led to complete loss in the activity , which is in contrast to the report available with metap <dig> of e. coli  <cit>
.
expression of all the variants, however, was authenticated by western blotting using anti-his antibody .
to examine the effect of mutations on the secondary and tertiary structures of the mtmetap1s, the cd spectral studies were performed.
far-uv cd spectrum of the mutant proteins of mtmetap1a revealed that h88a, d117a, e219v and w236l show reduction in helical content.
the trend of the data in the region of 200- <dig> nm suggests that the band with negative mean residue ellipticity at lower wavelengths has reduced signal strength presumably owing to the conversion of some helical regions to less ordered structures .
interestingly, mutant h193a shows greater helicity than the wild-type protein.
far-uv cd spectral studies of wild-type mtmetap1c and its variants revealed that in d131a although there is no significant reduction in the mean residue ellipticity at the 200- <dig> nm range, it decreased at the 220- <dig> nm range .
on the other hand, in h114a and e238a reduction in band intensity was observed at both 200- <dig> nm and 220- <dig> nm.
in h212a and w255l the band intensities at the 200- <dig> nm as well as in 220- <dig> nm were further reduced .
thus, the mutations affected secondary structure of both the proteins and changes observed in the near-uv cd spectra indicated alteration in their aromatic environment .
role of n-terminal extension of mtmetap1c on the enzyme activity
the presence of  <dig> amino acid n-terminal extension although not unusual among metap1s, its contribution towards the enzymatic activity is not known as yet  <cit> .
to elucidate this aspect, a series of n-terminal deletion variants for mtmetap1c were generated  and their enzyme activity was monitored .
surprisingly, compared to the wild-type, deletion constructs  displayed either drastic decrease or no enzymatic activity .
even use of  <dig> fold higher amounts of proteins in our assay, Δ2- <dig> or Δ2- <dig> exhibited only 30% activity while Δ2- <dig> or Δ2- <dig> or Δ2- <dig> had no activity .
thus, our results argue that the catalytic domain alone is not sufficient for the enzymatic function of mtmetap1c.
mtmetap1c variants, like Δ2- <dig> or Δ2- <dig> or Δ2- <dig>  which did not show any enzyme activity, when stained with coomassie brilliant blue after resolving in sds-page, displayed a progressive loss in the purity of the proteins with the increase in the length of deleted regions .
the authenticity of these mutant proteins was confirmed by western blotting using anti-his antibody .
the effect of mutation was evident by the presence of contaminating bands in the ni-nta purified samples  and even reduced protein yield .
analysis of its crystal structure revealed that the n-terminal extension present in mtmetap1c wraps around the surface of its catalytic domain in contiguous crevice  <cit> .
hence, its truncation as a result of deletion might have exposed the hydrophobic residues and thereby destabilized the protein.
this has been manifested presumably by exhibiting altered proteolytic property of mtmetap1c variants and/or led to acquiring the tendency to associate with other proteins  <cit> .
interestingly, reduction in the enzymatic activity was also observed in Δ2- <dig> and Δ2- <dig> variants despite the fact that the ni-nta purified proteins appear to be similar in profile as has been observed with wild-type.
the far-uv cd spectra showed that they  are folded like the wild-type protein .
a slight alteration observed in the near-uv cd spectra for the Δ2- <dig> variant  was very likely the result of alteration in the micro-environment of trp present at the amino acid position  <dig>  nonetheless, it is apparent from our results that the first  <dig> amino acids of mtmetap1c are important for the catalytic activity of the enzyme.
role of val- <dig> and pro- <dig> on mtmetap1c enzyme activity
to elucidate, the presence of any conserved residues between amino acid 15- <dig> of mtmetap1c, we aligned the amino acid sequence of the n-terminal extension region of the mtmetap proteins from gram positive bacteria.
multiple sequence analysis revealed that val- <dig> and pro- <dig> are two highly conserved residues in different metaps .
to investigate their role, mutants replacing these two residues, one at a time  or both at the same time  were generated and the enzymatic activities of the recombinant proteins were monitored.
relative to wild type, v18a protein had ~30% activity while p19a and p19g exhibited ~65% and 35% activity respectively.
on the other hand, v18g, v18ap19a and v18gp19g variants were devoid of any enzymatic activity .
use of 10-fold excess of protein in assays, v18a, p19a and p19g mutants displayed activities almost at par  with the wild-type.
however, use of v18g, v18ap19a and v18gp19g proteins even at increasing amounts in assays had hardly any activity .
these results indicated that val- <dig> and/or pro- <dig>  the residues present in n-terminal extension  may contribute towards the enzymatic activity of mtmetap1c.
further, to monitor whether variation in the activity of the mutant proteins could be due to structural changes, far-uv cd studies were performed.
the analysis of cd spectra revealed that mutations caused alteration in secondary structure of all the variants .
compared to the wild type, mutation of val- <dig> or pro- <dig>  resulted in the reduction of the negative band intensity in the region of 205- <dig> nm of the mutant proteins .
in the variant p19a, the reduction in band intensity was also observed at  <dig> nm , thereby indicating that the helical content was reduced.
in v18g significant reduction was observed both at 205- <dig> nm and  <dig> nm.
thus, these results suggested the importance of these two residues  exclusively and mutually towards the enzymatic activity of mtmetap1c.
to have an idea on how the val- <dig> and pro- <dig> of mtmetap1c influenced the activity of the enzyme, we used crystal structure of mtmetap1c to perform the molecular dynamic  simulation studies by introducing point mutation at the site of interest .
four md simulations were performed in explicit solvent conditions to examine the effect of specific mutations on overall structure and on the interactions of amino acids lining the active site of the protein.
comparison of the trajectories between wild-type and double mutant  showed that overall fold of mutant protein is retained .
however, specific and drastic changes in the positions of active site residues were observed .
the conformational changes that initiated from the site of mutation  propagated to the active site and displaced his- <dig> as well as affected his- <dig> and glu- <dig>  analysis of trajectories of single mutants  illustrated that stereo chemical structural changes similar to that observed in double mutant  were present in v18g .
on the contrary, md simulation results of p19a appeared much similar to that of the wild-type but the presence of differential population of the ensemble at different time could possibly explain the reduction in the activity of this mutant protein .
thus, the results of our md simulation studies support our biochemical observations.
discussion
metaps represent a unique class of metalloproteases that catalyze the co-translational removal of n-terminal methionine residue from the elongating polypeptide chain during the process of protein synthesis  <cit> .
since metap action in both prokaryotes and eukaryotes is mandatory during nascent protein synthesis, the importance of this enzyme has long been realized.
though the presence of two metap1s was known with the availability of the m. tuberculosis genome sequence, both of them  are active has been shown recently by others  <cit>  and also in this study.
however, it is still not known whether they are merely complementing each other for their functionality and therefore redundant within the genome or their presence is because of the specific needs of the bacterium.
recently, it has been reported that mtmetap1a knock down caused ~75% viability of mycobacteria while elimination of mtmetap1c resulted in 95% survival  <cit> .
since this enzyme from different sources has been considered as a potential drug target, identification of inhibitors against these enzymes, particularly for m. tuberculosis mtmetap1a is in progress  <cit> .
however, the initial step of structure-activity relationship between these two proteins has not been addressed properly.
in this context, to carry out systematic study of these two enzymes from m. tuberculosis, we have attempted here to analyze metap1s to elucidate subtle differences in their characteristics.
our results indicated that mtmetap1c had strikingly high enzyme turnover rate with the same substrate  compared to mtmetap1a , although they had different substrate preferences .
the outcome of our cd as well as activity assays with these mycobacterial enzymes illustrate that mtmetap1a can sustain high temperature  compared to mtmetap1c.
in fact, tm value calculated based on cd spectra for mtmetap1a was  <dig> °c as opposed to  <dig> °c for mtmetap1c .
to get an insight into the cause of such differential behaviour of the two enzymes, structure of mtmetap1a was modelled and compared with the available structure of mtmetap1c .
we observed that the active site residues in both the mtmetap1s are essentially same, though their arrangement in space is different .
furthermore, these amino acids are crucial because any alteration in them  yielded an inactive protein .
thus it is logical to presume that this difference in the arrangement may be responsible for the alteration in their activity/functionality.
sequence analysis revealed that the remarkable feature of mtmetap1c compared to mtmetap1a, is the presence of  <dig> amino acid long n-terminal extension.
it has been suggested that this extension may be involved in the interaction of mtmetap1c with the ribosome  <cit> .
in scmetap <dig> and hsmetap <dig> deletion of n-terminal extension did not significantly alter the catalytic efficiency of the enzymes  <cit> .
however, such deletion in scmetap <dig> reduced its efficiency in rescuing the slow growth phenotype of a map mutant  <cit> .
interestingly, the zinc finger motif present in the n-terminal extension of scmetap <dig> has been shown to be involved in its association with the ribosome  <cit> .
in hsmetap <dig>  on the other hand, these sequences have been implicated for the interaction of the enzyme with substrates and inhibitors  <cit> .
this led us to explore the role of the n-terminal extension present in mtmetap1c towards the activity of the enzyme.
we constructed a series of deletion mutants removing nine , fourteen , nineteen , twenty-nine  and thirty-nine  amino acids from the amino terminal end of the mtmetap1c .
enzyme assays with these constructs using even  <dig> fold excess of proteins compared to the wild-type revealed that there was a considerable loss in activity in Δ2- <dig> or Δ2- <dig> variants and no activity at all in mutants, like Δ2- <dig> or Δ2- <dig> or Δ2- <dig> .
intriguingly, following resolving in sds-page, analysis of the deletion constructs in coomassie brilliant blue stained gels reflected that purification profile of Δ2- <dig> or Δ2- <dig> mutants was similar to that of the wild-type but variants like Δ2- <dig>  Δ2- <dig> and Δ2- <dig> exhibited loss in purity of the proteins .
hence, it seems logical to postulate that the amino acid residues between  <dig> and  <dig> of mtmetap1c are crucial for its activity.
perhaps, deletion of residues beyond  <dig> from the n-terminal end of mtmetap1c renders the enzyme unstable or exposes its hydrophobic patches, thereby increasing its tendency to associate with the other proteins.
in fact, evaluation of structural changes through far-uv and near-uv cd spectra revealed that up to  <dig> residues from the n-terminal extension of mtmetap1c are dispensable for the folding of the protein  but required for its activity .
thus, our results argue that the n-terminal extension is important for the catalytic activity of mtmetap1c protein, which is unlike the reports available with scmetap <dig>  hsmetap <dig> and hsmetap <dig>  <cit> .
to identify the critical residue for the activity of the protein, we analyzed the sequence of metaps from different gram positive bacteria with n-terminal extension and found the presence of conserved val- <dig> and pro- <dig> in between amino acid residues  <dig> and  <dig> .
this observation, prompted us to create point mutations at 18th and 19th position of mtmetap1c  and examine their effect on the activity of the enzyme.
while v18a and p19a were partially active, v18ap19a did not show any activity.
on the other hand, p19g exhibited partial activity but neither v18g nor v18gp19g were active .
further, to assess if these mutations resulted in any structural alterations, far-uv cd studies were carried out.
interestingly the mutation caused alteration in the secondary structure of the protein.
thus, our results argue the importance of these two residues  exclusively and mutually towards the enzymatic activity of mtmetap1c.
it appears very likely that the structural alterations that occurred due to mutation at val- <dig> and/or pro- <dig> of mtmetap1c affected the active site conformation of the protein.
in fact, md simulations  indicated that the conformational changes generated at the site of mutation  propagated via connecting loops and helices to the enzyme active site resulting in the alteration in the movement/positioning of residues like his- <dig>  his- <dig> and glu- <dig> which are critical for the enzyme activity as revealed in our biochemical studies .
these variations in the v18gp19g or v18g mutant proteins presumably made the active site environment unfavourable for any activity.
thus, our results illustrate that residue without being in the active site of an enzyme is capable of modulating its activity, which does not seem to be unusual  <cit> .
CONCLUSIONS
our findings unequivocally emphasize that n-terminal extension of mtmetap1c contributes towards the functionality of the enzyme by regulating active site residues through "action-at-a-distance" mechanism and this is presumably its unique function in mtmetap1c, which we are reporting for the first time.
since val- <dig> and pro- <dig> are conserved residues throughout the gram positive bacterial metap1s with n-terminal extension, it remains to be seen whether they are universal in contributing towards the functionality of the enzyme.

 the n-terminal methionine excision is an essential co-translational proteolytic process responsible for the diversity of amino-termini of proteins in both prokaryotes and eukaryotes. it is an irreversible reaction, which occurs soon after n-terminal residues of the nascent polypeptide chain emerge from the ribosome exit tunnel before the commencement of protein folding. the enzyme involved in the process is known as methionine aminopeptidase, which specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. in prokaryotes and eukaryotic organelles where the initiator methionine is formylated, nme requires the action of another metalloprotease, peptide deformylase in addition to metap, which removes the n-formyl group present on all synthesized nascent polypeptides. in fact, removal of n-formyl group is a prerequisite for the subsequent action of metap. nevertheless, 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. metaps are ubiquitous in distribution and are highly conserved throughout the phylogeny. they were initially grouped in two classes, metap and metap on the basis of sequence comparison. the type enzymes, in contrast to type have an approximately amino acid long α helical domain inserted within the catalytic region of the enzyme. this helical sub-domain shares neither sequence nor structural homology with any other known protein. the metaps are further divided in two subclasses on the basis of absence or presence of n-terminal extension. the n-terminal extension present in type 1b contains two zinc finger motifs while that of type 2b has alternate stretches of polyacidic and polybasic residues. recently, two new subclasses, type 1c and 2c, have been introduced where ~ amino acid long n-terminal extension is present but zinc finger motif is absent. apart from several types of metaps, presence of more than one copy of functional genes encoding this enzyme has been reported in both prokaryotes and eukaryotes. in human, there are two such well characterized enzymes, which have been shown to be involved in cell proliferation. among four isoforms of metaps in plasmodium falciparum, three exhibited enzymatic activities. genetic studies further established the essentiality of this enzyme in both prokaryotes and eukaryotes. these findings led to the assumption that metap would be an ideal drug target and screening/designing of inhibitors against this enzyme might help in planning to combat the problem of rapid resurgence of diseases due to emergence of drug resistant strains of different pathogens. in this context, we focussed on tuberculosis, which is caused by mycobacterium tuberculosis, and responsible for considerable human mortality throughout the world in recent years. although bacteria generally have single metap mycobacterial genome revealed the presence of 2- putative genes for metap1; for instance m. tuberculosis has two genes mapa and mapb encoding mtmetap1a and mtmetap1c respectively. although, crystal structure of mtmetap1c was solved as an apoenzyme and in complex form, biochemical characterization of the two mtmetaps has not been reported until very recently. however, detailed structure-function analysis of these enzymes has not yet been carried out. in this scenario, we concentrated on both these mtmetap1s and in this article we report their distinct characteristics. furthermore, we provide here evidence that catalytic domain of mtmetap1c alone is not sufficient for the enzyme activity.  mtmetap1a and mtmetap1c exhibit subtle difference in their behaviour the mapa and mapb genes from m. tuberculosis were pcr amplified, cloned in pet-28c and expressed in e. coli strain bl the recombinant n-terminal his-tagged proteins were purified to near homogeneity by immobilized metal affinity chromatography using ni-nta resin. the purified proteins, on resolving in sds-page when visualized in coomassie brilliant blue stained gels, exhibited bands at ± kda and ± kda for mtmetap1a and mtmetap1c, respectively. the authenticity of the expressed proteins was further confirmed by western blotting using anti-his antibody. mtmetap1a and mtmetap1c displayed maximum activity with met-gly-met-met and met-ala-ser respectively. the enzymatic specificity for the terminal methionine residue was confirmed by using gly-gly-ala as the substrate and as expected both the enzymes were unable to hydrolyze it. furthermore, using different peptides as substrates revealed that at least a tripeptide is the prerequisite for their functionality, since both the enzymes showed almost negligible activity with the dipeptide met-gly. activities of purified enzymes as a function of increasing concentrations of substrate yielded typical michaelis-menten curves. interestingly, kinetic parameters determined by fitting the data to michaelis-menten equation for both the enzymes for their preferred substrate indicated that they were active. interestingly, using met-ala-ser as the substrate for both the enzymes the catalytic efficiency of mtmetap1c was strikingly high compared to that of the mtmetap1a. thus, our result is in consonance with a recent report that mtmetap1c is catalytically more efficient compared to mtmetap1a. expectedly, both the enzymes exhibited a linear increase in activity as a function of increasing protein concentrations. enzyme assays were carried out as described under'methods' following the incubation of purified mtmetap1a or mtmetap1c proteins with different concentrations of met-ala-ser or met-gly-met-met as substrate. km and vmax values were calculated from non-linear regression analysis by fitting the data to michaelis-menten equation, and the results are expressed as mean ± sd. for calculating kcat values, the molecular mass of recombinant enzyme was considered as kda or kda. mean ± sd for kcat/km was determined after calculating it for individual experiment. since metaps are known as dinuclear metalloproteases, we also monitored the mycobacterial enzyme activities in response to edta, a metal ion chelator. as expected, metal ion chelator affected the activities of both the enzymes. however, compared to that used for mtmetap1c enzyme activity of mtmetap1a was inhibited at a lower concentrations of edta. leucine aminopeptidase inhibitors like amastatin and bestatin, have already been shown to inhibit metap activity by binding to its metal centre. as shown in figure 1c, amastatin affected both mtmetap1a and mtmetap1c enzyme activities; magnitude of inhibition, however, varied considerably between them. bestatin, on the other hand, had no effect on the enzymatic activities of both the proteins within the concentration range tested in our experimental conditions. we further addressed the adaptability of mtmetap1a and mtmetap1c enzymes to different temperatures. for this purpose, both the enzymes following incubation at different temperatures were assayed at 30°c for their activities. the maximum activity for both the enzymes was noticed between 30°-37°c. interestingly, mtmetap1a retained ~85% of its activity at 50°c as opposed to ~25% by the mtmetap1c compared to that observed at 30°c. this observation led to the postulation that alteration in the enzyme activity might be occurring as a result of heating and this would very likely cause structural changes in both the proteins. this possibility was explored by incubating both the proteins to 50°c and comparing their far-uv cd spectra between 190- nm. mtmetap1a retained of its secondary structure on heating at 50°c while mtmetap1c was unfolded. in fact, determination of tm of both the proteins by cd spectroscopy yielded values of °c for mtmetap1a and °c for mtmetap1c. these findings, therefore, established the difference in the adaptive ability of mtmetap1a to high temperature compared to that of the mtmetap1c and bring an end to the argument about the temperature tolerance of the mtmetap1s. thus, all these lines of evidence implicated the existence of subtle differences in the behaviour of two mycobacterial metaps. contribution of active site residues on mtmetap enzyme activity nucleotide derived amino acid sequence alignment of mtmetap1a with mtmetap1c revealed ~41% identity and the crystal structure of mtmetap1c has already been resolved. since mtmetap1a did not show any significant identity with any other protein in the pdb database, we used mtmetap1c as the template for modelling the structure of this metalloprotease. the modelled structure of mtmetap1a was validated by procheck and it shows that 98% residues are in the most favoured regions of ramachandran plot. the monomer of each protein consists of two pairs of α-helices packed on the either side of the central antiparallel β-sheet. interestingly, ~55% of the structure comprised of loops connecting the secondary structural elements. however, the amino acid long n-terminal extension of mtmetap1c, which is devoid of any secondary structural element, lies across the surface of its catalytic domain. structural analysis predicted the presence of his- his- and glu- in the active site of mtmetap1a, while residues like asp- asp- glu- and trp- are lining the perimeter of the active site. the presence of homologous residues in mtmetap1c implies that the active site of both the proteins is essentially conserved. to gain an insight on their contribution towards the enzyme activity, we generated several point mutants. interestingly, the variants do not show any appreciable enzymatic activity even on using 10-fold excess protein compared to that of the wild-type. in fact, mutating w236l in mtmetap1a and w255l in mtmetap1c led to complete loss in the activity, which is in contrast to the report available with metap of e. coli. expression of all the variants, however, was authenticated by western blotting using anti-his antibody. to examine the effect of mutations on the secondary and tertiary structures of the mtmetap1s, the cd spectral studies were performed. far-uv cd spectrum of the mutant proteins of mtmetap1a revealed that h88a, d117a, e219v and w236l show reduction in helical content. the trend of the data in the region of 200- nm suggests that the band with negative mean residue ellipticity at lower wavelengths has reduced signal strength presumably owing to the conversion of some helical regions to less ordered structures. interestingly, mutant h193a shows greater helicity than the wild-type protein. far-uv cd spectral studies of wild-type mtmetap1c and its variants revealed that in d131a although there is no significant reduction in the mean residue ellipticity at the 200- nm range, it decreased at the 220- nm range. on the other hand, in h114a and e238a reduction in band intensity was observed at both 200- nm and 220- nm. in h212a and w255l the band intensities at the 200- nm as well as in 220- nm were further reduced. thus, the mutations affected secondary structure of both the proteins and changes observed in the near-uv cd spectra indicated alteration in their aromatic environment. role of n-terminal extension of mtmetap1c on the enzyme activity the presence of amino acid n-terminal extension although not unusual among metap1s, its contribution towards the enzymatic activity is not known as yet. to elucidate this aspect, a series of n-terminal deletion variants for mtmetap1c were generated and their enzyme activity was monitored. surprisingly, compared to the wild-type, deletion constructs displayed either drastic decrease or no enzymatic activity. even use of fold higher amounts of proteins in our assay, Δ2- or Δ2- exhibited only 30% activity while Δ2- or Δ2- or Δ2- had no activity. thus, our results argue that the catalytic domain alone is not sufficient for the enzymatic function of mtmetap1c. mtmetap1c variants, like Δ2- or Δ2- or Δ2- which did not show any enzyme activity, when stained with coomassie brilliant blue after resolving in sds-page, displayed a progressive loss in the purity of the proteins with the increase in the length of deleted regions. the authenticity of these mutant proteins was confirmed by western blotting using anti-his antibody. the effect of mutation was evident by the presence of contaminating bands in the ni-nta purified samples and even reduced protein yield. analysis of its crystal structure revealed that the n-terminal extension present in mtmetap1c wraps around the surface of its catalytic domain in contiguous crevice. hence, its truncation as a result of deletion might have exposed the hydrophobic residues and thereby destabilized the protein. this has been manifested presumably by exhibiting altered proteolytic property of mtmetap1c variants and/or led to acquiring the tendency to associate with other proteins. interestingly, reduction in the enzymatic activity was also observed in Δ2- and Δ2- variants despite the fact that the ni-nta purified proteins appear to be similar in profile as has been observed with wild-type. the far-uv cd spectra showed that they are folded like the wild-type protein. a slight alteration observed in the near-uv cd spectra for the Δ2- variant was very likely the result of alteration in the micro-environment of trp present at the amino acid position nonetheless, it is apparent from our results that the first amino acids of mtmetap1c are important for the catalytic activity of the enzyme. role of val- and pro- on mtmetap1c enzyme activity to elucidate, the presence of any conserved residues between amino acid 15- of mtmetap1c, we aligned the amino acid sequence of the n-terminal extension region of the mtmetap proteins from gram positive bacteria. multiple sequence analysis revealed that val- and pro- are two highly conserved residues in different metaps. to investigate their role, mutants replacing these two residues, one at a time or both at the same time were generated and the enzymatic activities of the recombinant proteins were monitored. relative to wild type, v18a protein had ~30% activity while p19a and p19g exhibited ~65% and 35% activity respectively. on the other hand, v18g, v18ap19a and v18gp19g variants were devoid of any enzymatic activity. use of 10-fold excess of protein in assays, v18a, p19a and p19g mutants displayed activities almost at par with the wild-type. however, use of v18g, v18ap19a and v18gp19g proteins even at increasing amounts in assays had hardly any activity. these results indicated that val- and/or pro- the residues present in n-terminal extension may contribute towards the enzymatic activity of mtmetap1c. further, to monitor whether variation in the activity of the mutant proteins could be due to structural changes, far-uv cd studies were performed. the analysis of cd spectra revealed that mutations caused alteration in secondary structure of all the variants. compared to the wild type, mutation of val- or pro- resulted in the reduction of the negative band intensity in the region of 205- nm of the mutant proteins. in the variant p19a, the reduction in band intensity was also observed at nm, thereby indicating that the helical content was reduced. in v18g significant reduction was observed both at 205- nm and nm. thus, these results suggested the importance of these two residues exclusively and mutually towards the enzymatic activity of mtmetap1c. to have an idea on how the val- and pro- of mtmetap1c influenced the activity of the enzyme, we used crystal structure of mtmetap1c to perform the molecular dynamic simulation studies by introducing point mutation at the site of interest. four md simulations were performed in explicit solvent conditions to examine the effect of specific mutations on overall structure and on the interactions of amino acids lining the active site of the protein. comparison of the trajectories between wild-type and double mutant showed that overall fold of mutant protein is retained. however, specific and drastic changes in the positions of active site residues were observed. the conformational changes that initiated from the site of mutation propagated to the active site and displaced his- as well as affected his- and glu- analysis of trajectories of single mutants illustrated that stereo chemical structural changes similar to that observed in double mutant were present in v18g. on the contrary, md simulation results of p19a appeared much similar to that of the wild-type but the presence of differential population of the ensemble at different time could possibly explain the reduction in the activity of this mutant protein. thus, the results of our md simulation studies support our biochemical observations. discussion metaps represent a unique class of metalloproteases that catalyze the co-translational removal of n-terminal methionine residue from the elongating polypeptide chain during the process of protein synthesis. since metap action in both prokaryotes and eukaryotes is mandatory during nascent protein synthesis, the importance of this enzyme has long been realized. though the presence of two metap1s was known with the availability of the m. tuberculosis genome sequence, both of them are active has been shown recently by others and also in this study. however, it is still not known whether they are merely complementing each other for their functionality and therefore redundant within the genome or their presence is because of the specific needs of the bacterium. recently, it has been reported that mtmetap1a knock down caused ~75% viability of mycobacteria while elimination of mtmetap1c resulted in 95% survival. since this enzyme from different sources has been considered as a potential drug target, identification of inhibitors against these enzymes, particularly for m. tuberculosis mtmetap1a is in progress. however, the initial step of structure-activity relationship between these two proteins has not been addressed properly. in this context, to carry out systematic study of these two enzymes from m. tuberculosis, we have attempted here to analyze metap1s to elucidate subtle differences in their characteristics. our results indicated that mtmetap1c had strikingly high enzyme turnover rate with the same substrate compared to mtmetap1a, although they had different substrate preferences. the outcome of our cd as well as activity assays with these mycobacterial enzymes illustrate that mtmetap1a can sustain high temperature compared to mtmetap1c. in fact, tm value calculated based on cd spectra for mtmetap1a was °c as opposed to °c for mtmetap1c. to get an insight into the cause of such differential behaviour of the two enzymes, structure of mtmetap1a was modelled and compared with the available structure of mtmetap1c. we observed that the active site residues in both the mtmetap1s are essentially same, though their arrangement in space is different. furthermore, these amino acids are crucial because any alteration in them yielded an inactive protein. thus it is logical to presume that this difference in the arrangement may be responsible for the alteration in their activity/functionality. sequence analysis revealed that the remarkable feature of mtmetap1c compared to mtmetap1a, is the presence of amino acid long n-terminal extension. it has been suggested that this extension may be involved in the interaction of mtmetap1c with the ribosome. in scmetap and hsmetap deletion of n-terminal extension did not significantly alter the catalytic efficiency of the enzymes. however, such deletion in scmetap reduced its efficiency in rescuing the slow growth phenotype of a map mutant. interestingly, the zinc finger motif present in the n-terminal extension of scmetap has been shown to be involved in its association with the ribosome. in hsmetap on the other hand, these sequences have been implicated for the interaction of the enzyme with substrates and inhibitors. this led us to explore the role of the n-terminal extension present in mtmetap1c towards the activity of the enzyme. we constructed a series of deletion mutants removing nine, fourteen, nineteen, twenty-nine and thirty-nine amino acids from the amino terminal end of the mtmetap1c. enzyme assays with these constructs using even fold excess of proteins compared to the wild-type revealed that there was a considerable loss in activity in Δ2- or Δ2- variants and no activity at all in mutants, like Δ2- or Δ2- or Δ2-. intriguingly, following resolving in sds-page, analysis of the deletion constructs in coomassie brilliant blue stained gels reflected that purification profile of Δ2- or Δ2- mutants was similar to that of the wild-type but variants like Δ2- Δ2- and Δ2- exhibited loss in purity of the proteins. hence, it seems logical to postulate that the amino acid residues between and of mtmetap1c are crucial for its activity. perhaps, deletion of residues beyond from the n-terminal end of mtmetap1c renders the enzyme unstable or exposes its hydrophobic patches, thereby increasing its tendency to associate with the other proteins. in fact, evaluation of structural changes through far-uv and near-uv cd spectra revealed that up to residues from the n-terminal extension of mtmetap1c are dispensable for the folding of the protein but required for its activity. thus, our results argue that the n-terminal extension is important for the catalytic activity of mtmetap1c protein, which is unlike the reports available with scmetap hsmetap and hsmetap. to identify the critical residue for the activity of the protein, we analyzed the sequence of metaps from different gram positive bacteria with n-terminal extension and found the presence of conserved val- and pro- in between amino acid residues and. this observation, prompted us to create point mutations at 18th and 19th position of mtmetap1c and examine their effect on the activity of the enzyme. while v18a and p19a were partially active, v18ap19a did not show any activity. on the other hand, p19g exhibited partial activity but neither v18g nor v18gp19g were active. further, to assess if these mutations resulted in any structural alterations, far-uv cd studies were carried out. interestingly the mutation caused alteration in the secondary structure of the protein. thus, our results argue the importance of these two residues exclusively and mutually towards the enzymatic activity of mtmetap1c. it appears very likely that the structural alterations that occurred due to mutation at val- and/or pro- of mtmetap1c affected the active site conformation of the protein. in fact, md simulations indicated that the conformational changes generated at the site of mutation propagated via connecting loops and helices to the enzyme active site resulting in the alteration in the movement/positioning of residues like his- his- and glu- which are critical for the enzyme activity as revealed in our biochemical studies. these variations in the v18gp19g or v18g mutant proteins presumably made the active site environment unfavourable for any activity. thus, our results illustrate that residue without being in the active site of an enzyme is capable of modulating its activity, which does not seem to be unusual.  our findings unequivocally emphasize that n-terminal extension of mtmetap1c contributes towards the functionality of the enzyme by regulating active site residues through "action-at-a-distance" mechanism and this is presumably its unique function in mtmetap1c, which we are reporting for the first time. since val- and pro- are conserved residues throughout the gram positive bacterial metap1s with n-terminal extension, it remains to be seen whether they are universal in contributing towards the functionality of the enzyme. 
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. 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. the metap action for exclusion of n-terminal methionine is mandatory in 50-70% of nascent proteins. we cloned genes encoding two such metalloproteases present in mycobacterium tuberculosis and expressed them as histidine-tagged proteins in escherichia coli. furthermore, mutational analysis revealed that val- and pro- of mtmetap1c are crucial for its enzymatic activity. it specifically removes the terminal methionine in all organisms, if the penultimate residue is non-bulky and uncharged. such an activity is required for proper sub cellular localization, additional processing and eventually for the degradation of proteins. 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. methionine aminopeptidase 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. strikingly, we observed that 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. circular dichroism spectroscopic studies as well as monitoring of enzyme activity indicated high temperature stability of mtmetap1a compared to that of the mtmetap1c. although they have different substrate preferences, for met-ala-ser, we found, mtmetap1c had significantly high enzyme turnover rate as opposed to mtmetap1a.
 methionine aminopeptidase 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.  we cloned genes encoding two such metalloproteases 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 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 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- and pro- 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.  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. 