BACKGROUND
aminoacylation is the process of adding an aminoacyl group to the 3' end  of the trna molecule.
trna is aminoacylated with a specific amino acid by aminoacyl-trna synthetase .
aarss are responsible for attaching correct amino acid onto the cognate trna molecule in a two-step reaction.
the amino acid is first activated with atp forming an aminoacyladenylate intermediate.
once activated, this amino acid is transferred to the 3' end of its corresponding trna molecule to be processed during protein synthesis.
all aarss require divalent cation mgcl <dig> for their aminoacylation reaction  <cit> .
reaction:
<dig>  amino acid + atp → aminoacyl-amp + ppi
<dig>  aminoacyl-amp + trna → aminoacyl-trna + amp
the aarss are divided into two major classes based on structural topology of their active sites.
class i aarss represent  <dig> amino acids, including arg, cys, gln, glu, ile, leu, lys, met, val, trp and tyr.
class ii aarss includes  <dig> amino acids - ala, asp, asn, gly, his, lys, phe, pro, ser and thr.
core domains of class i enzymes are characterized by a rossmann fold which consists of α-helices and β-pleated sheets.
this domain contains two conserved motifs  which are directly involved in atp binding.
catalytic domain of class ii enzymes has a unique fold with a central core of anti-parallel β strands flanked by α helices  <cit> .
there are three weakly conserved motifs, two of them are involved in atp binding while the third one plays a role in homo dimerization.
class i enzymes bind atp in an extended conformation while class ii do so in a bent conformation.
the two aars classes have different modes of aminoacylation - class i enzymes aminoacylate the 2'oh of the cognate trna whereas class ii enzymes aminoacylate 3'oh of the trna   <cit> .
all known aarss are multidomain proteins with complex modular architectures  <cit> .
in addition, eukaryotic aarss are distinguished by the presence of appended domains at either the n- or c-terminus which are generally absent from their bacterial/archaeal counterparts  <cit> .
these appendages to the catalytic cores of several aarss are non-catalytic and instead function to mediate protein- protein interactions or act as general rna-binding domains  <cit> .
in mammalian cells, some aarss are present as a larger multi- aars complex  composed of nine synthetases   <cit> .
the msc is composed of a mixture of class i and class ii aarss along with three non- aars proteins p <dig>  p <dig> and p <dig>  it is not clear why certain aarss exist as a complex while some are in free form.
msc might help in efficient protein synthesis by preventing mixing of charged trnas with cellular pool and by increasing local concentration of trna near the site of protein synthesis  <cit> .
the accuracy of trna aminoacylation reaction is critical in ensuring fidelity in protein translation  <cit> .
to achieve this accuracy, some aars enzymes possess a proofreading  mechanism that hydrolyzes trnas aminoacylated with the non-cognate amino acid  <cit> .
for example, editing domains may be found attached to alanyl-trna synthetase , leucyltrna synthetase  and so on  <cit> .
in other cases, the editing domain is not attached to aars but rather functions as an individual protein  <cit> .
for example, ybak protein from haemophilus influenza is capable of efficiently editing cys-trnapro  <cit> .
thrrs has been shown to have another editing domain called ntd which can cleave the bond between d-amino acid and trna  <cit> .
recently it has been shown that aarss are not only involved in protein synthesis but also perform many non-catalytic and non-canonical roles in rna processing/trafficking, apoptosis, rrna synthesis, angiogenesis and inflammation  <cit> .
these versatile properties of aarss are the outcome of their differential cellular localization, nucleic acid binding properties, protein-protein interactions and collaboration  with additional domains.
in case of malaria parasite, apicoplast proteins and pathways have already received particular attention as drug targets  <cit> .
in this work we present a study of aarss from p. falciparum - the most virulent agent of human malaria.
our aim for this study was to use bioinformatics tools to  discover special and unusual modules present in parasite aarss which are potentially absent from human homologues, and  to identify potential new drug targets based on this protein family.
RESULTS
sequence extraction and analysis
we exploited current annotation available in plasmodb  <cit>  to identify the repertoire of aarss in p. falciparum genome.
according to enzyme commission   <dig> proteins in plasmodb  are annotated as belonging to the ec group  <dig> .
<dig>  .
although in many cases current annotations allow an assignment to class i or ii of aarss, for some annotations are still preliminary.
due to this, we used hidden markov models  for identifying aarss in p. falciparum.
for each aars a set of known sequences was utilized to construct  <dig> hmms .
for each database search a score distribution was obtained and  <dig> cutoffs were considered to identify aars.
results are reported in table  <dig>  we observed that  <dig> proteins annotated as belonging to ec group  <dig> .
<dig> - in plasmodb are not found by hmms - pf14_ <dig> annotated as metrs is instead a generic trna binding protein as elucidated in the genome re-annotation process, while the second one  is still mis-annotated as valrs.
a total of  <dig> pf-aarss can be classified within the  <dig> aarss that define class i. all members of this class are represented in the p. falciparum proteome.
the annotations of these sequences are summarized in additional file  <dig>  similar to class i pf-aarss, the class ii pf-aarss have a total of  <dig> sequences for  <dig> different amino acid synthetases.
four genes are present in p. falciparum for phers but these likely encode for  <dig> heterodimeric and  <dig> monomeric versions of phers.
* hits with more than one assignment to hmm model.
@two proteins pf14_ <dig> and pfc0470w were not found by hmms which were annotated as trna binding protein and valrs in plasmodb respectively.
in order to carry out comparative analyses of aarss of p. falciparum with those of other species we considered aars sequences from several organisms representing three domains of life .
as expected, we found variable number of aarss in different species.
m. jannaschii  and m. tuberculosis  have the lowest aarss count amongst other organisms like e. coli, s. cerevisiae, d. discoidium, p. falciparum, o. sativa, r. norvegicus, d. melanogaster, and h. sapiens.
human bears the highest number of aarss in this analysis .
our analysis also shows that p. falciparum has the highest aars fraction  when compared with bacteria, yeast and human counterparts .
the number of individual aars varies in different species.
for example, when individual aarss from human and p. falciparum were compared it was evident that alars and thrrs were higher in number in humans .
presence of more than one copy of each aars in an organism may indicate additional biological, temporal or spatial roles for these enzymes as several aarss also perform non-canonical functions  <cit> .
in this work we describe in detail the  <dig> pf-aarss.
indirect pathways of aminoacylation
it was earlier believed that  <dig> aarss were necessary for the incorporation of  <dig> amino acids in proteins.
but surprisingly, some archaea, bacteria and chloroplasts lack glnrs and asnrs enzymes  <cit> .
interestingly, these organisms use an alternate pathway based on trna dependent amino acid transformation.
a non-discriminating glurs charges trnagln with glutamic amino acid and then a second enzyme called trna-dependent amidotransferase  amidates glutamate to make glutamine.
a corresponding reaction occurs in case of asparagine residues.
in case of p. falciparum, occurrence of glutamate-trna synthetase  and amidotransferase subunit a  & subunit b  together indicates presence of both direct and indirect pathways for aminoacylation  <cit> .
both subunits of amidotransferase have apicoplast targeting signals suggesting an indirect pathway for aminoacylation in p. falciparum apicoplast.
the expression of pf-adt subunit a is predicted in all life cycle stages of parasite based on proteomic and microarray data.
we therefore feel that this pathway must also be active in the parasite apicoplast.
we could not find sequence homologues of enzymes involved in indirect aminoacylation of cysteine residues  <cit>  in the proteome of p. falciparum.
the multi-synthetase complex
in mammalian cells, some aarss are present as a larger multi-aars complex .
a constituent of the msc - protein p <dig> - has sequence homologue  in p. falciparum although there is no evidence for presence of msc in malaria parasites.
interestingly, p <dig> is not only required for stability of the msc complex but also functions as a proinflammatory cytokine  <cit> .
role of p <dig> homolog in p. falciparum is unknown, but evidence from other organisms indicates that msc functions in protein stability, efficient protein translation and protein elongation  <cit> .
sequence identity between p. falciparum p <dig> and its human homolog is ~24% and based on microarray data p <dig> seems to be expressed at asexual life cycle stages of p. falciparum.
a mitochondrial targeting signal was also predicted for parasite p <dig> but the role of p <dig> in parasite remains to be explored experimentally.
targeting of aarss in the parasite
aarss are not only involved in protein synthesis but also in various other cellular activities including intron splicing, translational regulation and trna channeling.
diversified roles for aarss necessitate their presence  into various cellular compartments.
we therefore analyzed p. falciparum aars sequences for presence of putative signal sequences predicted by mitoprot, predictnls and pats for mitochondria, nucleus and apicoplast respectively.
we found that  <dig> p. falciparum aarss have signal peptides, possibly for directing them to different cellular organelles.
another  <dig> aarss from p. falciparum may be resident in the parasite cytoplasm .
apicoplast is known to have protein synthesis machinery which may use aarss  <cit> .
trafficking of nuclear encoded aarss to the apicoplast may explain why ~ <dig> out of  <dig> pf-aarss have apicoplast targeting signals.
our data indicate that out of total ~ <dig> pf-aarss bearing apicoplast targeting signals, ~ <dig> aarss may be exclusive to this organelle.
others are predicted to be shared between apicoplast, nucleus and mitochondria .
it has been earlier shown that some trnas need to be aminoacylated in the nucleus before they can be exported to the cytoplasm, an observation indicating occurrence of aminoacylation reaction  inside the nucleus  <cit> .
in p. falciparum, we found  <dig> aarss with nuclear localization signals but only one is predicted to be exclusively resident in the nucleus .
interestingly, we found no pf-aars sequences with specific pexel  motifs.
this motif is found in parasite proteins that are exported beyond the parasitophorous vacuole membrane  <cit> .
expression profiles of p. falciparum aarss
in order to study expression of aars during life cycle of the malaria parasite, we took advantage of available transcriptomics and proteomics data from plasmodb.
firstly, we analyzed proteomic data from several independent experiments and compared them with transcriptomics data by le roch  <cit> .
the latter sets of data were obtained using the affimetrix technology and hence provide a quantitative measure of mrna levels in the parasite.
our results are provided in table  <dig>  interestingly, we found that mrna levels of potential apicoplast proteins  are lower on average  than those of potential cytoplasmic proteins .
proteomic data confirmed that while the cytoplasmic aars are found in almost all stages, the apicoplast aars are rarely found in the parasite.
this could be in part due to experimental limits in the identification of apicoplast proteins by mass spectrometry.
indeed, when we carried out a chi-quadro test we found that proteins predicted to be targeted to apicoplast are significantly less represented  in the sample of proteins identified by mass spectrometry.
for these reasons we limited analysis of gene expression profiles only for putative cytoplasmic proteins.
we considered trascriptomics data for sexual stages and asexual stages  <cit> .
we considered a reduced set of the time course gene expression data  and normalized data by le roch .
this allowed us to analyse the expression of aars genes along all the intra-erythrocytic life cycle of the parasite .
further observations of the protein expression profiles indicated that some aarss were exclusively detected at specific stages like, leurs  and asprs  in sporozoites; ilers , serrs , glnrs , hisrs  and phers  in merozoites; asnrs , phers  and hisrs  in trophozoites and trprs  in gametocyte stages .
@ gene expression data are by le roch <cit> .
mean <dig> and mean <dig> asex refer to mean values of mrna abundance along asexual stages synchronized by sorbitol and by temperature respectively.
in the last two columns, mass spectrometry data from purified merozoites  and mosquito stages  are shown.
tg§ proteomic data are obtained by trophozoites, gametocytes early and gametocytes late by lasonder lab.
<cit> .
t , me , g , sp  columns refer to data by multi-dimensional protein identification technology in four stages of the parasite life cycle.
domain architecture of p. falciparum aarss
aarss are multi-domain proteins typically consisting of a conserved catalytic domain and an anti-codon binding domain.
in addition, some aarss have rna binding and editing domains that cleave incorrectly aminoacylated trna molecules  <cit> .
additional functional domains may be appended to aarss in the course of biological evolution  <cit> .
careful examination of  <dig> identified p. falciparum aarss using pfam database showed that most of them have a generic modular architecture that adheres to prototypical aarss .
the remaining p. falciparum aarss or related proteins like pf14_ <dig>  have complex domain architectures.
in several, concatenation of unusual domains such as ybak, gst, ser-thr kinase and dna binding domains is evident .
the functional relevance of these additional domains fused to typical aars in p. falciparum needs to be experimentally addressed.
intriguingly, two of the four pf-phers subunits contain dna binding domains .
it is likely that the phers, in addition to its aminoacylation function, influences other cellular processes via dna binding  <cit> .
consistent with its potential dna binding property, the p. falciparum phers  has a nuclear localization signal.
the cysrs of b. subtilis  is believed to play a role in initiating chromosomal replication  <cit> .
therefore, functional roles for p. falciparum pherss may extend from aminoacylation to dna recognition and replication - a suggestion that requires experimental investigation.
similarly, it has been shown that gst or gst homology domains can help in complex formation of aarss with multifunctional factors   <cit> .
additional data show that deletion of gst homology domain from the c-terminal region of p <dig> results in the dissociation of eprs  and metrs from the msc complex  <cit> .
mammalian valrs associated with elongation factor subunits also contain the gst homology domain  <cit> .
thus, the presence of gst domains might be a crucial feature of aarss.
p. falciparum proteome has two such proteins with gst domains appended to metrs  and glurs .
we also found a most interesting fusion of anticodon binding domain with a serine-threonine kinase  in p. falciparum.
this unusual kinase seems to be expressed throughout the life cycle of parasite  and interestingly is predicted to be localized to the parasite nucleus.
clearly, the presence of unusual domain fusions in p. falciparum aarss suggests multiple functional roles for many of these p. falciparum enzymes as has been shown in other organisms.
phylogenetics
overall the percentage identity between matching human and p. falciparum aars domains varies from  <dig> to  <dig>  clearly, pf-aarss which have low sequence identity with human counterparts might serve as good drug targets.
in order to study evolutionary relationships of p. falciparum aarss with other species, phylogenetic trees were developed in phyml using maximum likelihood method.
for each type of p. falciparum aars a separate tree was constructed .
aars sequences from  <dig> different species were used for multiple sequence alignments.
as an example, phylogenetic tree of tyrrs from various species  was constructed.
interestingly, one pf-tyrrs  clustered with human tyrrs whereas the second pf-tyrrs  clustered with bacterial tyrrs indicating different evolutionary origins .
based on distance matrices, several p. falciparum aars sequences clustered as being closer to plants  or to bacteria  .
it is already known that apicomplexan parasites like p. falciparum house a secondary endosymbiotic plastid, possibly hijacked by lateral genetic transfer from an alga.
therefore, the p. falciparum aars sequences which are evolutionary close to bacteria and plants are likely to be the outcome of horizontal gene transfer from the plastid.
p. falciparum contains ~ <dig> such aars sequences which cluster with bacterial or plant sequences.
functional and structural characterization of these bacterial/plant-like aars may be relevant in focusing efforts at using aars as drug targets.
homology modeling and structure comparisons
to date, no crystal structures have been obtained for any aars from p. falciparum.
hence, we performed homology modeling of several p. falciparum aarss using homologous structures available in pdb.
known structural templates  were used for molecular modeling of several p. falciparum aarss including the two tyrrss , the phers , thrrs , lysrs , metrs  and trprs .
the program align2d  was used to perform dynamic programming-based global alignments of the target and template sequences.
this program uses variable gap penalty for structural loops and core regions using information derived from template structures.
we found key differences in the conserved motifs in various aarss.
for example, the class i motif 'kysks' in p. falciparum tyrrs  and 'kmsks' in mal8p <dig>  differs from 'klgks' of human mitochondrial tyrrs  and 'kmsss' of human cytoplasmic  respectively.
similarly, class i motif 'high' has subtle sequences variations between p. falciparum and h. sapiens tyrrss .
using the above procedures, we could generate structural models for several pf-aarss.
stereo-chemical qualities of the generated protein models were assessed using procheck .
the overall superimposed three-dimension models were visualized in chimera and pymol .
many sequence insertions were observed for p. falciparum enzymes when compared to their homologous  <cit> .
location of insertions in p. falciparum tyrrs between well-conserved secondary structures suggests ability of tyrrs anticodon binding core to accommodate larger sequence inserts with minimum disruption to the catalytic domain.
direct comparison of modeled p. falciparum aarss with human aarss revealed several other important structural differences.
for example, numerous insertions are present in the loop regions linking various α-helices  in anticodon binding domain of p. falciparum tyrrss  when compared to its human homologous  respectively.
structural differences between tyrrs  and human counterparts are summarized in table  <dig> and shown in figure 7c.
these subtle structural changes that manifest as partial conservation of important motifs in p. falciparum aarss reflect evolutionary divergence, and may be useful for exploitation of parasite-specific features as drug targets.
* human mitochondrial
@ p. falciparum bacteria-like
!
human cytosolic
$ p. falciparum human-like
CONCLUSIONS
aminoacyl-trna synthetases  link rna with protein translation.
besides their key role in protein synthesis, aarss are also integral to various other cellular processes.
aars enzymes have been the focus for antimicrobial drug discovery  <cit> .
an example of clinical application of an aars inhibitor is provided by the antibiotic mupirocin , which selectively inactivates bacterial isoleucyl-trna synthetase  <cit> .
similarly, it has been shown that the broad-spectrum antifungal 5-fluoro- <dig> -dihydro-1-hydroxy- <dig> -benzoxaborole  inhibits yeast cytoplasmic leucyl-trna synthetase by blocking editing site of the enzyme  <cit> .
therefore, presence of distinct or tinkered p. falciparum aars lends an opportunity for their exploitation as new drug targets against malaria.
in this study, we have extensively analyzed aars sequences from plasmodium species in terms of their mrna/protein expression profiles, their cellular localization, their organelle targeting and their unique sequence/domain attributes.
we have discovered several distinct aarss in p. falciparum with no clear human counterparts in terms of their overall domain structures.
we have also highlighted deviations of some highly conserved sequence motifs and active site sequence clusters.
our analyses clearly show that a larger fraction of p. falciparum proteome is devoted to aars when compared with many other organisms.
the phylogenetic data hint at evolutionary closeness of some pf-aarss to bacteria and plants - this further supports the fact of secondary endosymbiosis in this apicomplexan.
we hope that our in-depth phylogenetic, protein targeting, domain architecture, protein expression profiling and homology modeling data on pf-aarss can be used as a platform for experimental studies of this important protein family in malaria parasites.

 aminoacylation is the process of adding an aminoacyl group to the 3' end of the trna molecule. trna is aminoacylated with a specific amino acid by aminoacyl-trna synthetase. aarss are responsible for attaching correct amino acid onto the cognate trna molecule in a two-step reaction. the amino acid is first activated with atp forming an aminoacyladenylate intermediate. once activated, this amino acid is transferred to the 3' end of its corresponding trna molecule to be processed during protein synthesis. all aarss require divalent cation mgcl for their aminoacylation reaction. reaction: amino acid + atp → aminoacyl-amp + ppi aminoacyl-amp + trna → aminoacyl-trna + amp the aarss are divided into two major classes based on structural topology of their active sites. class i aarss represent amino acids, including arg, cys, gln, glu, ile, leu, lys, met, val, trp and tyr. class ii aarss includes amino acids - ala, asp, asn, gly, his, lys, phe, pro, ser and thr. core domains of class i enzymes are characterized by a rossmann fold which consists of α-helices and β-pleated sheets. this domain contains two conserved motifs which are directly involved in atp binding. catalytic domain of class ii enzymes has a unique fold with a central core of anti-parallel β strands flanked by α helices. there are three weakly conserved motifs, two of them are involved in atp binding while the third one plays a role in homo dimerization. class i enzymes bind atp in an extended conformation while class ii do so in a bent conformation. the two aars classes have different modes of aminoacylation - class i enzymes aminoacylate the 2'oh of the cognate trna whereas class ii enzymes aminoacylate 3'oh of the trna. all known aarss are multidomain proteins with complex modular architectures. in addition, eukaryotic aarss are distinguished by the presence of appended domains at either the n- or c-terminus which are generally absent from their bacterial/archaeal counterparts. these appendages to the catalytic cores of several aarss are non-catalytic and instead function to mediate protein- protein interactions or act as general rna-binding domains. in mammalian cells, some aarss are present as a larger multi- aars complex composed of nine synthetases. the msc is composed of a mixture of class i and class ii aarss along with three non- aars proteins p p and p it is not clear why certain aarss exist as a complex while some are in free form. msc might help in efficient protein synthesis by preventing mixing of charged trnas with cellular pool and by increasing local concentration of trna near the site of protein synthesis. the accuracy of trna aminoacylation reaction is critical in ensuring fidelity in protein translation. to achieve this accuracy, some aars enzymes possess a proofreading mechanism that hydrolyzes trnas aminoacylated with the non-cognate amino acid. for example, editing domains may be found attached to alanyl-trna synthetase, leucyltrna synthetase and so on. in other cases, the editing domain is not attached to aars but rather functions as an individual protein. for example, ybak protein from haemophilus influenza is capable of efficiently editing cys-trnapro. thrrs has been shown to have another editing domain called ntd which can cleave the bond between d-amino acid and trna. recently it has been shown that aarss are not only involved in protein synthesis but also perform many non-catalytic and non-canonical roles in rna processing/trafficking, apoptosis, rrna synthesis, angiogenesis and inflammation. these versatile properties of aarss are the outcome of their differential cellular localization, nucleic acid binding properties, protein-protein interactions and collaboration with additional domains. in case of malaria parasite, apicoplast proteins and pathways have already received particular attention as drug targets. in this work we present a study of aarss from p. falciparum - the most virulent agent of human malaria. our aim for this study was to use bioinformatics tools to discover special and unusual modules present in parasite aarss which are potentially absent from human homologues, and to identify potential new drug targets based on this protein family.  sequence extraction and analysis we exploited current annotation available in plasmodb to identify the repertoire of aarss in p. falciparum genome. according to enzyme commission proteins in plasmodb are annotated as belonging to the ec group.. although in many cases current annotations allow an assignment to class i or ii of aarss, for some annotations are still preliminary. due to this, we used hidden markov models for identifying aarss in p. falciparum. for each aars a set of known sequences was utilized to construct hmms. for each database search a score distribution was obtained and cutoffs were considered to identify aars. results are reported in table we observed that proteins annotated as belonging to ec group. - in plasmodb are not found by hmms - pf14_ annotated as metrs is instead a generic trna binding protein as elucidated in the genome re-annotation process, while the second one is still mis-annotated as valrs. a total of pf-aarss can be classified within the aarss that define class i. all members of this class are represented in the p. falciparum proteome. the annotations of these sequences are summarized in additional file similar to class i pf-aarss, the class ii pf-aarss have a total of sequences for different amino acid synthetases. four genes are present in p. falciparum for phers but these likely encode for heterodimeric and monomeric versions of phers. * hits with more than one assignment to hmm model. @two proteins pf14_ and pfc0470w were not found by hmms which were annotated as trna binding protein and valrs in plasmodb respectively. in order to carry out comparative analyses of aarss of p. falciparum with those of other species we considered aars sequences from several organisms representing three domains of life. as expected, we found variable number of aarss in different species. m. jannaschii and m. tuberculosis have the lowest aarss count amongst other organisms like e. coli, s. cerevisiae, d. discoidium, p. falciparum, o. sativa, r. norvegicus, d. melanogaster, and h. sapiens. human bears the highest number of aarss in this analysis. our analysis also shows that p. falciparum has the highest aars fraction when compared with bacteria, yeast and human counterparts. the number of individual aars varies in different species. for example, when individual aarss from human and p. falciparum were compared it was evident that alars and thrrs were higher in number in humans. presence of more than one copy of each aars in an organism may indicate additional biological, temporal or spatial roles for these enzymes as several aarss also perform non-canonical functions. in this work we describe in detail the pf-aarss. indirect pathways of aminoacylation it was earlier believed that aarss were necessary for the incorporation of amino acids in proteins. but surprisingly, some archaea, bacteria and chloroplasts lack glnrs and asnrs enzymes. interestingly, these organisms use an alternate pathway based on trna dependent amino acid transformation. a non-discriminating glurs charges trnagln with glutamic amino acid and then a second enzyme called trna-dependent amidotransferase amidates glutamate to make glutamine. a corresponding reaction occurs in case of asparagine residues. in case of p. falciparum, occurrence of glutamate-trna synthetase and amidotransferase subunit a & subunit b together indicates presence of both direct and indirect pathways for aminoacylation. both subunits of amidotransferase have apicoplast targeting signals suggesting an indirect pathway for aminoacylation in p. falciparum apicoplast. the expression of pf-adt subunit a is predicted in all life cycle stages of parasite based on proteomic and microarray data. we therefore feel that this pathway must also be active in the parasite apicoplast. we could not find sequence homologues of enzymes involved in indirect aminoacylation of cysteine residues in the proteome of p. falciparum. the multi-synthetase complex in mammalian cells, some aarss are present as a larger multi-aars complex. a constituent of the msc - protein p - has sequence homologue in p. falciparum although there is no evidence for presence of msc in malaria parasites. interestingly, p is not only required for stability of the msc complex but also functions as a proinflammatory cytokine. role of p homolog in p. falciparum is unknown, but evidence from other organisms indicates that msc functions in protein stability, efficient protein translation and protein elongation. sequence identity between p. falciparum p and its human homolog is ~24% and based on microarray data p seems to be expressed at asexual life cycle stages of p. falciparum. a mitochondrial targeting signal was also predicted for parasite p but the role of p in parasite remains to be explored experimentally. targeting of aarss in the parasite aarss are not only involved in protein synthesis but also in various other cellular activities including intron splicing, translational regulation and trna channeling. diversified roles for aarss necessitate their presence into various cellular compartments. we therefore analyzed p. falciparum aars sequences for presence of putative signal sequences predicted by mitoprot, predictnls and pats for mitochondria, nucleus and apicoplast respectively. we found that p. falciparum aarss have signal peptides, possibly for directing them to different cellular organelles. another aarss from p. falciparum may be resident in the parasite cytoplasm. apicoplast is known to have protein synthesis machinery which may use aarss. trafficking of nuclear encoded aarss to the apicoplast may explain why ~ out of pf-aarss have apicoplast targeting signals. our data indicate that out of total ~ pf-aarss bearing apicoplast targeting signals, ~ aarss may be exclusive to this organelle. others are predicted to be shared between apicoplast, nucleus and mitochondria. it has been earlier shown that some trnas need to be aminoacylated in the nucleus before they can be exported to the cytoplasm, an observation indicating occurrence of aminoacylation reaction inside the nucleus. in p. falciparum, we found aarss with nuclear localization signals but only one is predicted to be exclusively resident in the nucleus. interestingly, we found no pf-aars sequences with specific pexel motifs. this motif is found in parasite proteins that are exported beyond the parasitophorous vacuole membrane. expression profiles of p. falciparum aarss in order to study expression of aars during life cycle of the malaria parasite, we took advantage of available transcriptomics and proteomics data from plasmodb. firstly, we analyzed proteomic data from several independent experiments and compared them with transcriptomics data by le roch. the latter sets of data were obtained using the affimetrix technology and hence provide a quantitative measure of mrna levels in the parasite. our results are provided in table interestingly, we found that mrna levels of potential apicoplast proteins are lower on average than those of potential cytoplasmic proteins. proteomic data confirmed that while the cytoplasmic aars are found in almost all stages, the apicoplast aars are rarely found in the parasite. this could be in part due to experimental limits in the identification of apicoplast proteins by mass spectrometry. indeed, when we carried out a chi-quadro test we found that proteins predicted to be targeted to apicoplast are significantly less represented in the sample of proteins identified by mass spectrometry. for these reasons we limited analysis of gene expression profiles only for putative cytoplasmic proteins. we considered trascriptomics data for sexual stages and asexual stages. we considered a reduced set of the time course gene expression data and normalized data by le roch. this allowed us to analyse the expression of aars genes along all the intra-erythrocytic life cycle of the parasite. further observations of the protein expression profiles indicated that some aarss were exclusively detected at specific stages like, leurs and asprs in sporozoites; ilers, serrs, glnrs, hisrs and phers in merozoites; asnrs, phers and hisrs in trophozoites and trprs in gametocyte stages. @ gene expression data are by le roch. mean and mean asex refer to mean values of mrna abundance along asexual stages synchronized by sorbitol and by temperature respectively. in the last two columns, mass spectrometry data from purified merozoites and mosquito stages are shown. tg§ proteomic data are obtained by trophozoites, gametocytes early and gametocytes late by lasonder lab.. t, me, g, sp columns refer to data by multi-dimensional protein identification technology in four stages of the parasite life cycle. domain architecture of p. falciparum aarss aarss are multi-domain proteins typically consisting of a conserved catalytic domain and an anti-codon binding domain. in addition, some aarss have rna binding and editing domains that cleave incorrectly aminoacylated trna molecules. additional functional domains may be appended to aarss in the course of biological evolution. careful examination of identified p. falciparum aarss using pfam database showed that most of them have a generic modular architecture that adheres to prototypical aarss. the remaining p. falciparum aarss or related proteins like pf14_ have complex domain architectures. in several, concatenation of unusual domains such as ybak, gst, ser-thr kinase and dna binding domains is evident. the functional relevance of these additional domains fused to typical aars in p. falciparum needs to be experimentally addressed. intriguingly, two of the four pf-phers subunits contain dna binding domains. it is likely that the phers, in addition to its aminoacylation function, influences other cellular processes via dna binding. consistent with its potential dna binding property, the p. falciparum phers has a nuclear localization signal. the cysrs of b. subtilis is believed to play a role in initiating chromosomal replication. therefore, functional roles for p. falciparum pherss may extend from aminoacylation to dna recognition and replication - a suggestion that requires experimental investigation. similarly, it has been shown that gst or gst homology domains can help in complex formation of aarss with multifunctional factors. additional data show that deletion of gst homology domain from the c-terminal region of p results in the dissociation of eprs and metrs from the msc complex. mammalian valrs associated with elongation factor subunits also contain the gst homology domain. thus, the presence of gst domains might be a crucial feature of aarss. p. falciparum proteome has two such proteins with gst domains appended to metrs and glurs. we also found a most interesting fusion of anticodon binding domain with a serine-threonine kinase in p. falciparum. this unusual kinase seems to be expressed throughout the life cycle of parasite and interestingly is predicted to be localized to the parasite nucleus. clearly, the presence of unusual domain fusions in p. falciparum aarss suggests multiple functional roles for many of these p. falciparum enzymes as has been shown in other organisms. phylogenetics overall the percentage identity between matching human and p. falciparum aars domains varies from to clearly, pf-aarss which have low sequence identity with human counterparts might serve as good drug targets. in order to study evolutionary relationships of p. falciparum aarss with other species, phylogenetic trees were developed in phyml using maximum likelihood method. for each type of p. falciparum aars a separate tree was constructed. aars sequences from different species were used for multiple sequence alignments. as an example, phylogenetic tree of tyrrs from various species was constructed. interestingly, one pf-tyrrs clustered with human tyrrs whereas the second pf-tyrrs clustered with bacterial tyrrs indicating different evolutionary origins. based on distance matrices, several p. falciparum aars sequences clustered as being closer to plants or to bacteria. it is already known that apicomplexan parasites like p. falciparum house a secondary endosymbiotic plastid, possibly hijacked by lateral genetic transfer from an alga. therefore, the p. falciparum aars sequences which are evolutionary close to bacteria and plants are likely to be the outcome of horizontal gene transfer from the plastid. p. falciparum contains ~ such aars sequences which cluster with bacterial or plant sequences. functional and structural characterization of these bacterial/plant-like aars may be relevant in focusing efforts at using aars as drug targets. homology modeling and structure comparisons to date, no crystal structures have been obtained for any aars from p. falciparum. hence, we performed homology modeling of several p. falciparum aarss using homologous structures available in pdb. known structural templates were used for molecular modeling of several p. falciparum aarss including the two tyrrss, the phers, thrrs, lysrs, metrs and trprs. the program align2d was used to perform dynamic programming-based global alignments of the target and template sequences. this program uses variable gap penalty for structural loops and core regions using information derived from template structures. we found key differences in the conserved motifs in various aarss. for example, the class i motif'kysks' in p. falciparum tyrrs and'kmsks' in mal8p differs from'klgks' of human mitochondrial tyrrs and'kmsss' of human cytoplasmic respectively. similarly, class i motif'high' has subtle sequences variations between p. falciparum and h. sapiens tyrrss. using the above procedures, we could generate structural models for several pf-aarss. stereo-chemical qualities of the generated protein models were assessed using procheck. the overall superimposed three-dimension models were visualized in chimera and pymol. many sequence insertions were observed for p. falciparum enzymes when compared to their homologous. location of insertions in p. falciparum tyrrs between well-conserved secondary structures suggests ability of tyrrs anticodon binding core to accommodate larger sequence inserts with minimum disruption to the catalytic domain. direct comparison of modeled p. falciparum aarss with human aarss revealed several other important structural differences. for example, numerous insertions are present in the loop regions linking various α-helices in anticodon binding domain of p. falciparum tyrrss when compared to its human homologous respectively. structural differences between tyrrs and human counterparts are summarized in table and shown in figure 7c. these subtle structural changes that manifest as partial conservation of important motifs in p. falciparum aarss reflect evolutionary divergence, and may be useful for exploitation of parasite-specific features as drug targets. * human mitochondrial @ p. falciparum bacteria-like! human cytosolic $ p. falciparum human-like  aminoacyl-trna synthetases link rna with protein translation. besides their key role in protein synthesis, aarss are also integral to various other cellular processes. aars enzymes have been the focus for antimicrobial drug discovery. an example of clinical application of an aars inhibitor is provided by the antibiotic mupirocin, which selectively inactivates bacterial isoleucyl-trna synthetase. similarly, it has been shown that the broad-spectrum antifungal 5-fluoro- -dihydro-1-hydroxy- -benzoxaborole inhibits yeast cytoplasmic leucyl-trna synthetase by blocking editing site of the enzyme. therefore, presence of distinct or tinkered p. falciparum aars lends an opportunity for their exploitation as new drug targets against malaria. in this study, we have extensively analyzed aars sequences from plasmodium species in terms of their mrna/protein expression profiles, their cellular localization, their organelle targeting and their unique sequence/domain attributes. we have discovered several distinct aarss in p. falciparum with no clear human counterparts in terms of their overall domain structures. we have also highlighted deviations of some highly conserved sequence motifs and active site sequence clusters. our analyses clearly show that a larger fraction of p. falciparum proteome is devoted to aars when compared with many other organisms. the phylogenetic data hint at evolutionary closeness of some pf-aarss to bacteria and plants - this further supports the fact of secondary endosymbiosis in this apicomplexan. we hope that our in-depth phylogenetic, protein targeting, domain architecture, protein expression profiling and homology modeling data on pf-aarss can be used as a platform for experimental studies of this important protein family in malaria parasites. 
our key observations are: fraction of proteome dedicated to aarss in p. falciparum is very high compared to many other organisms; out of pf-aars sequences contain signal peptides possibly directing them to different cellular organelles; expression profiles of pf-aarss vary considerably at various life cycle stages of the parasite; several pfaarss posses very unusual domain architectures; phylogenetic analyses reveal evolutionary relatedness of several parasite aarss to bacterial and plants aarss; three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aarss. using various computational and bioinformatics tools, we have identified aarss in p. falciparum. aminoacyl-trna synthetases are pivotal enzymes for protein translation and other vital cellular processes. we have annotated all pf-aarss based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. our data reveal several unique attributes in this protein family.
 plasmodium parasites are causative agents of malaria which affects > million people and claims ~ million lives annually. the completion of plasmodium genome sequencing and availability of plasmodb database has provided a platform for systematic study of parasite genome. aminoacyl-trna synthetases are pivotal enzymes for protein translation and other vital cellular processes. we report an extensive analysis of the plasmodium falciparum genome to identify and classify aarss in this organism.  using various computational and bioinformatics tools, we have identified aarss in p. falciparum. our key observations are: fraction of proteome dedicated to aarss in p. falciparum is very high compared to many other organisms; out of pf-aars sequences contain signal peptides possibly directing them to different cellular organelles; expression profiles of pf-aarss vary considerably at various life cycle stages of the parasite; several pfaarss posses very unusual domain architectures; phylogenetic analyses reveal evolutionary relatedness of several parasite aarss to bacterial and plants aarss; three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasite aarss.  we have identified pf-aarss based on our bioinformatics analysis. our data reveal several unique attributes in this protein family. we have annotated all pf-aarss based on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. the sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria. 