BACKGROUND
phosphatidylinositols are important metabolites present in animal cell membranes.
they can be phosphorylated at three main positions of the inositol head  to generate seven different phosphoinositides .
their metabolism is tightly controlled by phospholipases that in some instances generate second messengers such as inositol  <dig> ,5-trisphosphate p3) and diacylglycerol .
in addition, kinases and phosphatases control the levels of the mono, di- or tri-phosphorylated forms  <cit> .
pi phosphatases belong to different families of enzymes; pten and myotubularin inositol 3-phosphatases  belong to the protein tyrosine phosphatase  superfamily and have both protein and lipid phosphatase activities  <cit> .
inositol 4-phosphatases , share with ptps the conserved active site signature cx5r  where the cys is the nucleophile in catalysis and arg binds the phosphate group in the substrate.
the inositol 5-phosphatases  are mg2+-dependent enzymes related to endonucleases .
pis are implicated in the regulation of a wide variety of cellular functions, including signal transduction, membrane dynamics, cytoskeleton arrangements, endocytosis, endosome trafficking, permeability and transport across membranes  <cit> .
pis are also important in facilitating phagocytosis of pathogenic bacteria and the subsequent phagosomal maturation leading to bacteria decay.
this process is mediated by pip and pip <dig>  <cit> .
pathogenic bacteria have evolved sophisticated strategies to escape the innate immune response, using pi metabolising proteins such as phosphatases and kinases to block phagosome maturation.
some of these bacterial phosphatases like sigd/sopb, ipgd and sapm have phosphoinositide activity resulting in the alteration of pi levels in the host membranes  <cit> .
mycobacterium tuberculosis  is an exceptionally successful pathogen that is able to survive in the host for a lifetime.
mtb enters macrophages in a phosphoinositide-3-kinase  dependent manner and uses lipid analogues   <cit>  and pi metabolising enzymes  to keep low levels of pip that prevent recruitment of rab <dig> and phagosomal maturation.
mptpb is a secreted phosphatase  <cit>  critical for mtb survival in host cells  <cit> .
we have recently shown that it has protein and pi phosphatase activity  <cit>  and could act together with sapm to control pip levels in the host.
mptpb has a unique active site p-loop motif : hcfagkdr, which is strictly conserved in other mycobacterial species, where the asp <dig> is an essential catalytic residue and lys <dig> is important for pi binding  <cit> .
this signature shares similarities with that of eukaryotic 3-ptases myotubularin , hcsdgwdr, and pten, hckagkgr, despite a low overall sequence homology .
the mtm/pten family of 3-ptases is conserved in mammals, drosophila melanogaster, caenorhabditis elegans, arabidopsis thaliana, yeast, kinetoplastids and several protozoa  <cit> .
however, homologous 3-ptases have not been identified in bacteria.
instead, some pathogenic bacteria have multifunctional 5-ptases such as sopb from salmonella and ipgd from shigella  <cit> .
the p-loop features and catalytic profile of mptpb, together with the lack of mtm and pten orthologues in bacteria suggested that mptpb like phosphatases may constitute an alternative type of pi phosphatase in prokaryotes.
this hypothesis prompted us to further investigate the existence of mptpb related protein sequences in other organisms.
in this study, bioinformatics explorations revealed a large family of mptpb related sequences present mostly in bacteria, with some in fungi and protozoa.
several of the sequences identified are found in recognised intracellular pathogens.
no mptpb homologues were found in archaea, plants or animals.
we characterised biochemically, selected proteins from different organisms  and confirmed that they have phosphatase activity towards both phosphorylated peptides and pis.
mutagenesis of conserved p-loop residues confirmed a cys based mechanism of catalysis, where the conserved asp is an essential catalytic residue.
furthermore, we provide evidence for the importance of key residues in the p-loop as determinants in pi substrate specificity.
substitution of p-loop residues to mimic that of eukaryotic 3-ptases, resulted in mutant enzymes with enhanced phosphatase activity and new poly-phosphate pi specificity.
phylogenetic analysis shows that this new family of pi phosphatases is not related to the classic eukaryotic lipid phosphatases like mtms/pten.
moreover, these phosphatases exhibit distinct and unusual features in their catalytic profile, sequence and domain organisation to other known pi and inositol phosphatases, for which we designated them as atypical lipid phosphatases .
RESULTS
bioinformatics analysis reveals a large family of mptpb related sequences
initial sequence homology searches were done using the full-length mptpb sequence.
these searches found  <dig> protein sequences in bacteria and one single sequence from fungi  .
no sequences were found in archaea, plants or animals.
some bacterial species contained more than one gene, like listeria monocytogenes, lactobacillus brevis and rhodopseudomonas palustris.
these  <dig> proteins are still uncharacterised functionally and one third of them are annotated as hypothetical proteins.
the remaining are annotated as dual-specificity phosphatases   or ptps  based on the presence of the cx5r motif, although there is not experimental evidence in the literature to support this assignment.
cyanobacteria
verrucomicrobia
parabasalidea
trypanosomatide
metazoa
sequences were identified in both gram positive  and gram negative  bacteria.
the mptpb p-loop signature is conserved in all sequences, except  <dig>  of which the cys residue is absent in  <dig> sequences  and the asp residue is absent in  <dig> sequences .
as both residues are important for catalysis  <cit>  these  <dig> sequences are probably inactive phosphatases and were excluded in subsequent analyses.
a multiple sequence alignment of the bacterial alps, highlights five main regions of conservation in the family .
the active site region shows the highest conservation, with the p-loop: hcxxgkdrg, and a contiguous extended signature, reminiscent of that found in dsps  <cit>  .
the p-loop motif is the landmark feature of this family of sequences and retains the unusual composition found in mptpb with the strictly conserved lys and asp, suggesting likely similarities in catalytic activity.
the four additional regions, c1-c <dig> and c <dig>  show good conservation  with some residues showing greater than 50% conservation, in agreement with those regions forming part of the main structural core of the phosphatase domain.
although the p-loop shares the cx5r signature with ptps, other key functional motifs in ptps are not present in the alps sequences.
these include the wpd motif, which contains the catalytic asp in ptps, the knry motif  and the q-loop, characteristic of tyrosine specific phosphatases  <cit> .
the absence of the wpd loop suggests that an alternative asp residue in alps may function as the cognate general acid in the dephosphorylation reaction.
the most likely candidate is the conserved asp in the p-loop, which is a catalytic residue in mptpb  <cit>
the alignment of alps highlighted that mycobacterial sequences contain two unique insertions flanking the active site motif: insertion  <dig>  and insertion  <dig>  .
in m. tuberculosis and m. bovis there is a single residue alteration in insertion  <dig>  while the corresponding region in other mycobacterial species is also unique but unrelated.
insertion  <dig> also has deviations between m. tuberculosis and m. bovis sequences and the remaining mycobacterial sequences.
these insertions appear to be important for functional regulation of mptpb as suggested from the crystallographic structures.
insertion  <dig> forms a lid that covers the active site and could control substrate access   <cit> , and insertion  <dig> changes conformation upon ligand binding  <cit> .
another distinct structural feature of mptpb is an alpha helix in the conserved motif c <dig> , which is not present in the structures of ptps  or dsps.
this helix provides a secondary binding pocket for inhibitory compounds  <cit>  and may be important in conferring selectivity.
the c <dig> motif is relatively conserved in the alp family with a high proportion of basic and acidic residues throughout, suggesting a good potential for helical content in this region.
the domain organisation of this new family shows a main difference with other eukaryotic lipid phosphatases, which generally contain a number of characteristic modules for membrane targeting and lipid binding .
in contrast, alps contain the phosphatase domain only and lack any classic lipid binding modules , suggesting that they use alternative mechanisms for lipid binding and membrane anchoring, maybe mediated by effector proteins.
phylogenetic analysis of mptpb related sequences
the occurrence of one single eukaryotic sequence in the blast search was intriguing, but the high conservation of the p-loop motif, suggested that perhaps other eukaryotic sequences may still retain this signature albeit with a low overall homology for the rest of the sequence.
to test this, we carried out a scanprosite search using the active site motif "hcxxgkdr".
the search returned a total of  <dig> sequences, of which  <dig> were bacterial and  <dig> were from lower eukaryotes .
the majority of eukaryotic sequences identified were fungi , with  <dig> leishmania sequences ,  <dig> sequence from caenorhabditis elegans and  <dig> sequence from trichomonas vaginalis.
a total of 31% of the sequences identified in scanprosite were also present in the blast hit list, indicating that in the remaining sequences the overall sequence identity was less than 30% to mptpb.
interestingly, more than  <dig> sequences of the total list belong to pathogens.
other known bacterial lipid and inositol phosphatase sequences were missing from our hit list.
this is the case of sopb/sigd and ipgd, with a slight different motif: ncksgkdr, and low sequence identity to mptpb .
also absent are the mammalian 4-ptases  and 5-ptases that lack the cx5r consensus motif.
a phylogenetic tree was compiled using the bacterial alp sequences from the blast search together with eukaryotic sequences from the scanprosite motif search, the phosphatase domain of classical eukaryotic lipid phosphatases  and the recently reported pip <dig> phosphatases from plants and kinetoplastids pfas  <cit> .
the analysis identified eleven clusters , with mtms, pten sequences  and pfas,  clustering independently from the alp sequences.
the bacterial and eukaryotic alps form separate clusters with bacterial sequences in groups 1- <dig>  and eukaryotic sequences in groups 7- <dig> .
all mycobacterial sequences cluster together in group  <dig> reflecting a highest homology within this subgroup.
gram positive  and gram negative  bacteria group separately with the exception of group  <dig>  which contains a mix of gram positive and negative bacteria.
group  <dig> is formed by yeast species, while the other groups contained a mix of species.
only aspergillus nidulans has sequences in different groups .
a number of sequences fail to cluster into any group , but remain independent of classic eukaryotic lipid phosphatases and pfas.
overall the phylogenetic tree emphasises the diversity of the apl sequences with clear separation between the bacterial and fungal/protozoan clusters and confirms that none of them are closely related to classical eukaryotic 3-ptases typified by mtms and pten.
mptpb related sequences exhibit both protein phosphatase and lipid phosphatase activity
we previously reported that mptpb had both protein phosphatase and lipid phosphatase activity.
we also established that cys <dig>  lys <dig>  asp <dig> and arg <dig> in the p-loop, are important residues involved in catalysis or substrate binding  <cit> .
the conservation of these residues in the alp sequences, suggested they could have a similar enzymatic profile, which could be relevant to pi metabolism.
to test this hypothesis, two prokaryotic sequences and one eukaryotic sequence from our hit list were selected for enzymatic analysis, all of them belonging to intracellular pathogens; lmo <dig> and lmo <dig> from l. monocytogenes and lm <dig> from l. major.
these sequences occupy different locations within the phylogenetic tree  and had sequence identities of only 22-24% to mptpb.
despite their relatively poor sequence homology the p-loop motif is almost identical to mptpb with only one substitution of f for t in position two.
biochemical analyses confirmed that all three proteins had phosphatase activity, with the ability to dephosphorylate the generic phosphatase substrate pnpp.
lmo <dig> shows the highest specific activity  of  <dig>  nmoles min- <dig> mg- <dig>  followed by lmo <dig> of  <dig>  nmoles min- <dig> mg- <dig> and lm <dig> of  <dig>  nmoles min- <dig> mg- <dig>  comparable to mptpb of  <dig>  nmoles min- <dig> mg- <dig> and the control protein phosphatase, tbptp <dig>   <dig>  nmoles min- <dig> mg- <dig>  in addition, they all dephosphorylate p-tyr peptides, with lmo <dig> showing the greatest sa of  <dig>  nmoles min- <dig> mg- <dig>  whilst lm <dig> showed considerably lower activity,  <dig>  nmoles min- <dig> mg- <dig> .
dephosphorylation of p-ser and p-thr peptides was significantly lower for all phosphatases, with mptpb having the highest sa of  <dig>  nmoles min- <dig> mg- <dig>  and lm <dig> the lowest at  <dig>  nmoles min- <dig> mg- <dig> .
the dephosphorylation ratio of py: ps/t peptides for mptpb and lmo <dig> is  <dig> :  <dig> and  <dig> :  <dig>  respectively, comparable to the dsps such as vhr , in contrast to tyr specific ptps like hptp1b that show a ratio greater than  <dig>  ×  <dig> :  <dig>  <cit> .
based on the results it appears that lmo <dig> and lm <dig> have a stronger preference for p-tyr peptides while lmo <dig> exhibits a similar activity profile for the phospho-peptides to mptpb.
we further tested if these proteins had phosphatase activity towards pis, as the p-loop signature seem to predict.
we tested all seven pi forms and we found that the control protein tyrosine phosphatase, tbptp <dig>  had little activity of towards any of the pis .
lmo <dig> exhibited high activity towards pip <dig>  and mono-phosphate pis pip , pip  and pip , with no detectable activity towards the remaining pis .
both lmo <dig> and lm <dig> show also activity towards mono-phosphorylated pis  but not for pip <dig> .
although the activity of lm <dig> towards pis is lower than mptpb, it was of a similar magnitude to p-tyr peptides indicating no preferred substrate.
this is in contrast to lmo <dig>  which has a stronger preference for peptide substrates .
none of the phosphatases were able to dephosphorylate contiguous di-phosphate pis: pip <dig>  pip <dig> and the tri-phosphate pip <dig>  showing a similar catalytic profile to mptpb  <cit> .
other phosphoester substrates were tested and confirmed the preference of mptpb and lmo <dig> for the p-tyr amino acid over p-ser and p-thr and showed modest activity for 5'amp and 5'imp .
dephosphorylation of pnpp is shown as a reference.
n.d, no activity detected.
p-loop composition in lipid phosphatases: identification of determinants for substrate specificity
comparison of the p-loop composition between different pi phosphatases reveals two distinct groups.
group  <dig>  with pten, arabidopsis thaliana pfa , 4-ptases and sopb/igpd, which have three or more basic residues, and group  <dig>  with mptpb, mtms, lmo <dig> and lm <dig>  that have only one or two basic residues and the conserved catalytic asp in position six .
all of them, except mtms, have a conserved lys residue in position  <dig>  important in substrate binding  <cit> .
group  <dig> enzymes have an additional lys in position  <dig>  which appears to correlate with the ability to dephosphorylate pip <dig> and poly-phosphoinositols.
group  <dig> enzymes lack this extra lys and are unable to dephosphorylate pip <dig> and most di-phosphorylated pis, favouring instead mono-phosphorylated pi substrates  <cit> .
the absence of this extra basic residue, presumably important to compensate for additional negative charge in poly-phosphate substrates, may account for the lack of activity towards those pis in group  <dig>  to test this hypothesis and to corroborate the functional role of the conserved lys and asp at positions  <dig> and  <dig> we produced a number of p-loop single mutants.
we previously established the function of asp <dig> in mptpb as an essential residue in catalysis  <cit> .
mutagenesis of the homologue asp residue  in lmo <dig> and lm <dig> to ala resulted in a significant loss in activity, with < 10% of wt activity .
another residue in the p-loop, lys <dig> in mptpb, is involved in substrate binding and particularly important for dephosphorylation of pip <dig>  <cit> .
mutation of the cognates lmo1800-lys <dig> and lm1-lys <dig> to ala resulted in a significant loss in activity towards pip <dig> but retained 10% of the wt activity for pip .
having established the importance of residues in position  <dig> and  <dig>  we then wanted to explore the importance of lys in position  <dig> in substrate specificity.
we mutated position  <dig> residues in mptpb and related enzymes to lys to see if we could convert a group  <dig> lipid phosphatase into a group  <dig> phosphatase with similar substrate specificity.
we found that in mptpb, the residue in position  <dig>  phe <dig>  when mutated to lys, results in an mutant enzyme with new activity for di-phosphorylated substrates pip <dig>  and pip <dig>  and even shows activity towards the tri-phosphorylated pip <dig>  .
note that the wt enzyme does not dephosphorylate any of these substrates.
mutation of the equivalent position in lmo <dig>  to lys resulted in increased dephosphorylation of pip , a 3-fold increase of activity towards and pip <dig> with low activity for pip <dig> .
for lm <dig>  mutation of thr <dig> to lys, also shows an increase  in activity for pip but not towards di- or tri-phosphorylated pis, suggesting that other factors and possibly conformational arrangements in the active site of lmo <dig> and lm <dig> determine substrate preference.
discussion
in this study, we have used the p-loop signature of the bacterial lipid phosphatase mptpb as a "diagnostic motif" to identify related putative lipid phosphatases.
we found a whole new family of predicted phosphatases in bacteria, with some instances in fungi and protozoa.
interestingly, no homologues were found in other organisms such as archaea, plants or animals.
these proteins have not previously been characterised functionally and at least one third of them are not annotated.
the existence of such a large family of putative lipid phosphatases in bacteria is unexpected.
very few bacteria, with the exception of actinobacteria, produce inositols or phosphoinositides, therefore they generally lack the enzymes involved in inositol metabolism  <cit> .
on the other hand, some pathogenic bacteria secrete pi metabolising enzymes, like kinases and phosphatases, that they use to subvert pi signalling in the host cells, promoting virulence and survival in the infected cells  <cit> .
although the biological role of the alps identified in this study remains unknown, it is interesting to note that many of the sequences belong to pathogenic microorganisms, such as listeria, yersinia, klebsiella, clostridium, leishmania, candida, and aspergillus.
phylogenetic analysis showed a number of clusters, distinct for the bacterial and fungal/protozoan species, and that alps are clearly different to classic mammalian lipid 3-ptases, mtms and pten or the pfas.
additionally, alps lack the typical lipid anchoring domains commonly found in mammalian lipid phosphatases.
biochemical characterisation of selected sequences from the intracellular pathogens l. monocytogenes and l. major, confirmed our prediction that these are active phosphatases, able to dephosphorylate phosphoinositides.
a broader pi activity in alps is also in contrast with the more restrictive specificity observed in mtms/pten 3-ptases and the capability to dephosphorylate di- and tri-phosphoinositides found in the sopb/ipgd 5-ptases.
clear differences were observed in the activity profile between the two proteins in l. monocytogenes, lmo <dig> and lmo <dig>  while lmo <dig> has preference for peptide substrates, lmo <dig> exhibits a similar activity profile as mptpb, with dsp activity and broad pi specificity.
this suggests that the two lmo proteins could have distinct functional roles and provides an explanation for the presence of the two genes in listeria.
the catalytic function of critical p-loop residues in classic eukaryotic lipid phosphatases has previously been determined.
in mtms, position  <dig> in the p-loop is occupied by an asp residue , which functions as the general acid in catalysis  <cit> .
similar findings were reported for mptpb  <cit> .
here we established the importance of the conserved asp in the alp proteins as mutation to ala results in mutant enzymes with compromised activity, suggesting a similar role of this residue in catalysis as shown for mptpb and mtms.
in pten, the presence of several basic residues  facilitates the binding of highly negatively charged pi substrates and in particular of pip <dig>  its preferred substrate  <cit> .
a similar motif, cksxkdr, is also found in sopb/ipgd inositol and mammalian 4-ptases that have a preference for poly-phosphate pis.
however, alps, contain only two basic residues  in the p-loop and they dephosphorylate preferably mono-phosphoralyted pis with no activity towards pip <dig>  here we have shown that firstly, the conserved lys in position  <dig> of the p-loop is important for efficient dephosphorylation of pip <dig>  and probably involved in substrate binding, consistent with the proposed role of the cognate lys <dig> in mptpb  <cit>  and lys <dig> in pten  <cit> .
secondly, we showed that the number of basic residues is critical to dephosphorylate poly-phosphate pis.
we mutated the residues in position  <dig> of the p-loop to lys to mimic the extra basic residue in pten and sopb.
mutation of phe <dig> in mptpb, thr <dig> in lmo <dig> and thr <dig> in lm <dig> to lys resulted in enhanced activity towards mono- and di-phospho pis in lmo <dig>  and mptpb, and new activity towards pip <dig> for mptpb .
this is an important finding as, historically, the role of p-loop residues has been restricted to enzyme catalysis and catalytic efficiency.
in this study we demonstrate that single amino acid substitutions can result in subtle changes in substrate preferences and supports the notion that the active site loop sequence can be used as diagnostic of substrate specificity.
new insights regarding the structural constrains that determine substrate binding in this new family of phosphatases should be revealed when the three-dimensional structures are determined.
CONCLUSIONS
overall, this new family of enzymes shows both distinct sequence features and biochemical characteristics.
alps have a unique p-loop signature and exhibit broad pi specificity.
in addition, they have low sequence homology and different domain organisation to classic lipid phosphatases.
the biological role of these proteins remains to be elucidated, but their potential role in pi metabolism suggests interesting links with established mechanisms of pathogenesis in other microorganisms such as m. tuberculosis, leishmania, listeria, salmonella, and shigella  <cit> , and a potential to be exploited as targets in anti-infective treatments given their low homology to any other human phosphatases.

 phosphatidylinositols are important metabolites present in animal cell membranes. they can be phosphorylated at three main positions of the inositol head to generate seven different phosphoinositides. their metabolism is tightly controlled by phospholipases that in some instances generate second messengers such as inositol,5-trisphosphate p3) and diacylglycerol. in addition, kinases and phosphatases control the levels of the mono, di- or tri-phosphorylated forms. pi phosphatases belong to different families of enzymes; pten and myotubularin inositol 3-phosphatases belong to the protein tyrosine phosphatase superfamily and have both protein and lipid phosphatase activities. inositol 4-phosphatases, share with ptps the conserved active site signature cx5r where the cys is the nucleophile in catalysis and arg binds the phosphate group in the substrate. the inositol 5-phosphatases are mg2+-dependent enzymes related to endonucleases. pis are implicated in the regulation of a wide variety of cellular functions, including signal transduction, membrane dynamics, cytoskeleton arrangements, endocytosis, endosome trafficking, permeability and transport across membranes. pis are also important in facilitating phagocytosis of pathogenic bacteria and the subsequent phagosomal maturation leading to bacteria decay. this process is mediated by pip and pip. pathogenic bacteria have evolved sophisticated strategies to escape the innate immune response, using pi metabolising proteins such as phosphatases and kinases to block phagosome maturation. some of these bacterial phosphatases like sigd/sopb, ipgd and sapm have phosphoinositide activity resulting in the alteration of pi levels in the host membranes. mycobacterium tuberculosis is an exceptionally successful pathogen that is able to survive in the host for a lifetime. mtb enters macrophages in a phosphoinositide-3-kinase dependent manner and uses lipid analogues and pi metabolising enzymes to keep low levels of pip that prevent recruitment of rab and phagosomal maturation. mptpb is a secreted phosphatase critical for mtb survival in host cells. we have recently shown that it has protein and pi phosphatase activity and could act together with sapm to control pip levels in the host. mptpb has a unique active site p-loop motif: hcfagkdr, which is strictly conserved in other mycobacterial species, where the asp is an essential catalytic residue and lys is important for pi binding. this signature shares similarities with that of eukaryotic 3-ptases myotubularin, hcsdgwdr, and pten, hckagkgr, despite a low overall sequence homology. the mtm/pten family of 3-ptases is conserved in mammals, drosophila melanogaster, caenorhabditis elegans, arabidopsis thaliana, yeast, kinetoplastids and several protozoa. however, homologous 3-ptases have not been identified in bacteria. instead, some pathogenic bacteria have multifunctional 5-ptases such as sopb from salmonella and ipgd from shigella. the p-loop features and catalytic profile of mptpb, together with the lack of mtm and pten orthologues in bacteria suggested that mptpb like phosphatases may constitute an alternative type of pi phosphatase in prokaryotes. this hypothesis prompted us to further investigate the existence of mptpb related protein sequences in other organisms. in this study, bioinformatics explorations revealed a large family of mptpb related sequences present mostly in bacteria, with some in fungi and protozoa. several of the sequences identified are found in recognised intracellular pathogens. no mptpb homologues were found in archaea, plants or animals. we characterised biochemically, selected proteins from different organisms and confirmed that they have phosphatase activity towards both phosphorylated peptides and pis. mutagenesis of conserved p-loop residues confirmed a cys based mechanism of catalysis, where the conserved asp is an essential catalytic residue. furthermore, we provide evidence for the importance of key residues in the p-loop as determinants in pi substrate specificity. substitution of p-loop residues to mimic that of eukaryotic 3-ptases, resulted in mutant enzymes with enhanced phosphatase activity and new poly-phosphate pi specificity. phylogenetic analysis shows that this new family of pi phosphatases is not related to the classic eukaryotic lipid phosphatases like mtms/pten. moreover, these phosphatases exhibit distinct and unusual features in their catalytic profile, sequence and domain organisation to other known pi and inositol phosphatases, for which we designated them as atypical lipid phosphatases.  bioinformatics analysis reveals a large family of mptpb related sequences initial sequence homology searches were done using the full-length mptpb sequence. these searches found protein sequences in bacteria and one single sequence from fungi. no sequences were found in archaea, plants or animals. some bacterial species contained more than one gene, like listeria monocytogenes, lactobacillus brevis and rhodopseudomonas palustris. these proteins are still uncharacterised functionally and one third of them are annotated as hypothetical proteins. the remaining are annotated as dual-specificity phosphatases or ptps based on the presence of the cx5r motif, although there is not experimental evidence in the literature to support this assignment. cyanobacteria verrucomicrobia parabasalidea trypanosomatide metazoa sequences were identified in both gram positive and gram negative bacteria. the mptpb p-loop signature is conserved in all sequences, except of which the cys residue is absent in sequences and the asp residue is absent in sequences. as both residues are important for catalysis these sequences are probably inactive phosphatases and were excluded in subsequent analyses. a multiple sequence alignment of the bacterial alps, highlights five main regions of conservation in the family. the active site region shows the highest conservation, with the p-loop: hcxxgkdrg, and a contiguous extended signature, reminiscent of that found in dsps. the p-loop motif is the landmark feature of this family of sequences and retains the unusual composition found in mptpb with the strictly conserved lys and asp, suggesting likely similarities in catalytic activity. the four additional regions, c1-c and c show good conservation with some residues showing greater than 50% conservation, in agreement with those regions forming part of the main structural core of the phosphatase domain. although the p-loop shares the cx5r signature with ptps, other key functional motifs in ptps are not present in the alps sequences. these include the wpd motif, which contains the catalytic asp in ptps, the knry motif and the q-loop, characteristic of tyrosine specific phosphatases. the absence of the wpd loop suggests that an alternative asp residue in alps may function as the cognate general acid in the dephosphorylation reaction. the most likely candidate is the conserved asp in the p-loop, which is a catalytic residue in mptpb the alignment of alps highlighted that mycobacterial sequences contain two unique insertions flanking the active site motif: insertion and insertion. in m. tuberculosis and m. bovis there is a single residue alteration in insertion while the corresponding region in other mycobacterial species is also unique but unrelated. insertion also has deviations between m. tuberculosis and m. bovis sequences and the remaining mycobacterial sequences. these insertions appear to be important for functional regulation of mptpb as suggested from the crystallographic structures. insertion forms a lid that covers the active site and could control substrate access, and insertion changes conformation upon ligand binding. another distinct structural feature of mptpb is an alpha helix in the conserved motif c, which is not present in the structures of ptps or dsps. this helix provides a secondary binding pocket for inhibitory compounds and may be important in conferring selectivity. the c motif is relatively conserved in the alp family with a high proportion of basic and acidic residues throughout, suggesting a good potential for helical content in this region. the domain organisation of this new family shows a main difference with other eukaryotic lipid phosphatases, which generally contain a number of characteristic modules for membrane targeting and lipid binding. in contrast, alps contain the phosphatase domain only and lack any classic lipid binding modules, suggesting that they use alternative mechanisms for lipid binding and membrane anchoring, maybe mediated by effector proteins. phylogenetic analysis of mptpb related sequences the occurrence of one single eukaryotic sequence in the blast search was intriguing, but the high conservation of the p-loop motif, suggested that perhaps other eukaryotic sequences may still retain this signature albeit with a low overall homology for the rest of the sequence. to test this, we carried out a scanprosite search using the active site motif "hcxxgkdr". the search returned a total of sequences, of which were bacterial and were from lower eukaryotes. the majority of eukaryotic sequences identified were fungi, with leishmania sequences, sequence from caenorhabditis elegans and sequence from trichomonas vaginalis. a total of 31% of the sequences identified in scanprosite were also present in the blast hit list, indicating that in the remaining sequences the overall sequence identity was less than 30% to mptpb. interestingly, more than sequences of the total list belong to pathogens. other known bacterial lipid and inositol phosphatase sequences were missing from our hit list. this is the case of sopb/sigd and ipgd, with a slight different motif: ncksgkdr, and low sequence identity to mptpb. also absent are the mammalian 4-ptases and 5-ptases that lack the cx5r consensus motif. a phylogenetic tree was compiled using the bacterial alp sequences from the blast search together with eukaryotic sequences from the scanprosite motif search, the phosphatase domain of classical eukaryotic lipid phosphatases and the recently reported pip phosphatases from plants and kinetoplastids pfas. the analysis identified eleven clusters, with mtms, pten sequences and pfas, clustering independently from the alp sequences. the bacterial and eukaryotic alps form separate clusters with bacterial sequences in groups 1- and eukaryotic sequences in groups 7-. all mycobacterial sequences cluster together in group reflecting a highest homology within this subgroup. gram positive and gram negative bacteria group separately with the exception of group which contains a mix of gram positive and negative bacteria. group is formed by yeast species, while the other groups contained a mix of species. only aspergillus nidulans has sequences in different groups. a number of sequences fail to cluster into any group, but remain independent of classic eukaryotic lipid phosphatases and pfas. overall the phylogenetic tree emphasises the diversity of the apl sequences with clear separation between the bacterial and fungal/protozoan clusters and confirms that none of them are closely related to classical eukaryotic 3-ptases typified by mtms and pten. mptpb related sequences exhibit both protein phosphatase and lipid phosphatase activity we previously reported that mptpb had both protein phosphatase and lipid phosphatase activity. we also established that cys lys asp and arg in the p-loop, are important residues involved in catalysis or substrate binding. the conservation of these residues in the alp sequences, suggested they could have a similar enzymatic profile, which could be relevant to pi metabolism. to test this hypothesis, two prokaryotic sequences and one eukaryotic sequence from our hit list were selected for enzymatic analysis, all of them belonging to intracellular pathogens; lmo and lmo from l. monocytogenes and lm from l. major. these sequences occupy different locations within the phylogenetic tree and had sequence identities of only 22-24% to mptpb. despite their relatively poor sequence homology the p-loop motif is almost identical to mptpb with only one substitution of f for t in position two. biochemical analyses confirmed that all three proteins had phosphatase activity, with the ability to dephosphorylate the generic phosphatase substrate pnpp. lmo shows the highest specific activity of nmoles min- mg- followed by lmo of nmoles min- mg- and lm of nmoles min- mg- comparable to mptpb of nmoles min- mg- and the control protein phosphatase, tbptp nmoles min- mg- in addition, they all dephosphorylate p-tyr peptides, with lmo showing the greatest sa of nmoles min- mg- whilst lm showed considerably lower activity, nmoles min- mg-. dephosphorylation of p-ser and p-thr peptides was significantly lower for all phosphatases, with mptpb having the highest sa of nmoles min- mg- and lm the lowest at nmoles min- mg-. the dephosphorylation ratio of py: ps/t peptides for mptpb and lmo is: and: respectively, comparable to the dsps such as vhr, in contrast to tyr specific ptps like hptp1b that show a ratio greater than ×:. based on the results it appears that lmo and lm have a stronger preference for p-tyr peptides while lmo exhibits a similar activity profile for the phospho-peptides to mptpb. we further tested if these proteins had phosphatase activity towards pis, as the p-loop signature seem to predict. we tested all seven pi forms and we found that the control protein tyrosine phosphatase, tbptp had little activity of towards any of the pis. lmo exhibited high activity towards pip and mono-phosphate pis pip, pip and pip, with no detectable activity towards the remaining pis. both lmo and lm show also activity towards mono-phosphorylated pis but not for pip. although the activity of lm towards pis is lower than mptpb, it was of a similar magnitude to p-tyr peptides indicating no preferred substrate. this is in contrast to lmo which has a stronger preference for peptide substrates. none of the phosphatases were able to dephosphorylate contiguous di-phosphate pis: pip pip and the tri-phosphate pip showing a similar catalytic profile to mptpb. other phosphoester substrates were tested and confirmed the preference of mptpb and lmo for the p-tyr amino acid over p-ser and p-thr and showed modest activity for 5'amp and 5'imp. dephosphorylation of pnpp is shown as a reference. n.d, no activity detected. p-loop composition in lipid phosphatases: identification of determinants for substrate specificity comparison of the p-loop composition between different pi phosphatases reveals two distinct groups. group with pten, arabidopsis thaliana pfa, 4-ptases and sopb/igpd, which have three or more basic residues, and group with mptpb, mtms, lmo and lm that have only one or two basic residues and the conserved catalytic asp in position six. all of them, except mtms, have a conserved lys residue in position important in substrate binding. group enzymes have an additional lys in position which appears to correlate with the ability to dephosphorylate pip and poly-phosphoinositols. group enzymes lack this extra lys and are unable to dephosphorylate pip and most di-phosphorylated pis, favouring instead mono-phosphorylated pi substrates. the absence of this extra basic residue, presumably important to compensate for additional negative charge in poly-phosphate substrates, may account for the lack of activity towards those pis in group to test this hypothesis and to corroborate the functional role of the conserved lys and asp at positions and we produced a number of p-loop single mutants. we previously established the function of asp in mptpb as an essential residue in catalysis. mutagenesis of the homologue asp residue in lmo and lm to ala resulted in a significant loss in activity, with < 10% of wt activity. another residue in the p-loop, lys in mptpb, is involved in substrate binding and particularly important for dephosphorylation of pip. mutation of the cognates lmo1800-lys and lm1-lys to ala resulted in a significant loss in activity towards pip but retained 10% of the wt activity for pip. having established the importance of residues in position and we then wanted to explore the importance of lys in position in substrate specificity. we mutated position residues in mptpb and related enzymes to lys to see if we could convert a group lipid phosphatase into a group phosphatase with similar substrate specificity. we found that in mptpb, the residue in position phe when mutated to lys, results in an mutant enzyme with new activity for di-phosphorylated substrates pip and pip and even shows activity towards the tri-phosphorylated pip. note that the wt enzyme does not dephosphorylate any of these substrates. mutation of the equivalent position in lmo to lys resulted in increased dephosphorylation of pip, a 3-fold increase of activity towards and pip with low activity for pip. for lm mutation of thr to lys, also shows an increase in activity for pip but not towards di- or tri-phosphorylated pis, suggesting that other factors and possibly conformational arrangements in the active site of lmo and lm determine substrate preference. discussion in this study, we have used the p-loop signature of the bacterial lipid phosphatase mptpb as a "diagnostic motif" to identify related putative lipid phosphatases. we found a whole new family of predicted phosphatases in bacteria, with some instances in fungi and protozoa. interestingly, no homologues were found in other organisms such as archaea, plants or animals. these proteins have not previously been characterised functionally and at least one third of them are not annotated. the existence of such a large family of putative lipid phosphatases in bacteria is unexpected. very few bacteria, with the exception of actinobacteria, produce inositols or phosphoinositides, therefore they generally lack the enzymes involved in inositol metabolism. on the other hand, some pathogenic bacteria secrete pi metabolising enzymes, like kinases and phosphatases, that they use to subvert pi signalling in the host cells, promoting virulence and survival in the infected cells. although the biological role of the alps identified in this study remains unknown, it is interesting to note that many of the sequences belong to pathogenic microorganisms, such as listeria, yersinia, klebsiella, clostridium, leishmania, candida, and aspergillus. phylogenetic analysis showed a number of clusters, distinct for the bacterial and fungal/protozoan species, and that alps are clearly different to classic mammalian lipid 3-ptases, mtms and pten or the pfas. additionally, alps lack the typical lipid anchoring domains commonly found in mammalian lipid phosphatases. biochemical characterisation of selected sequences from the intracellular pathogens l. monocytogenes and l. major, confirmed our prediction that these are active phosphatases, able to dephosphorylate phosphoinositides. a broader pi activity in alps is also in contrast with the more restrictive specificity observed in mtms/pten 3-ptases and the capability to dephosphorylate di- and tri-phosphoinositides found in the sopb/ipgd 5-ptases. clear differences were observed in the activity profile between the two proteins in l. monocytogenes, lmo and lmo while lmo has preference for peptide substrates, lmo exhibits a similar activity profile as mptpb, with dsp activity and broad pi specificity. this suggests that the two lmo proteins could have distinct functional roles and provides an explanation for the presence of the two genes in listeria. the catalytic function of critical p-loop residues in classic eukaryotic lipid phosphatases has previously been determined. in mtms, position in the p-loop is occupied by an asp residue, which functions as the general acid in catalysis. similar findings were reported for mptpb. here we established the importance of the conserved asp in the alp proteins as mutation to ala results in mutant enzymes with compromised activity, suggesting a similar role of this residue in catalysis as shown for mptpb and mtms. in pten, the presence of several basic residues facilitates the binding of highly negatively charged pi substrates and in particular of pip its preferred substrate. a similar motif, cksxkdr, is also found in sopb/ipgd inositol and mammalian 4-ptases that have a preference for poly-phosphate pis. however, alps, contain only two basic residues in the p-loop and they dephosphorylate preferably mono-phosphoralyted pis with no activity towards pip here we have shown that firstly, the conserved lys in position of the p-loop is important for efficient dephosphorylation of pip and probably involved in substrate binding, consistent with the proposed role of the cognate lys in mptpb and lys in pten. secondly, we showed that the number of basic residues is critical to dephosphorylate poly-phosphate pis. we mutated the residues in position of the p-loop to lys to mimic the extra basic residue in pten and sopb. mutation of phe in mptpb, thr in lmo and thr in lm to lys resulted in enhanced activity towards mono- and di-phospho pis in lmo and mptpb, and new activity towards pip for mptpb. this is an important finding as, historically, the role of p-loop residues has been restricted to enzyme catalysis and catalytic efficiency. in this study we demonstrate that single amino acid substitutions can result in subtle changes in substrate preferences and supports the notion that the active site loop sequence can be used as diagnostic of substrate specificity. new insights regarding the structural constrains that determine substrate binding in this new family of phosphatases should be revealed when the three-dimensional structures are determined.  overall, this new family of enzymes shows both distinct sequence features and biochemical characteristics. alps have a unique p-loop signature and exhibit broad pi specificity. in addition, they have low sequence homology and different domain organisation to classic lipid phosphatases. the biological role of these proteins remains to be elucidated, but their potential role in pi metabolism suggests interesting links with established mechanisms of pathogenesis in other microorganisms such as m. tuberculosis, leishmania, listeria, salmonella, and shigella, and a potential to be exploited as targets in anti-infective treatments given their low homology to any other human phosphatases. 
we used this p-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. mptpb, a virulence factor from mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site p-loop signature hcxxgkdr that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. furthermore, we provide experimental evidence that the number of basic residues in the p-loop is critical in determining activity towards poly-phosphoinositides. however, biochemical characterisation of those from listeria monocytogenes and leishmania major, demonstrated that, like mptpb, they have phosphatase activity towards phosphoinositides. phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. this study provides a foundation for examining the biological role of this new family of phosphatases and their potential as pharmaceutical targets against infectious diseases. phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. this new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases and they have no homologues in humans. modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. mutagenesis studies established that the conserved asp and lys in the p-loop signature are important in catalysis and substrate binding respectively.
 phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. mptpb, a virulence factor from mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site p-loop signature hcxxgkdr that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. we used this p-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms.  we found more than uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. many of the sequences identified belong to recognised human pathogens. interestingly, no homologues were found in any other organisms including archaea, plants, or animals. phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. however, biochemical characterisation of those from listeria monocytogenes and leishmania major, demonstrated that, like mptpb, they have phosphatase activity towards phosphoinositides. mutagenesis studies established that the conserved asp and lys in the p-loop signature are important in catalysis and substrate binding respectively. furthermore, we provide experimental evidence that the number of basic residues in the p-loop is critical in determining activity towards poly-phosphoinositides.  this new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases and they have no homologues in humans. this study provides a foundation for examining the biological role of this new family of phosphatases and their potential as pharmaceutical targets against infectious diseases. 