BACKGROUND
reversible phosphorylation of proteins is a ubiquitous mechanism, indispensable for regulation of virtually any cellular function.
therefore, protein kinases and phosphatases are of paramount importance for normal functioning of all metabolic and signalling pathways.
in eukaryotes, ppp family is one of the two known protein phosphatase families specific for ser and thr  <cit> .
unlike eukaryotic  ppp phosphatases, bacterial members of the family have broad substrate specificity  <cit>  or may even be tyr-specific  <cit> .
moreover, one group of bacterial ppps are diadenosine tetraphosphatases  <cit> .
unlike eukaryotes, in prokaryotes ppp phosphatases appear to be facultative, since entirely sequenced genomes of some bacteria and archaea do not encode them  <cit> .
nevertheless, when present, they appear to play essential roles  <cit> .
three motifs ), highly conserved in the n-terminal subdomains of the catalytic domains of all ppp phosphatases  <cit> , contain most residues which coordinate metal ions in the active centre  <cit>  and are considered as the signature of the ppp family.
in a previous work  <cit>  we identified an unusual cdna fragment from a moss physcomitrella patens, showing no similarities to the known ppp phosphatases beyond the presence of the gdxhg and gdxxdrg motifs.
detection of homologous cdna sequences from arabidopsis and rice suggested the presence of an unknown ppp group in plants, distinct from "conventional" eukaryotic ppp phosphatases  <cit> .
we have now taken advantage of a much greater representation  of sequence databases for various species to further explore this initial observation.
we present the evidence for the existence in eukaryotes of three "non-conventional" branches of the ppp family.
we also identify a previously unrecognised conserved motif in the ppp catalytic domain, which can be used as a signature of "bacterial"-type ppp phosphatases.
RESULTS
"rhizobiales / rhodobacterales / rhodospirillaceae – like" ppp phosphatases in plants
two arabidopsis sequences, at3g <dig> and at3g <dig>  were retrieved using the p. patens fragment  <cit>  as a query in tblastn searches.
they share 85% identity with each other at the protein level .
both genes are transcribed, since full-length cdnas have been detected in a large-scale transcription study  <cit> .
they are arranged on chromosome  <dig> in tandem, suggesting their origin by a recent duplication.
a number of ests from other plant species  were also detected by tblastn searches, which in most cases provide no evidence for the existence of more than one isoform.
among prokaryotes, related sequences were detected in some α-proteobacteria, the closest matches being with rhizobiales, rhodobacterales and rhodospirillaceae.
therefore, we designate this group as rhizobiales / rhodobacterales / rhodospirillaceae-like phosphatases, or rhilphs , and the two arabidopsis genes products at3g <dig> and at3g <dig> as "rhizobiales-like" phosphatases  <dig>  and  <dig> , respectively.
* this phosphatase is unlikely to be catalytically active due to replacements of essential residues in the active centre.
structural features of rhilphs
all residues that are expected to bind metal ions in the catalytic centre are conserved in rhilphs .
rhilphs do not have n- or c-terminal extensions beyond their catalytic domains, which in many ppp phosphatases have regulatory function and / or interact with regulatory proteins / subunits.
instead, they have characteristic inserts between the conserved motifs gnh and hag .
notably, much shorter inserts are found at a similar position in α-proteobacterial phosphatases .
inserts in both plant rhilphs and α-proteobacterial phosphatases contain a conserved motif lxxaxpxxh .
similarly to bacteriophage λphosphatase , rhilphs lack a region corresponding to β <dig>  β <dig> and α <dig> of eukaryotic ppps  <cit> .
like bacterial ppp phosphatases, rhilphs do not have a sapny motif, conserved in the β12-β <dig> loop of eukaryotic ppps.
analysis of rhilph sequences did not reveal targeting or signal peptides.
while this work was in progress, a phosphatase encoded by an arabidopsis gene at1g <dig> was reported in an independent study  <cit> .
"shewanella-like" ppp phosphatases in plants, red algae, fungi and unicellular parasites
we undertook further tblastn searches using full-length arabidopsis rlp <dig> as a query to see whether arabidopsis genome encodes additional "bacterial-like" ppps.
these searches identified two more genes for putative ppp phosphatases, only distantly related to rhilphs and to any other members of the family .
one of these genes is at1g <dig>  at least three different predicted products of this gene could be found in protein databases.
on the basis of comparison with est sequences, we consider as the correct structure that of q8ry <dig> with asp and gly at positions  <dig> and  <dig>  respectively .
the other detected gene, at1g <dig>  is also represented in the databases by three distinct deduced proteins.
comparison with a. thaliana ests confirms q944l <dig> as the correct structure.
genomic and est database searches provided ample evidence for the presence of related phosphatases in a number of green plants, including multiple angiosperm species, pine and a unicellular green alga chlamydomonas reinhardtii .
related sequences were also identified in some fungi , in apicomplexa, trypanosomatidae, and in a red alga porphyra yezoensis .
the most closely related prokaryotic phosphatases were detected in myxococcus xanthus  and psychrophilic bacteria alteromonadales : uncharacterised phosphatases from shewanella oneidensis and colwellia psychrerythraea and a tyr-specific phosphatase ppi from shewanella sp.
<cit> .
therefore, we designate this phosphatase group as "shewanella-like" phosphatases, or shelphs, and the products of the two prototype arabidopsis genes at1g <dig> and at1g <dig> as "shewanella-like" phosphatases  <dig>  and  <dig> , respectively.
structural features of shelphs
like in rhilphs, all residues that are expected to bind metal ions in the catalytic centre are conserved in shelphs .
another feature common with rhilphs is the presence of inserts  between the gnhe and hg motifs , which are especially long in plant shelphs.
however, these inserts share no sequence similarity between rhilphs and shelphs and probably appeared in the two phosphatase groups independently.
like in rhilphs, a region corresponding to β <dig>  β <dig> and α <dig> of eukaryotic ppps is absent in shelphs, and the primary structure of the region corresponding to the β12-β <dig> loop is similar to that of typical bacterial ppps.
a.thaliana slp <dig> and corresponding shelph isoform from rice have chloroplast targeting sequence, which could not be detected in a.thaliana slp <dig> and corresponding isoform from medicago truncatula.
eukaryotic ppps distantly related to bacterial diadenosine tetraphosphatases
identification of rhilphs and shelphs prompted us to perform extensive searches of eukaryotic sequence databases.
these searches revealed the existence of other "bacterial-like" ppp phosphatases throughout eukaryotes.
sequences only distantly related to rhilphs, shelphs or any other ppp phosphatases were detected in several fungi , in trypanosomatidae, a tick amblyomma, an ascidian ciona, chlamydomonas, pine and diatoms fragilariopsis cylindrus and phaeodactylum tricornutum.
blast searches using these sequences revealed that all of them share higher similarity to bacterial diadenosine tetraphosphatases  than to other ppp groups.
therefore, we tentatively designate them as apah-like phosphatases, or alphs .
alphs share a distinctive common structural feature.
in the gdxxdrg motif, absolutely conserved in other ppps, the second asp  is replaced by a neutral amino acid, and the arg residue  is replaced, with one exception, by lys .
the former of these replacements is also found in apah, while the latter is unique to alphs.
while higher overall sequence similarity and a common alteration in the gdxxdrg motif are compatible with closer relatedness of alphs to bacterial diadenosine tetraphosphatases, phylogenetic analysis using full length sequences failed to produce a robust tree due to high sequence diversity .
relationship of novel eukaryotic ppp groups to known ppp phosphatases
in order to better understand the relationship of "bacterial-like" ppp phosphatases in eukaryotes to each other and to bacterial ppps, we attempted to extend our previous phylogenetic analysis of eukaryotic ppp phosphatases  <cit>  by including ppp sequences from a number of bacteria and archaea.
primary structures of bacterial ppp phosphatases are extremely diverse and, outside the relatively conserved n-terminal subdomain of about  <dig> amino acids containing the gdxhg, gdxxdrg and gnh motifs, they share only a few conserved residues.
moreover, many of the sequences have long insertions at different positions.
this leads to the failure to produce informative alignments of full-length catalytic domains.
therefore, we aligned more conserved n-terminal subdomains only, an approach applied previously by kennelly  <cit>  to a much smaller set of ppp sequences available at that time.
phylogenetic reconstruction was attempted with either neighbor-joining  or maximum likelihood analysis using quartet puzzling ; in the latter case a smaller dataset consisting of with consisting of  <dig> representative sequences was analyzed due to the inability of the algorithm to handle large datasets.
due to the relatively short length of the sequences and their high diversity, some of the major clades did not receive significant bootstrap support and were different depending on the method used, although most major clades, including rhilphs and shelphs, were recovered by both methods.
alphs tended to be grouped together by neighbor-joining but were split into smaller clades when maximum likelihood analysis was used.
however, we still tentatively consider alphs as a single group due to the characteristic replacements in their catalytic centre.
to circumvent the ambiguity of the results, we used neighbor-net  <cit> , a neighbor-joining based method that constructs phylogenetic networks rather than trees and thus represents conflicting signals and visualises feasible trees in a single plot .
the neighbor-net analysis accurately identified the major clades such as eukaryotic and archaeal phosphatases, as well as their closer relationship to each other than to bacterial ppps  <cit> .
separation of "conventional" eukaryotic ppps into two branches, suggested previously from the analysis of the full-length catalytic domains  <cit> , was also recovered.
as it was suggested by initial sequence similarity searches, rhilphs, shelphs and alphs represent distinct major clades of the ppp family, as divergent from "conventional" eukaryotic and archaeal ppp phosphatases as they are from major bacterial clades .
common structural elements in all "bacterial-like" ppp phosphatases from eukaryotes and bacterial phosphatases
c-terminal regions of the catalytic domain of all "conventional" eukaryotic ppp phosphatases share a highly conserved  sapny motif, located in the β12-β <dig> loop.
this loop and the tyr residue of the sapny motif in particular are implicated in interaction with regulators and inhibitors  <cit> .
β strands  corresponding to β <dig> and β <dig> are conserved in λpp  <cit> .
however, the sequence on the c-terminal side of β <dig> is dissimilar to sapny in λpp and in bacterial ppps.
a conservative replacement of the first ser of sapny by thr is found in many bacterial sequences .
the two adjacent positions are occupied by highly conserved asp and gly residues, respectively, thus defining a previously unrecognised motif dg.
this motif is present in all examined bacterial ppps, as well as in all "bacterial-like" phosphatases from eukaryotes described above .
in addition, we note the presence of another characteristic feature of bacterial and "bacterial-like" ppps: the his residue  coordinating one of the metal ions in the catalytic centre is preceded by an absolutely conserved gly; this residue is conserved in some archaeal but not in "conventional" eukaryotic ppps .
discussion
in this report, we have documented the presence in different eukaryotic lineages of the genes that encode ppp phosphatases resembling those of bacterial origin, rather than "conventional" eukaryotic members of the family.
catalytic domains of these "bacterial-like" phosphatases are characterised by relatively conserved structure of the n-terminal subdomains, but very diverse organisation of the c-terminal subdomains, where conserved motifs and residues forming the active centre are separated by sequences of various length that share little or no similarity between different clades .
in most cases, corresponding est sequences could be detected, which confirms that these genes are expressed.
the most conspicuous presence of "bacterial-like" ppps has been detected in plants.
plants possess phosphatases from all three novel groups described in this work.
rhilphs are most closely related to ppp phosphatases from a number of α-proteobacteria, including purple photosynthetic bacteria and rhizobiales.
the absence of related sequences in eukaryotes other than land plants suggests that rhilphs may have been acquired after plants started colonising land.
although bacterial lineage that could be the source for plant rhilphs could not be unambiguously identified by phylogenetic reconstruction, rhizobiales  appear to be likely candidates.
indeed, rhizobiales have transmissible chromosomal elements and some, like agrobacterium, are able to integrate their plasmid genes into plant genome  <cit>  or even transform animal cells  <cit> , a situation that would be ideally suited for a horizontal gene transfer to occur.
interestingly, the presence of genes of rhizobial origin has been detected in plant parasitic nematodes  <cit> .
possible origin of plant rhilphs from α-proteobacterial phosphatases is also supported by the presence in the enzymes of both groups of characteristic inserts in similar positions, which share some sequence similarity .
phosphatases of another group, designated as shelphs, are found in green plants, in a red alga, in apicomplexa, trypanosomatidae, as well as in some fungi.
the similarity between proteins from apicomplexa and trypanosomatidae and those from plants is well documented.
trypanosomatidae are related to photosynthetic euglenoids and are thought to have lost plastids secondarily  <cit> .
apicomplexan parasites have a relict plastid, originated from the engulfment of a red alga  <cit> .
thus, the presence of phosphatases shared by plants, red algae, apicomplexa and trypanosomatidae is not surprising and probably reflects the presence of shelphs in a common ancestor of photosynthetic eukaryotes.
the presence of chloroplast targeting sequence in slp <dig> suggests a possible origin of shelphs from a bacterial precursor of the chloroplast ; alternatively, this sequence may have appeared secondarily.
protein ser/thr phosphorylation / dephosphorylation is essential for regulation of photosynthesis, and unidentified okadaic acid-insensitive protein phosphatases in chloroplasts have been reported  <cit> .
slp <dig> appears to be a good candidate for such a phosphatase.
the origin of fungal shelphs is unclear.
curiously, they are found in basidiomycetes and in an ascomycete s. pombe, but not in a number of other ascomycetes, whose genomes have been completed.
current data do not permit to discriminate between  the presence of shelphs in a common ancestor of eukaryotes and their loss in such lineages as animals and many fungi, and  independent acquisition of shelphs from bacteria by an ancestor of photosynthetic eukaryotes and by fungi.
further sequencing of eukaryotic genomes may shed light on the evolutionary history of this ppp group.
the third group of "bacterial-like" phosphatases detected in eukaryotes, designated here as alphs, appears to be distantly related to bacterial diadenosine tetraphosphatases apah.
patchy distribution in several eukaryotic kingdoms suggests that alphs were probably present in the common ancestor of eukaryotes, but were independently lost in many lineages, including insects, vertebrates and flowering plants.
a characteristic modification of the conserved gdxxdrg motif shared only with apah further supports a suggestion that alphs may represent a divergent branch of diadenosine tetraphosphatases, rather than protein phosphatases.
however, relatedness of eukaryotic alphs to bacterial diadenosine tetraphosphatases remains hypothetical, since alph sequences are too divergent from apah, as well as from each other, to permit a reliable phylogenetic reconstruction.
diadenosine oligophosphates are considered as emerging signalling molecules in both intra- and intercellular signalling in eukaryotes  <cit> .
in particular, human diadenosine oligophosphate hydrolase fhit has been identified as a tumor suppressor  <cit> .
it seems plausible that appearance of eukaryotic diadenosine oligophosphate hydrolases  may have made bacterial-type diadenosine tetraphosphatases redundant, leading to their loss in many eukaryotic lineages.
it would be interesting to test experimentally whether alphs are indeed diadenosine oligophosphatases.
more generally, an important implication of our findings is that many eukaryotes possess ppp phosphatases with yet undetermined substrate specificity.
eukaryotic ppp phosphatases are generally considered as ser/thr specific in vivo, although they may be able to dephosphorylate phosphotyr-containing substrates in vitro .
this is probably true for archaeal ppps as well  <cit> .
however, ser/thr specificity is not a feature of bacterial ppp phosphatases  <cit> .
thus, it would not be possible to predict substrate specificity of uncharacterised "bacterial-like" ppp phosphatases without experimental evidence.
in particular, since shewanella ppi is tyr-specific  <cit> , it would be interesting to determine substrate specificity of eukaryotic shelphs.
it is also worth noting that interest in tyrosine phosphorylation in plants has recently been stimulated by identification of plant tyr phosphatase genes and by the finding that tyr phosphorylation is involved in the regulation of stomatal movement .
three motifs, gdxhg, gdxxdrg and gnh form the diagnostic signature of all ppp phosphatases  <cit> .
we detected a dg motif, which appears to be a characteristic signature of "bacterial"-type ppps.
the existence of such a motif is striking per se, taking into account extreme structural diversity of bacterial ppp phosphatases.
it indicates that dg was probably present as the fourth "universal" signature motif in the common ancestor of ppp phosphatases, and was lost in the common lineage of archaeal and "conventional" eukaryotic ppps.
an alternative possibility could be that the dg motif was acquired by a bacterium and propagated by lateral gene transfer, replacing the ancestral sapny-related motif.
however this scenario seems less likely, since the dg motif is present, with minor variations, in virtually all bacterial phosphatases, despite their great diversity, and is replaced by sapny-related sequences only in archaeal and "conventional" eukaryotic ppps.
the asp residue in the 2nd position of dg is highly conserved and can only be replaced by glu, indicating that the negative charge is essential.
the presence of a highly conserved gly in the 4th position indicates that flexibility of the polypeptide chain is likely to be important.
the crystal structure of bacteriophage λ phosphatase  shows that the asp residue of dg  is just downstream of the β <dig> strand, which corresponds to the β <dig> strand in mammalian pp <dig>  in ppλ, asp <dig> is hydrogen bonded to a water molecule coordinated to one of the metal ions in the catalytic centre, which probably accounts for its conservation.
in eukaryotic or archaeal ppps, corresponding position is occupied by neutral residues .
it would be tempting to speculate that this difference in the region just downstream of the β <dig>  may be responsible for a feature that is common to all "bacterial"-type but not to eukaryotic / archaeal ppps.
one such feature is the ser/thr specificity of the latter group.
the tyr residue of the sapny motif has been suggested to provide a bulky phenol ring in the β12-β <dig> loop, sufficient to sterically block access of phosphotyr-containing substrates to the active site  <cit> .
however this is unlikely to be the sole determinant of ser/thr specificity, since residues containing bulky aromatic rings  are found in the same or adjacent positions in many bacterial phosphatases .
since the dg motif, absent in eukaryotic and archaeal ppps, is involved in organisation of the catalytic centre  <cit> , it is possible that this difference in the catalytic centre organisation may be one of the determinants of broad substrate specificity vs. ser/thr specificity.
CONCLUSIONS
so far, eukaryotic ppp phosphatases were considered as a well-defined monophyletic group of enzymes, specifically dephosphorylating phosphoser and phosphothr, while a much more structurally and enzymatically diverse ppp phosphatases were known to be present in prokaryotes.
our findings demonstrate that, in addition to "conventional" eukaryotic ppp ser/thr-specific protein phosphatases, many eukaryotes possess very diverse "bacterial-like" ppp phosphatases.
enzymatic characteristics, physiological roles and evolutionary history of these phosphatases have yet to be revealed.

 reversible phosphorylation of proteins is a ubiquitous mechanism, indispensable for regulation of virtually any cellular function. therefore, protein kinases and phosphatases are of paramount importance for normal functioning of all metabolic and signalling pathways. in eukaryotes, ppp family is one of the two known protein phosphatase families specific for ser and thr. unlike eukaryotic ppp phosphatases, bacterial members of the family have broad substrate specificity or may even be tyr-specific. moreover, one group of bacterial ppps are diadenosine tetraphosphatases. unlike eukaryotes, in prokaryotes ppp phosphatases appear to be facultative, since entirely sequenced genomes of some bacteria and archaea do not encode them. nevertheless, when present, they appear to play essential roles. three motifs ), highly conserved in the n-terminal subdomains of the catalytic domains of all ppp phosphatases, contain most residues which coordinate metal ions in the active centre and are considered as the signature of the ppp family. in a previous work we identified an unusual cdna fragment from a moss physcomitrella patens, showing no similarities to the known ppp phosphatases beyond the presence of the gdxhg and gdxxdrg motifs. detection of homologous cdna sequences from arabidopsis and rice suggested the presence of an unknown ppp group in plants, distinct from "conventional" eukaryotic ppp phosphatases. we have now taken advantage of a much greater representation of sequence databases for various species to further explore this initial observation. we present the evidence for the existence in eukaryotes of three "non-conventional" branches of the ppp family. we also identify a previously unrecognised conserved motif in the ppp catalytic domain, which can be used as a signature of "bacterial"-type ppp phosphatases.  "rhizobiales / rhodobacterales / rhodospirillaceae – like" ppp phosphatases in plants two arabidopsis sequences, at3g and at3g were retrieved using the p. patens fragment as a query in tblastn searches. they share 85% identity with each other at the protein level. both genes are transcribed, since full-length cdnas have been detected in a large-scale transcription study. they are arranged on chromosome in tandem, suggesting their origin by a recent duplication. a number of ests from other plant species were also detected by tblastn searches, which in most cases provide no evidence for the existence of more than one isoform. among prokaryotes, related sequences were detected in some α-proteobacteria, the closest matches being with rhizobiales, rhodobacterales and rhodospirillaceae. therefore, we designate this group as rhizobiales / rhodobacterales / rhodospirillaceae-like phosphatases, or rhilphs, and the two arabidopsis genes products at3g and at3g as "rhizobiales-like" phosphatases and, respectively. * this phosphatase is unlikely to be catalytically active due to replacements of essential residues in the active centre. structural features of rhilphs all residues that are expected to bind metal ions in the catalytic centre are conserved in rhilphs. rhilphs do not have n- or c-terminal extensions beyond their catalytic domains, which in many ppp phosphatases have regulatory function and / or interact with regulatory proteins / subunits. instead, they have characteristic inserts between the conserved motifs gnh and hag. notably, much shorter inserts are found at a similar position in α-proteobacterial phosphatases. inserts in both plant rhilphs and α-proteobacterial phosphatases contain a conserved motif lxxaxpxxh. similarly to bacteriophage λphosphatase, rhilphs lack a region corresponding to β β and α of eukaryotic ppps. like bacterial ppp phosphatases, rhilphs do not have a sapny motif, conserved in the β12-β loop of eukaryotic ppps. analysis of rhilph sequences did not reveal targeting or signal peptides. while this work was in progress, a phosphatase encoded by an arabidopsis gene at1g was reported in an independent study. "shewanella-like" ppp phosphatases in plants, red algae, fungi and unicellular parasites we undertook further tblastn searches using full-length arabidopsis rlp as a query to see whether arabidopsis genome encodes additional "bacterial-like" ppps. these searches identified two more genes for putative ppp phosphatases, only distantly related to rhilphs and to any other members of the family. one of these genes is at1g at least three different predicted products of this gene could be found in protein databases. on the basis of comparison with est sequences, we consider as the correct structure that of q8ry with asp and gly at positions and respectively. the other detected gene, at1g is also represented in the databases by three distinct deduced proteins. comparison with a. thaliana ests confirms q944l as the correct structure. genomic and est database searches provided ample evidence for the presence of related phosphatases in a number of green plants, including multiple angiosperm species, pine and a unicellular green alga chlamydomonas reinhardtii. related sequences were also identified in some fungi, in apicomplexa, trypanosomatidae, and in a red alga porphyra yezoensis. the most closely related prokaryotic phosphatases were detected in myxococcus xanthus and psychrophilic bacteria alteromonadales: uncharacterised phosphatases from shewanella oneidensis and colwellia psychrerythraea and a tyr-specific phosphatase ppi from shewanella sp.. therefore, we designate this phosphatase group as "shewanella-like" phosphatases, or shelphs, and the products of the two prototype arabidopsis genes at1g and at1g as "shewanella-like" phosphatases and, respectively. structural features of shelphs like in rhilphs, all residues that are expected to bind metal ions in the catalytic centre are conserved in shelphs. another feature common with rhilphs is the presence of inserts between the gnhe and hg motifs, which are especially long in plant shelphs. however, these inserts share no sequence similarity between rhilphs and shelphs and probably appeared in the two phosphatase groups independently. like in rhilphs, a region corresponding to β β and α of eukaryotic ppps is absent in shelphs, and the primary structure of the region corresponding to the β12-β loop is similar to that of typical bacterial ppps. a.thaliana slp and corresponding shelph isoform from rice have chloroplast targeting sequence, which could not be detected in a.thaliana slp and corresponding isoform from medicago truncatula. eukaryotic ppps distantly related to bacterial diadenosine tetraphosphatases identification of rhilphs and shelphs prompted us to perform extensive searches of eukaryotic sequence databases. these searches revealed the existence of other "bacterial-like" ppp phosphatases throughout eukaryotes. sequences only distantly related to rhilphs, shelphs or any other ppp phosphatases were detected in several fungi, in trypanosomatidae, a tick amblyomma, an ascidian ciona, chlamydomonas, pine and diatoms fragilariopsis cylindrus and phaeodactylum tricornutum. blast searches using these sequences revealed that all of them share higher similarity to bacterial diadenosine tetraphosphatases than to other ppp groups. therefore, we tentatively designate them as apah-like phosphatases, or alphs. alphs share a distinctive common structural feature. in the gdxxdrg motif, absolutely conserved in other ppps, the second asp is replaced by a neutral amino acid, and the arg residue is replaced, with one exception, by lys. the former of these replacements is also found in apah, while the latter is unique to alphs. while higher overall sequence similarity and a common alteration in the gdxxdrg motif are compatible with closer relatedness of alphs to bacterial diadenosine tetraphosphatases, phylogenetic analysis using full length sequences failed to produce a robust tree due to high sequence diversity. relationship of novel eukaryotic ppp groups to known ppp phosphatases in order to better understand the relationship of "bacterial-like" ppp phosphatases in eukaryotes to each other and to bacterial ppps, we attempted to extend our previous phylogenetic analysis of eukaryotic ppp phosphatases by including ppp sequences from a number of bacteria and archaea. primary structures of bacterial ppp phosphatases are extremely diverse and, outside the relatively conserved n-terminal subdomain of about amino acids containing the gdxhg, gdxxdrg and gnh motifs, they share only a few conserved residues. moreover, many of the sequences have long insertions at different positions. this leads to the failure to produce informative alignments of full-length catalytic domains. therefore, we aligned more conserved n-terminal subdomains only, an approach applied previously by kennelly to a much smaller set of ppp sequences available at that time. phylogenetic reconstruction was attempted with either neighbor-joining or maximum likelihood analysis using quartet puzzling; in the latter case a smaller dataset consisting of with consisting of representative sequences was analyzed due to the inability of the algorithm to handle large datasets. due to the relatively short length of the sequences and their high diversity, some of the major clades did not receive significant bootstrap support and were different depending on the method used, although most major clades, including rhilphs and shelphs, were recovered by both methods. alphs tended to be grouped together by neighbor-joining but were split into smaller clades when maximum likelihood analysis was used. however, we still tentatively consider alphs as a single group due to the characteristic replacements in their catalytic centre. to circumvent the ambiguity of the results, we used neighbor-net, a neighbor-joining based method that constructs phylogenetic networks rather than trees and thus represents conflicting signals and visualises feasible trees in a single plot. the neighbor-net analysis accurately identified the major clades such as eukaryotic and archaeal phosphatases, as well as their closer relationship to each other than to bacterial ppps. separation of "conventional" eukaryotic ppps into two branches, suggested previously from the analysis of the full-length catalytic domains, was also recovered. as it was suggested by initial sequence similarity searches, rhilphs, shelphs and alphs represent distinct major clades of the ppp family, as divergent from "conventional" eukaryotic and archaeal ppp phosphatases as they are from major bacterial clades. common structural elements in all "bacterial-like" ppp phosphatases from eukaryotes and bacterial phosphatases c-terminal regions of the catalytic domain of all "conventional" eukaryotic ppp phosphatases share a highly conserved sapny motif, located in the β12-β loop. this loop and the tyr residue of the sapny motif in particular are implicated in interaction with regulators and inhibitors. β strands corresponding to β and β are conserved in λpp. however, the sequence on the c-terminal side of β is dissimilar to sapny in λpp and in bacterial ppps. a conservative replacement of the first ser of sapny by thr is found in many bacterial sequences. the two adjacent positions are occupied by highly conserved asp and gly residues, respectively, thus defining a previously unrecognised motif dg. this motif is present in all examined bacterial ppps, as well as in all "bacterial-like" phosphatases from eukaryotes described above. in addition, we note the presence of another characteristic feature of bacterial and "bacterial-like" ppps: the his residue coordinating one of the metal ions in the catalytic centre is preceded by an absolutely conserved gly; this residue is conserved in some archaeal but not in "conventional" eukaryotic ppps. discussion in this report, we have documented the presence in different eukaryotic lineages of the genes that encode ppp phosphatases resembling those of bacterial origin, rather than "conventional" eukaryotic members of the family. catalytic domains of these "bacterial-like" phosphatases are characterised by relatively conserved structure of the n-terminal subdomains, but very diverse organisation of the c-terminal subdomains, where conserved motifs and residues forming the active centre are separated by sequences of various length that share little or no similarity between different clades. in most cases, corresponding est sequences could be detected, which confirms that these genes are expressed. the most conspicuous presence of "bacterial-like" ppps has been detected in plants. plants possess phosphatases from all three novel groups described in this work. rhilphs are most closely related to ppp phosphatases from a number of α-proteobacteria, including purple photosynthetic bacteria and rhizobiales. the absence of related sequences in eukaryotes other than land plants suggests that rhilphs may have been acquired after plants started colonising land. although bacterial lineage that could be the source for plant rhilphs could not be unambiguously identified by phylogenetic reconstruction, rhizobiales appear to be likely candidates. indeed, rhizobiales have transmissible chromosomal elements and some, like agrobacterium, are able to integrate their plasmid genes into plant genome or even transform animal cells, a situation that would be ideally suited for a horizontal gene transfer to occur. interestingly, the presence of genes of rhizobial origin has been detected in plant parasitic nematodes. possible origin of plant rhilphs from α-proteobacterial phosphatases is also supported by the presence in the enzymes of both groups of characteristic inserts in similar positions, which share some sequence similarity. phosphatases of another group, designated as shelphs, are found in green plants, in a red alga, in apicomplexa, trypanosomatidae, as well as in some fungi. the similarity between proteins from apicomplexa and trypanosomatidae and those from plants is well documented. trypanosomatidae are related to photosynthetic euglenoids and are thought to have lost plastids secondarily. apicomplexan parasites have a relict plastid, originated from the engulfment of a red alga. thus, the presence of phosphatases shared by plants, red algae, apicomplexa and trypanosomatidae is not surprising and probably reflects the presence of shelphs in a common ancestor of photosynthetic eukaryotes. the presence of chloroplast targeting sequence in slp suggests a possible origin of shelphs from a bacterial precursor of the chloroplast; alternatively, this sequence may have appeared secondarily. protein ser/thr phosphorylation / dephosphorylation is essential for regulation of photosynthesis, and unidentified okadaic acid-insensitive protein phosphatases in chloroplasts have been reported. slp appears to be a good candidate for such a phosphatase. the origin of fungal shelphs is unclear. curiously, they are found in basidiomycetes and in an ascomycete s. pombe, but not in a number of other ascomycetes, whose genomes have been completed. current data do not permit to discriminate between the presence of shelphs in a common ancestor of eukaryotes and their loss in such lineages as animals and many fungi, and independent acquisition of shelphs from bacteria by an ancestor of photosynthetic eukaryotes and by fungi. further sequencing of eukaryotic genomes may shed light on the evolutionary history of this ppp group. the third group of "bacterial-like" phosphatases detected in eukaryotes, designated here as alphs, appears to be distantly related to bacterial diadenosine tetraphosphatases apah. patchy distribution in several eukaryotic kingdoms suggests that alphs were probably present in the common ancestor of eukaryotes, but were independently lost in many lineages, including insects, vertebrates and flowering plants. a characteristic modification of the conserved gdxxdrg motif shared only with apah further supports a suggestion that alphs may represent a divergent branch of diadenosine tetraphosphatases, rather than protein phosphatases. however, relatedness of eukaryotic alphs to bacterial diadenosine tetraphosphatases remains hypothetical, since alph sequences are too divergent from apah, as well as from each other, to permit a reliable phylogenetic reconstruction. diadenosine oligophosphates are considered as emerging signalling molecules in both intra- and intercellular signalling in eukaryotes. in particular, human diadenosine oligophosphate hydrolase fhit has been identified as a tumor suppressor. it seems plausible that appearance of eukaryotic diadenosine oligophosphate hydrolases may have made bacterial-type diadenosine tetraphosphatases redundant, leading to their loss in many eukaryotic lineages. it would be interesting to test experimentally whether alphs are indeed diadenosine oligophosphatases. more generally, an important implication of our findings is that many eukaryotes possess ppp phosphatases with yet undetermined substrate specificity. eukaryotic ppp phosphatases are generally considered as ser/thr specific in vivo, although they may be able to dephosphorylate phosphotyr-containing substrates in vitro. this is probably true for archaeal ppps as well. however, ser/thr specificity is not a feature of bacterial ppp phosphatases. thus, it would not be possible to predict substrate specificity of uncharacterised "bacterial-like" ppp phosphatases without experimental evidence. in particular, since shewanella ppi is tyr-specific, it would be interesting to determine substrate specificity of eukaryotic shelphs. it is also worth noting that interest in tyrosine phosphorylation in plants has recently been stimulated by identification of plant tyr phosphatase genes and by the finding that tyr phosphorylation is involved in the regulation of stomatal movement. three motifs, gdxhg, gdxxdrg and gnh form the diagnostic signature of all ppp phosphatases. we detected a dg motif, which appears to be a characteristic signature of "bacterial"-type ppps. the existence of such a motif is striking per se, taking into account extreme structural diversity of bacterial ppp phosphatases. it indicates that dg was probably present as the fourth "universal" signature motif in the common ancestor of ppp phosphatases, and was lost in the common lineage of archaeal and "conventional" eukaryotic ppps. an alternative possibility could be that the dg motif was acquired by a bacterium and propagated by lateral gene transfer, replacing the ancestral sapny-related motif. however this scenario seems less likely, since the dg motif is present, with minor variations, in virtually all bacterial phosphatases, despite their great diversity, and is replaced by sapny-related sequences only in archaeal and "conventional" eukaryotic ppps. the asp residue in the 2nd position of dg is highly conserved and can only be replaced by glu, indicating that the negative charge is essential. the presence of a highly conserved gly in the 4th position indicates that flexibility of the polypeptide chain is likely to be important. the crystal structure of bacteriophage λ phosphatase shows that the asp residue of dg is just downstream of the β strand, which corresponds to the β strand in mammalian pp in ppλ, asp is hydrogen bonded to a water molecule coordinated to one of the metal ions in the catalytic centre, which probably accounts for its conservation. in eukaryotic or archaeal ppps, corresponding position is occupied by neutral residues. it would be tempting to speculate that this difference in the region just downstream of the β may be responsible for a feature that is common to all "bacterial"-type but not to eukaryotic / archaeal ppps. one such feature is the ser/thr specificity of the latter group. the tyr residue of the sapny motif has been suggested to provide a bulky phenol ring in the β12-β loop, sufficient to sterically block access of phosphotyr-containing substrates to the active site. however this is unlikely to be the sole determinant of ser/thr specificity, since residues containing bulky aromatic rings are found in the same or adjacent positions in many bacterial phosphatases. since the dg motif, absent in eukaryotic and archaeal ppps, is involved in organisation of the catalytic centre, it is possible that this difference in the catalytic centre organisation may be one of the determinants of broad substrate specificity vs. ser/thr specificity.  so far, eukaryotic ppp phosphatases were considered as a well-defined monophyletic group of enzymes, specifically dephosphorylating phosphoser and phosphothr, while a much more structurally and enzymatically diverse ppp phosphatases were known to be present in prokaryotes. our findings demonstrate that, in addition to "conventional" eukaryotic ppp ser/thr-specific protein phosphatases, many eukaryotes possess very diverse "bacterial-like" ppp phosphatases. enzymatic characteristics, physiological roles and evolutionary history of these phosphatases have yet to be revealed. 
in addition, comparison of primary structures revealed a previously unrecognised dg motif, conserved in all bacterial and "bacterial-like" eukaryotic ppps, but not in "conventional" eukaryotic and archaeal ppps. moreover, one group of bacterial ppps are diadenosine tetraphosphatases, indicating that bacterial ppp phosphatases may not necessarily function as protein phosphatases. phosphatases of the third group are distantly related to apah, bacterial diadenosine tetraphosphatases, and are termed apah-like phosphatases, or alphs. unlike eukaryotic ppp phosphatases, bacterial members of the family have broad substrate specificity or may even be tyr-specific. in eukaryotes, ppp family is one of the two known protein phosphatase families specific for ser and thr. patchy distribution of alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. we describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the ppp family. our findings demonstrate that many eukaryotes possess diverse "bacterial-like" ppp phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined. this group is therefore termed rhizobiales / rhodobacterales / rhodospirillaceae-like phosphatases, or rhilphs. these enzymes are more closely related to bacterial ppp phosphatases than to the known eukaryotic members of the family. the role of ppp phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. one group, found exclusively in land plants, is most closely related to ppp phosphatases from some α-proteobacteria, including rhizobiales, rhodobacterales and rhodospirillaceae. rhilphs, shelphs and alphs form ppp clades, as divergent from "conventional" eukaryotic ppp phosphatases as they are from each other and from major bacterial clades.
 in eukaryotes, ppp family is one of the two known protein phosphatase families specific for ser and thr. the role of ppp phosphatases in multiple signaling pathways in eukaryotic cell has been extensively studied. unlike eukaryotic ppp phosphatases, bacterial members of the family have broad substrate specificity or may even be tyr-specific. moreover, one group of bacterial ppps are diadenosine tetraphosphatases, indicating that bacterial ppp phosphatases may not necessarily function as protein phosphatases.  we describe the presence in eukaryotes of three groups of expressed genes encoding "non-conventional" phosphatases of the ppp family. these enzymes are more closely related to bacterial ppp phosphatases than to the known eukaryotic members of the family. one group, found exclusively in land plants, is most closely related to ppp phosphatases from some α-proteobacteria, including rhizobiales, rhodobacterales and rhodospirillaceae. this group is therefore termed rhizobiales / rhodobacterales / rhodospirillaceae-like phosphatases, or rhilphs. phosphatases of the other group are found in viridiplantae, rhodophyta, trypanosomatidae, plasmodium and some fungi. they are structurally related to phosphatases from psychrophilic bacteria shewanella and colwellia, and are termed shewanella-like phosphatases, or shelphs. phosphatases of the third group are distantly related to apah, bacterial diadenosine tetraphosphatases, and are termed apah-like phosphatases, or alphs. patchy distribution of alphs in animals, plants, fungi, diatoms and kinetoplasts suggests that these phosphatases were present in the common ancestor of eukaryotes but were independently lost in many lineages. rhilphs, shelphs and alphs form ppp clades, as divergent from "conventional" eukaryotic ppp phosphatases as they are from each other and from major bacterial clades. in addition, comparison of primary structures revealed a previously unrecognised dg motif, conserved in all bacterial and "bacterial-like" eukaryotic ppps, but not in "conventional" eukaryotic and archaeal ppps.  our findings demonstrate that many eukaryotes possess diverse "bacterial-like" ppp phosphatases, the enzymatic characteristics, physiological roles and precise evolutionary history of which have yet to be determined. 