BACKGROUND
flavin mononucleotide  and flavin adenine dinucleotide  are essential cofactors for numerous enzymes  that participate in one- and two-electron oxidation-reduction processes critical to the major metabolic routes in all living organisms  <cit> .
riboflavin , the precursor of fmn and fad can be de novo synthesized by plants, fungi and many bacteria, but in mammals the only known rf source is the exogenous riboflavin  obtained through the diet  <cit> .
in most prokaryotes, the synthesis of fmn and fad is catalyzed from rf and atp by a single bifunctional enzyme, usually known as fad-synthetase , through the sequential action of its two enzymatic activities: an atp:riboflavin kinase  that transforms rf and atp into fmn, and an atp:fmn adenylyltransferase  that catalyzes the subsequent adenylylation of fmn to fad.
thus, fads is a bifunctional rfk/fmnat enzyme  <cit> .
fadss are typically 310- <dig> residues in length and are folded in two modules  <cit> , each one mainly involved in one of the activities.
the rfk reaction has been related with the c-terminal module , while the n-terminal module is mainly related to the fmnat activity ; hence, two independent substrate binding and catalytic sites are in charge of each activity  <cit> .
in one hand, the rfk-module  folds in a globular domain and its overall topology is similar to that found in the rfks from homo sapiens  and schizosaccharomyzes pombe , with differences only observed in the loops connecting secondary structure elements  <cit> .
furthermore, the substrate binding motifs ptan and gxy of the rfk-module are conserved among fadss and eukaryotic rfks.
in the other hand, the fmnat-module consists of an α/β dinucleotide binding domain with a typical rossmann fold topology   <cit> .
moreover, it seems to be remotely similar to the nucleotidyltransferase  superfamily and contains the typical xgh and xsst/sxxr motifs involved in binding nucleotide and phosphate groups.
interestingly, monofunctional enzymes with only rfk activity have been described in bacillus subtilis  <cit>  and streptococcus agalactiae  <cit>  but no monofunctional fmnat enzymes have been reported in prokaryotes.
a different scenario is found in eukaryotes, where both activities are generally split in two different enzymes with either rfk or fmnat activity  <cit> .
as mentioned above, the rfk enzymes show sequence and structure similarity to the rfk-module of prokaryotic fads  <cit> .
however, eukaryotic fmnats share little or no sequence similarity to the fmnat-module of fads, as these enzymes belong to two different protein superfamilies, which are thought to require different sets of active-site residues to carry out the same chemistry  <cit> .
the eukaryotic fmnat-module is currently classified as a member of the 3'-phosphoadenosine 5'-phosphosulfate  reductase-like family belonging to the "adenosine nucleotide α-hydrolase-like" superfamily, which has motifs different from those of nts.
in plants only a few efforts have been devoted to this system.
early studies characterized apparently monofunctional enzymes with either rfk or fmnat activities in several plant species  <cit> .
in those studies the subcellular localization of rfk and fads was not addressed although it is known that plants use flavin nucleotides in mitochondria, plastids and in the cytosol.
in an earlier work rfk activity was associated to the cytosol and to an organellar fraction containing chloroplasts and mitochondria  <cit> .
more recently, a bifunctional enzyme with both fmn hydrolase and rfk activities has been described in arabidopsis thaliana   <cit> , whose n-terminal module responsible for the fmn hydrolase activity, shares sequence similarity with members of the haloacid dehalogenase  superfamily.
atfhy/rfk enzyme was predicted to be cytosolic  <cit> .
additionally, two more enzymes with fmnat activity have been identified, cloned and characterized in the same species  <cit> .
these atribf <dig> and atribf <dig> enzymes, herein plant-like fads proteins, have an n-terminal module which is found to be homologous to the fmnat-module of fads, but instead its c-terminal module does not catalyze rf phosphorylation.
atribf <dig> and atribf <dig> were localized to the chloroplast  <cit> .
in mitochondria, the catalytically conversion of rf into fmn and fad has been reported, due to the existence of mitochondrial rfk and fads enzymes  <cit> , but nevertheless fads activity was much lower than in chloroplasts.
these results agree with the cited confocal microscopy studies  <cit> , but the hypothesis for the localization of fads isoforms  in mitochondria cannot be ruled out on the basis of bioinformatics  analysis  <cit> .
the mitochondrial fad-forming enzymes reside in two distinct monofunctional enzymes, which can be separated in soluble and membrane-enriched fractions.
it is worth mentioning that the genes encoding organellar rfk activity remains unidentified.
in order to investigate rfk and fmnat activities in plants we have conducted an extensive bioinformatics survey using the available genomes in public databases.
here we report the identification of a conserved c-terminal module in plant fads enzymes, which does not contain the typical rfk active site sequence, suggesting that it belongs to a new family of fads proteins.
the activity of this module is discussed.
RESULTS
sequence and evolutionary analysis
as shown in table  <dig>  most prokaryotic genomes  surveyed in this study, including cyanobacteria, contain a single gene encoding for a bifunctional fads enzyme, hereafter identified as fads-type i protein.
sequence searches in a variety of repositories of green plant sequences allowed us to identify a related group of genes, which contain two domains with high similarity  to fads-type i sequences , and are also present in a single copy in most cases with currently available complete genome .
this result agrees with previous work by sandoval et al.
<cit> .
the n-terminal module of these proteins displays high similarity to the fmnat-module of prokaryotic fads-type i, showing the typical motifs hxgh and xsst/sxxr involved in fmnat activity, also common to other nts.
however, several observations can be made with respect to the c-terminal module in plant proteins: i) its length is  <dig> to  <dig> residues shorter; ii) the ptan motif characteristic of the rfk activity mutates to pxs; iii) a lnxpp motif is found conserved in plants, next to the invariant gxy motif.
atribf <dig> and atribf <dig> belong to this group of proteins and were recently characterized by sandoval et al.
<cit> , who did not detect any rfk activity in these enzymes.
this experimental observation, together with the absence of the ptan motif suggests a different enzymatic activity for this module.
therefore we named these proteins as plant-like fads.
fmnat
mesoplasma florum l1
mycoplasma capricolum subsp.
capricolum
fads-type i
bacillus cereus atcc 10987
bacillus turingiensis str.
alhakam
geobacillus thermodenitrificans ng80-2
fads-type i
fads-type i
bacillus cereus subsp.
cytotoxis nvh 391-98
lactobacillus plantarum jdm1
lactobacillus plantarum wcfs1
listeria monocytogenes hcc23
listeria welshimeri serovar
oceanobacillus iheyensis hte831
mycoplasma conjunctivae
fads-type ii
<dig> check details in section "sequence analysis" of methods.
furthermore, a few bacterial parasites and pathogens isolated from plant, human or soil material and belonging to phyla firmicutes, actinobacteria, tenericutes and spirochaetes contain extra sequences with significant similarity to fads-type i .
however, as shown in figure  <dig> and  <dig>  these sequences do not conserve the catalytic ptan motifs, and have shorter c-terminal modules similar in length to plant like-fads, suggesting that they might constitute another divergent type of fads, which we label as fads-type ii .
in our sequence searches, plant-like fads proteins are distributed across a variety of green plant lineages.
among land plants we found  <dig> matches .
other than land plants, plant-like fads proteins were restricted to unicellular photosynthetic organisms belonging to phylum chlorophyta  .
all plant genomes surveyed encode these proteins in the nuclear genome .
our search strategies did not find plant-like fads proteins in any other eukaryotic genomes.
these observations might imply that these genes have a prokaryotic origin, somewhat related to the endosymbiotic origin of chloroplasts.
on the other hand, all green plant genomes explored have a copy of the cytosolic bifunctional fhy/rfk protein except two micromonas species that have a monofunctional rfk enzyme like in most eukaryotes.
proteins related to the had domain of fhy/rfk have been found in either bacteria or eukaryotes but this enzyme has been suggested to be unique in plant lineages probably being originated by fusion of a had to an eukaryotic-type rfk  <cit> .
babesia bovis
caenorhabditis japonica strain df5081
caernorhabditis remanei strain pb4641
culex pipiens quienquefasciatus
plasmodium knowlesi
trichoplax adhaerens
plant-like fads
plant-like fads
chlamydomonas reinhardtii
coccomyxa sp.
<dig> check details in section "sequence analysis" of methods.
<dig> fmn hydrolase fused to rfk in ref.
29
<dig> includes unigenes compiled from ests.
with the aim of further exploring the origin of plant-like fads proteins, we carried out a phylogenetic analysis, which is summarized in the phylogram in figure  <dig>  according to this tree, which was rooted by taking the sequence of the cytosolic protein atfhy/rfk from arabidopsis thaliana as an outgroup, plant-like fads proteins are closer to the group of cyanobacteria than any other bacterial species, which were selected to represent taxa included in table  <dig>  indeed proteins from both cyanobacteria and green plants are enclosed in a clade with an associated approximate likelihood ratio  of  <dig> .
these observations suggest that plant-like fads proteins have a prokaryotic origin closely related with cyanobacteria, although shaping a divergent group of sequences, as illustrated in figure  <dig>  note that atfhy/rfk clusters apart from prokaryotic fads, confirming a different origin for this enzyme.
the tree also suggests that plant-like fads proteins diverged from bacterial fads probably before the separation of the two major plant phyla , since they are present in species from both.
in order to further investigate this, we searched for putative plant-like fads homologues in mesostigma viride, proposed to be the earliest plant lineage and anterior to the divergence of the streptophyta and chlorophyta  <cit> .
unfortunately, the nuclear genome of this species is not available and the chloroplast and mitochondrial genomes yielded no sequence matches.
our findings could indicate that plant-like fads indeed derived from cyanobacterial fads, despite the fact that they are now encoded in the nuclear genome  <cit> .
moreover, these results reveal that most bacteria containing fads-type ii sequences have also typical fads-type i proteins  and the tree in figure  <dig> shows that these two types of sequences cluster together, implying that they might actually be paralogous genes.
only the genomes of eubacterium saphenum atcc  <dig>  mycoplasma conjunctivae, treponema pallidum subsp.
pallidum contain exclusively fads-type ii proteins.
although the tree does not support that fads-type ii proteins constitute a distinct evolutionary class, their shorter and non-conserved c-terminal domains still clusters them clearly as a distinct functional group, which might have lost the c-terminal activity typical of fads-type i proteins.
we also note the observed variability in terms of rfk and fmnat enzymatic activities in bacterial genomes.
while most prokaryotes have a single copy of a typical fads-type i sequence, in  <dig> species both enzymatic activities are separated in monofunctional proteins, which correspond to rfk or fmnat modules, respectively.
in other cases the fads-type i sequence was accompanied by either monofunctional prokaryotic fmnat  or monofunctional prokaryotic rfk .
for instance, the genome of alistipes putredinis contains both a monofunctional rfk and a fads-type ii sequence.
furthermore, although most bacterial fads and rfk proteins include the conserved ptan motif, some sequence variants can be found, including ptlk, ptln, ptin or ktan, which nevertheless conserve the c-terminal module length.
as these genomes do not contain any other rfk related proteins, these sequence variants are supposed still to be responsible for the rfk activity.
fads-type i sequences were also found in  <dig> eukaryotic species , including anopheles gambiae, caenorhabditis sp., trichoplax adhaerens, which is considered to be the most primitive multi-cellular animal known, or the freshwater amoeba paulinella chromatophora, which harbours a cyanobacterial endosymbiont.
it has been proposed that the double enzymatic activity of fads proteins might be the result of a gene fusion event that genetically perpetuated an ancient protein-protein interaction  <cit> .
if this hypothesis holds true, it is remarkable that  <dig> out of  <dig> bacterial genomes have a copy of this fused gene  while monofunctional rfks are vastly predominant  across eukaryotic genomes .
this observed unbalance suggests that this fusion event, or functional coupling, would have been evolutionary favoured only in unicellular organisms, from which chloroplasts are thought to be derived.
we would like to remark that fads proteins are annotated in sequence databases with confusing or contradictory names such as riboflavin biosynthesis protein ribf , fmn adenylylate transferase , fmn adenylyltransferase , fad synthase , riboflavin kinase/fmn adenylyltransferase , flavokinase/fad synthetase , riboflavin kinase/fad synthetase .
indeed, non-strictly fads sequences are also named as that .
in the case of plants, plant-like fads sequences are found as riboflavin kinase  or protein-s isoprenylcysteine o-methyltransferase |, cassava1385).
this misleading variability in names is of no benefit to users, and clearly so a consensus in the nomenclature would be desirable.
we hope this work makes a contribution in this direction.
a putative molecular function for the c-terminal module in plant-like fads proteins
psi-blast searches of both the complete sequence of the plant-like fads atribf <dig> and its c-terminus matched only nts and rfks  from bacteria, cyanobacteria, yeast and human.
no other family was identified as related to the c-terminus of plant-like fads.
the similarity between the newly identified c-terminal module and nts was further explored in the pdb <dig> structural library using the fold-recognition algorithm hhpred in local and global mode.
local searches provided significant matches  to: the rfk-module of tmfads , sprfk , hsrfk  and trypanosoma brucei rfk .
apart from these hits, global searches with the c-terminal domain yielded significant matches  to: nicotinamide mononucleotide  adenylyl transferase/ribosylnicotinamide kinase from haemophilus influenzae , ethanolamine-phosphate cytidylyltransferase from h. sapiens , nicotinamide-nucleotide adenylyltransferase  from francisella tularensis and the c-terminal module of bifunctional nicotinamide mononucleotide  adenylyltransferase/nudix hydrolase from synechocystis sp.
.
to evaluate the possible role of the c-terminal module of plant-like fadss and the molecular arrangement of this protein region we modelled the atribf <dig> structure using all the above mentioned protein structures as templates.
the best predicted models, as expected in terms of sequence similarity and alignment quality in putative binding and catalytic regions were obtained with the x-ray structure of tmfads, sprfk and hsrfk.
in order to annotate putative functional residues, the comparative models were superposed to the original templates, including all ligands present in the experimental coordinates.
the superposition in figure  <dig> indicate that plant conserved residues 290p×s <dig> and 302gvy <dig> in the model occupy equivalent positions with respect to the ligands  present in the crystallographic structure of tmfads.
similar results were obtained with sprfk and hsrfk .
the predicted secondary structure of atribf <dig> corresponds well with that of tmfads , although it lacks the last α-helix at the n-terminal end, which is conserved also in sprfk and hsrfk.
this helix appears to be crucial for a correct orientation of the bound flavin substrate  <cit>  and its absence in atribf <dig> is in agreement with the observed lack of rfk activity  <cit> .
as mentioned above, remote similarity of the c-terminal module of plant-like fadss was found with the c-terminal domain of other families such as synadm-nudix , which belongs to a large superfamily of pyrophosphohydrolases .
in arabidopsis  <dig> nudix hydrolase genes have been found and the proteins they encode are able to hydrolyze various types of nucleoside diphosphates derivatives such as adp-glucose, adp-ribose and a wide range of its derivatives, fad, nadh, nadph, and diadenosine polyphosphates  <cit> .
moreover, a remote sequence consensus of this protein region including the lnxpp motif was found with serine/threonine phosphatases 2c and members of the hydrolase superfamily.
these observations suggest that the c-terminal module of atribf <dig> could have a function other than rfk enzymatic activity.
sandoval et al.
<cit>  showed that purified recombinant atribf <dig> and atribf <dig> enzymes only display fads activity, with undetectable rfk activity and hence assumed that these are indeed monofunctional enzymes.
however, they were able to measure fmn hydrolase, fad pyrophosphatase and rfk activities in percoll-isolated chloroplasts.
as mentioned above, our bioinformatic analyses point out that plant-like fads proteins could be bifunctional enzymes.
more precisely, structural similarities predict a hydrolase and phosphatase activity for the c-terminal module, although the possibility to have a non-enzymatic regulatory role or to be a simple evolutionary relic should not be dismissed.
nevertheless, considering the results of sandoval et al.
<cit>  and ours, we could speculate that some of the measured activities in isolated chloroplasts  could be associated to this c-terminal module.
in order to test this hypothesis we have designed some experiments with recombinant plant-like fads from soybean  and preliminary results seem to indicate that its c-terminal module might have a hydrolytic activity since gmfads was able to convert fmn into rf .
interestingly, this activity was not detected in purified fads from c. ammoniagenes, a typical fads-type i protein  <cit> .
while these preliminary results seem to be in agreement with our theoretical analyses, clearly further investigations are necessary to confirm the possible enzymatic role of the c-terminal module of plant-like fads.
future work will be done by using recombinant plant-like fads  in order to confirm this observed hydrolytic enzymatic activity.
CONCLUSIONS
plant-like fads enzymes are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin.
homology models predict that plant-specific conserved residues are orientated towards a cavity, building a distinct active site when compared to that involved in substrate binding and catalysis in the c-terminus of typical fads-type i enzymes.
the remote relationship reported here between plant-like fads proteins and members of pyrophosphohydrolase or phosphatase superfamilies as well as preliminary experimental results suggest that the c-terminal module of these proteins, clearly of bacterial origin, might be involved in a catalytic function.

 flavin mononucleotide and flavin adenine dinucleotide are essential cofactors for numerous enzymes that participate in one- and two-electron oxidation-reduction processes critical to the major metabolic routes in all living organisms. riboflavin, the precursor of fmn and fad can be de novo synthesized by plants, fungi and many bacteria, but in mammals the only known rf source is the exogenous riboflavin obtained through the diet. in most prokaryotes, the synthesis of fmn and fad is catalyzed from rf and atp by a single bifunctional enzyme, usually known as fad-synthetase, through the sequential action of its two enzymatic activities: an atp:riboflavin kinase that transforms rf and atp into fmn, and an atp:fmn adenylyltransferase that catalyzes the subsequent adenylylation of fmn to fad. thus, fads is a bifunctional rfk/fmnat enzyme. fadss are typically 310- residues in length and are folded in two modules, each one mainly involved in one of the activities. the rfk reaction has been related with the c-terminal module, while the n-terminal module is mainly related to the fmnat activity; hence, two independent substrate binding and catalytic sites are in charge of each activity. in one hand, the rfk-module folds in a globular domain and its overall topology is similar to that found in the rfks from homo sapiens and schizosaccharomyzes pombe, with differences only observed in the loops connecting secondary structure elements. furthermore, the substrate binding motifs ptan and gxy of the rfk-module are conserved among fadss and eukaryotic rfks. in the other hand, the fmnat-module consists of an α/β dinucleotide binding domain with a typical rossmann fold topology. moreover, it seems to be remotely similar to the nucleotidyltransferase superfamily and contains the typical xgh and xsst/sxxr motifs involved in binding nucleotide and phosphate groups. interestingly, monofunctional enzymes with only rfk activity have been described in bacillus subtilis and streptococcus agalactiae but no monofunctional fmnat enzymes have been reported in prokaryotes. a different scenario is found in eukaryotes, where both activities are generally split in two different enzymes with either rfk or fmnat activity. as mentioned above, the rfk enzymes show sequence and structure similarity to the rfk-module of prokaryotic fads. however, eukaryotic fmnats share little or no sequence similarity to the fmnat-module of fads, as these enzymes belong to two different protein superfamilies, which are thought to require different sets of active-site residues to carry out the same chemistry. the eukaryotic fmnat-module is currently classified as a member of the 3'-phosphoadenosine 5'-phosphosulfate reductase-like family belonging to the "adenosine nucleotide α-hydrolase-like" superfamily, which has motifs different from those of nts. in plants only a few efforts have been devoted to this system. early studies characterized apparently monofunctional enzymes with either rfk or fmnat activities in several plant species. in those studies the subcellular localization of rfk and fads was not addressed although it is known that plants use flavin nucleotides in mitochondria, plastids and in the cytosol. in an earlier work rfk activity was associated to the cytosol and to an organellar fraction containing chloroplasts and mitochondria. more recently, a bifunctional enzyme with both fmn hydrolase and rfk activities has been described in arabidopsis thaliana, whose n-terminal module responsible for the fmn hydrolase activity, shares sequence similarity with members of the haloacid dehalogenase superfamily. atfhy/rfk enzyme was predicted to be cytosolic. additionally, two more enzymes with fmnat activity have been identified, cloned and characterized in the same species. these atribf and atribf enzymes, herein plant-like fads proteins, have an n-terminal module which is found to be homologous to the fmnat-module of fads, but instead its c-terminal module does not catalyze rf phosphorylation. atribf and atribf were localized to the chloroplast. in mitochondria, the catalytically conversion of rf into fmn and fad has been reported, due to the existence of mitochondrial rfk and fads enzymes, but nevertheless fads activity was much lower than in chloroplasts. these results agree with the cited confocal microscopy studies, but the hypothesis for the localization of fads isoforms in mitochondria cannot be ruled out on the basis of bioinformatics analysis. the mitochondrial fad-forming enzymes reside in two distinct monofunctional enzymes, which can be separated in soluble and membrane-enriched fractions. it is worth mentioning that the genes encoding organellar rfk activity remains unidentified. in order to investigate rfk and fmnat activities in plants we have conducted an extensive bioinformatics survey using the available genomes in public databases. here we report the identification of a conserved c-terminal module in plant fads enzymes, which does not contain the typical rfk active site sequence, suggesting that it belongs to a new family of fads proteins. the activity of this module is discussed.  sequence and evolutionary analysis as shown in table most prokaryotic genomes surveyed in this study, including cyanobacteria, contain a single gene encoding for a bifunctional fads enzyme, hereafter identified as fads-type i protein. sequence searches in a variety of repositories of green plant sequences allowed us to identify a related group of genes, which contain two domains with high similarity to fads-type i sequences, and are also present in a single copy in most cases with currently available complete genome. this result agrees with previous work by sandoval et al.. the n-terminal module of these proteins displays high similarity to the fmnat-module of prokaryotic fads-type i, showing the typical motifs hxgh and xsst/sxxr involved in fmnat activity, also common to other nts. however, several observations can be made with respect to the c-terminal module in plant proteins: i) its length is to residues shorter; ii) the ptan motif characteristic of the rfk activity mutates to pxs; iii) a lnxpp motif is found conserved in plants, next to the invariant gxy motif. atribf and atribf belong to this group of proteins and were recently characterized by sandoval et al., who did not detect any rfk activity in these enzymes. this experimental observation, together with the absence of the ptan motif suggests a different enzymatic activity for this module. therefore we named these proteins as plant-like fads. fmnat mesoplasma florum l1 mycoplasma capricolum subsp. capricolum fads-type i bacillus cereus atcc 10987 bacillus turingiensis str. alhakam geobacillus thermodenitrificans ng80-2 fads-type i fads-type i bacillus cereus subsp. cytotoxis nvh 391-98 lactobacillus plantarum jdm1 lactobacillus plantarum wcfs1 listeria monocytogenes hcc23 listeria welshimeri serovar oceanobacillus iheyensis hte831 mycoplasma conjunctivae fads-type ii check details in section "sequence analysis" of methods. furthermore, a few bacterial parasites and pathogens isolated from plant, human or soil material and belonging to phyla firmicutes, actinobacteria, tenericutes and spirochaetes contain extra sequences with significant similarity to fads-type i. however, as shown in figure and these sequences do not conserve the catalytic ptan motifs, and have shorter c-terminal modules similar in length to plant like-fads, suggesting that they might constitute another divergent type of fads, which we label as fads-type ii. in our sequence searches, plant-like fads proteins are distributed across a variety of green plant lineages. among land plants we found matches. other than land plants, plant-like fads proteins were restricted to unicellular photosynthetic organisms belonging to phylum chlorophyta. all plant genomes surveyed encode these proteins in the nuclear genome. our search strategies did not find plant-like fads proteins in any other eukaryotic genomes. these observations might imply that these genes have a prokaryotic origin, somewhat related to the endosymbiotic origin of chloroplasts. on the other hand, all green plant genomes explored have a copy of the cytosolic bifunctional fhy/rfk protein except two micromonas species that have a monofunctional rfk enzyme like in most eukaryotes. proteins related to the had domain of fhy/rfk have been found in either bacteria or eukaryotes but this enzyme has been suggested to be unique in plant lineages probably being originated by fusion of a had to an eukaryotic-type rfk. babesia bovis caenorhabditis japonica strain df5081 caernorhabditis remanei strain pb4641 culex pipiens quienquefasciatus plasmodium knowlesi trichoplax adhaerens plant-like fads plant-like fads chlamydomonas reinhardtii coccomyxa sp. check details in section "sequence analysis" of methods. fmn hydrolase fused to rfk in ref. 29 includes unigenes compiled from ests. with the aim of further exploring the origin of plant-like fads proteins, we carried out a phylogenetic analysis, which is summarized in the phylogram in figure according to this tree, which was rooted by taking the sequence of the cytosolic protein atfhy/rfk from arabidopsis thaliana as an outgroup, plant-like fads proteins are closer to the group of cyanobacteria than any other bacterial species, which were selected to represent taxa included in table indeed proteins from both cyanobacteria and green plants are enclosed in a clade with an associated approximate likelihood ratio of. these observations suggest that plant-like fads proteins have a prokaryotic origin closely related with cyanobacteria, although shaping a divergent group of sequences, as illustrated in figure note that atfhy/rfk clusters apart from prokaryotic fads, confirming a different origin for this enzyme. the tree also suggests that plant-like fads proteins diverged from bacterial fads probably before the separation of the two major plant phyla, since they are present in species from both. in order to further investigate this, we searched for putative plant-like fads homologues in mesostigma viride, proposed to be the earliest plant lineage and anterior to the divergence of the streptophyta and chlorophyta. unfortunately, the nuclear genome of this species is not available and the chloroplast and mitochondrial genomes yielded no sequence matches. our findings could indicate that plant-like fads indeed derived from cyanobacterial fads, despite the fact that they are now encoded in the nuclear genome. moreover, these results reveal that most bacteria containing fads-type ii sequences have also typical fads-type i proteins and the tree in figure shows that these two types of sequences cluster together, implying that they might actually be paralogous genes. only the genomes of eubacterium saphenum atcc mycoplasma conjunctivae, treponema pallidum subsp. pallidum contain exclusively fads-type ii proteins. although the tree does not support that fads-type ii proteins constitute a distinct evolutionary class, their shorter and non-conserved c-terminal domains still clusters them clearly as a distinct functional group, which might have lost the c-terminal activity typical of fads-type i proteins. we also note the observed variability in terms of rfk and fmnat enzymatic activities in bacterial genomes. while most prokaryotes have a single copy of a typical fads-type i sequence, in species both enzymatic activities are separated in monofunctional proteins, which correspond to rfk or fmnat modules, respectively. in other cases the fads-type i sequence was accompanied by either monofunctional prokaryotic fmnat or monofunctional prokaryotic rfk. for instance, the genome of alistipes putredinis contains both a monofunctional rfk and a fads-type ii sequence. furthermore, although most bacterial fads and rfk proteins include the conserved ptan motif, some sequence variants can be found, including ptlk, ptln, ptin or ktan, which nevertheless conserve the c-terminal module length. as these genomes do not contain any other rfk related proteins, these sequence variants are supposed still to be responsible for the rfk activity. fads-type i sequences were also found in eukaryotic species, including anopheles gambiae, caenorhabditis sp., trichoplax adhaerens, which is considered to be the most primitive multi-cellular animal known, or the freshwater amoeba paulinella chromatophora, which harbours a cyanobacterial endosymbiont. it has been proposed that the double enzymatic activity of fads proteins might be the result of a gene fusion event that genetically perpetuated an ancient protein-protein interaction. if this hypothesis holds true, it is remarkable that out of bacterial genomes have a copy of this fused gene while monofunctional rfks are vastly predominant across eukaryotic genomes. this observed unbalance suggests that this fusion event, or functional coupling, would have been evolutionary favoured only in unicellular organisms, from which chloroplasts are thought to be derived. we would like to remark that fads proteins are annotated in sequence databases with confusing or contradictory names such as riboflavin biosynthesis protein ribf, fmn adenylylate transferase, fmn adenylyltransferase, fad synthase, riboflavin kinase/fmn adenylyltransferase, flavokinase/fad synthetase, riboflavin kinase/fad synthetase. indeed, non-strictly fads sequences are also named as that. in the case of plants, plant-like fads sequences are found as riboflavin kinase or protein-s isoprenylcysteine o-methyltransferase |, cassava1385). this misleading variability in names is of no benefit to users, and clearly so a consensus in the nomenclature would be desirable. we hope this work makes a contribution in this direction. a putative molecular function for the c-terminal module in plant-like fads proteins psi-blast searches of both the complete sequence of the plant-like fads atribf and its c-terminus matched only nts and rfks from bacteria, cyanobacteria, yeast and human. no other family was identified as related to the c-terminus of plant-like fads. the similarity between the newly identified c-terminal module and nts was further explored in the pdb structural library using the fold-recognition algorithm hhpred in local and global mode. local searches provided significant matches to: the rfk-module of tmfads, sprfk, hsrfk and trypanosoma brucei rfk. apart from these hits, global searches with the c-terminal domain yielded significant matches to: nicotinamide mononucleotide adenylyl transferase/ribosylnicotinamide kinase from haemophilus influenzae, ethanolamine-phosphate cytidylyltransferase from h. sapiens, nicotinamide-nucleotide adenylyltransferase from francisella tularensis and the c-terminal module of bifunctional nicotinamide mononucleotide adenylyltransferase/nudix hydrolase from synechocystis sp.. to evaluate the possible role of the c-terminal module of plant-like fadss and the molecular arrangement of this protein region we modelled the atribf structure using all the above mentioned protein structures as templates. the best predicted models, as expected in terms of sequence similarity and alignment quality in putative binding and catalytic regions were obtained with the x-ray structure of tmfads, sprfk and hsrfk. in order to annotate putative functional residues, the comparative models were superposed to the original templates, including all ligands present in the experimental coordinates. the superposition in figure indicate that plant conserved residues 290p×s and 302gvy in the model occupy equivalent positions with respect to the ligands present in the crystallographic structure of tmfads. similar results were obtained with sprfk and hsrfk. the predicted secondary structure of atribf corresponds well with that of tmfads, although it lacks the last α-helix at the n-terminal end, which is conserved also in sprfk and hsrfk. this helix appears to be crucial for a correct orientation of the bound flavin substrate and its absence in atribf is in agreement with the observed lack of rfk activity. as mentioned above, remote similarity of the c-terminal module of plant-like fadss was found with the c-terminal domain of other families such as synadm-nudix, which belongs to a large superfamily of pyrophosphohydrolases. in arabidopsis nudix hydrolase genes have been found and the proteins they encode are able to hydrolyze various types of nucleoside diphosphates derivatives such as adp-glucose, adp-ribose and a wide range of its derivatives, fad, nadh, nadph, and diadenosine polyphosphates. moreover, a remote sequence consensus of this protein region including the lnxpp motif was found with serine/threonine phosphatases 2c and members of the hydrolase superfamily. these observations suggest that the c-terminal module of atribf could have a function other than rfk enzymatic activity. sandoval et al. showed that purified recombinant atribf and atribf enzymes only display fads activity, with undetectable rfk activity and hence assumed that these are indeed monofunctional enzymes. however, they were able to measure fmn hydrolase, fad pyrophosphatase and rfk activities in percoll-isolated chloroplasts. as mentioned above, our bioinformatic analyses point out that plant-like fads proteins could be bifunctional enzymes. more precisely, structural similarities predict a hydrolase and phosphatase activity for the c-terminal module, although the possibility to have a non-enzymatic regulatory role or to be a simple evolutionary relic should not be dismissed. nevertheless, considering the results of sandoval et al. and ours, we could speculate that some of the measured activities in isolated chloroplasts could be associated to this c-terminal module. in order to test this hypothesis we have designed some experiments with recombinant plant-like fads from soybean and preliminary results seem to indicate that its c-terminal module might have a hydrolytic activity since gmfads was able to convert fmn into rf. interestingly, this activity was not detected in purified fads from c. ammoniagenes, a typical fads-type i protein. while these preliminary results seem to be in agreement with our theoretical analyses, clearly further investigations are necessary to confirm the possible enzymatic role of the c-terminal module of plant-like fads. future work will be done by using recombinant plant-like fads in order to confirm this observed hydrolytic enzymatic activity.  plant-like fads enzymes are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. homology models predict that plant-specific conserved residues are orientated towards a cavity, building a distinct active site when compared to that involved in substrate binding and catalysis in the c-terminus of typical fads-type i enzymes. the remote relationship reported here between plant-like fads proteins and members of pyrophosphohydrolase or phosphatase superfamilies as well as preliminary experimental results suggest that the c-terminal module of these proteins, clearly of bacterial origin, might be involved in a catalytic function. 
the c-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the n-terminal module is broadly conserved. in fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the c-terminus. using a variety of bioinformatics methods we have found that fads enzymes localized to the chloroplasts, which we term as plant-like fads proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. flavin adenine dinucleotide synthetases - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide and its subsequent adenylation to generate fad in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history. this work is a contribution to our understanding of the evolutionary history of plant-like fads enzymes, which constitute a new family of fads proteins whose c-terminal module might be involved in a distinct catalytic activity. these results agree with a previous work reported by sandoval et al. in furthermore, our observations and preliminary experimental results indicate that the c-terminus of plant-like fads proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. a structure-based sequence alignment and an in-depth evolutionary survey of fads proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies.
 flavin adenine dinucleotide synthetases - a group of bifunctional enzymes that carry out the dual functions of riboflavin phosphorylation to produce flavin mononucleotide and its subsequent adenylation to generate fad in most prokaryotes - were studied in plants in terms of sequence, structure and evolutionary history.  using a variety of bioinformatics methods we have found that fads enzymes localized to the chloroplasts, which we term as plant-like fads proteins, are distributed across a variety of green plant lineages and constitute a divergent protein family clearly of cyanobacterial origin. the c-terminal module of these enzymes does not contain the typical riboflavin kinase active site sequence, while the n-terminal module is broadly conserved. these results agree with a previous work reported by sandoval et al. in furthermore, our observations and preliminary experimental results indicate that the c-terminus of plant-like fads proteins may contain a catalytic activity, but different to that of their prokaryotic counterparts. in fact, homology models predict that plant-specific conserved residues constitute a distinct active site in the c-terminus.  a structure-based sequence alignment and an in-depth evolutionary survey of fads proteins, thought to be crucial in plant metabolism, are reported, which will be essential for the correct annotation of plant genomes and further structural and functional studies. this work is a contribution to our understanding of the evolutionary history of plant-like fads enzymes, which constitute a new family of fads proteins whose c-terminal module might be involved in a distinct catalytic activity. 