BACKGROUND
the arabidopsis thaliana transthyretin-like protein  was first identified as a potential substrate of brassinosteroid-insensitive  <dig> , the principal brassinosteroid  receptor, playing a negative role in br-mediated plant growth  <cit> .
sequence analysis shows that ttl displays an n-terminal domain corresponding to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline  decarboxylase, and a c-terminal domain that has approximately 42% sequence identity with transthyretin , a vertebrate-specific transport protein  <cit> .
ttr is a plasma protein which transports the thyroid hormones  <dig> ,3',5'-tetraiodo-l-thyronin  and  <dig> ,3-triiodo-l-thyronin  as well as retinol by association with the retinol-binding protein  <cit> .
ttrs are homotetrameric proteins, each monomer containing two four-stranded β-sheets and one short α-helix.
they have a channel that runs through the dimer-dimer interface, where two identical thyroid hormone binding sites are located  <cit> .
proteins with sequence homology to ttr and with the characteristic c-terminal sequence motif tyr-arg-gly-ser, which is absent in ttrs, are named transthyretin-related proteins  and are found in bacteria, fungi, animals  and plants  <cit> .
trps are thought to be ttr ancestors  <cit> .
three ttl splice variants were reported; two of them are cytoplasmic  and the other one is peroxisomal   <cit> .
in all three variants the n-terminal domain, ohcu decarboxylase, is composed of  <dig> amino acids, whereas the c-terminal domain varies in length, having  <dig>   <dig> and  <dig> amino acids for the 324-, 311- and 286-residues isoforms, respectively  <cit> .
the 324-residues isoform contains a type- <dig> peroxisomal targeting sequence , which is deleted in the other two isoforms  <cit> .
in most eukaryotes, 5-hiu hydrolases contain a pts- <dig> and ohcu decarboxylases contain a type- <dig> peroxisomal targeting sequence , except in plants  <cit> .
in a. thaliana these two enzymes are fused into a single polypeptide chain, containing a pts- <dig> sequence, between the two domains, in the 324-residues isoform.
in a previous study, the c-terminal domain of ttl was expressed and studied separately.
this single-domain was named transthyretin-like protein   <cit>  and is distinct from ttl, which contains both this c-terminal domain, with 5-hiu hydrolase activity, and a n-terminal domain, with ohcu decarboxylase activity, fused into the same polypeptide chain  <cit> .
tlp was described as a tetramer  <cit> .
the three-dimensional structure of trps from salmonella dublin, escherichia coli, zebrafish and the homologous domain from bacillus subtilis were determined  <cit> .
the overall structures of these trps and also the model predicted for tlp  <cit>  are very similar to those of ttrs, as expected due to sequence homology, although functionally they are different proteins.
trps are hydrolases with a role in uric acid degradation  <cit>  and they do not bind thyroid hormones  <cit> .
ttl is the a. thaliana trp member, which contains an extra domain of  <dig> amino acids as compared to other trps.
until now, only trps from magnetospirillum magnetotacticum, bradyrhizobium japonicum,bacillus subtilis and plant species have been described as composed of two sequence domains, one related to ttr and the other with features common to ohcu decarboxylases  <cit> .
ttl was predicted by sequence homology as being a bifunctional enzyme with 5-hydroxyisourate  hydrolase and ohcu decarboxylase activities, catalysing the two final steps in the uric acid degradation pathway   <cit> .
in most organisms, uric acid, the end product of purine degradation, is catabolised to allantoin.
the process is initiated by uricase, which oxidizes uric acid into 5-hiu; this intermediate compound is then hydrolysed by 5-hiu hydrolase, leading to ohcu.
finally, a third enzyme, ohcu decarboxylase, catalyses the decarboxylation of ohcu producing -allantoin  <cit> .
the spontaneous degradation of uric acid follows the same pathway, resulting into 5-hiu and ohcu intermediates, which can be further oxidized, producing potentially harmful reactive species.
however, many organisms have an enzymatic pathway, possibly to convert these intermediates more rapidly into allantoin, a much less reactive species.
the spontaneous pathway produces racemic allantoin, whereas the enzymatic pathway produces exclusively -allantoin  <cit> .
so far, two different functions have been assigned to ttl: i) a signalling role to the cytoplasmic isoforms and ii) an enzymatic role to the peroxisomal isoform.
while the signalling role has already been studied  <cit> , it is not known if the two enzymatic sequence domains that are in this protein fused into a long polypeptide chain do not compromise the role of each domain in an isolated form.
in this work we studied the 311-residue cytoplasmic isoform, referred as ttl throughout the manuscript, to address the protein enzymatic role.
RESULTS
ttl oligomerization state
recombinant ttl was purified and used to investigate the protein oligomerization state.
we decided to use the method developed by siegel and monty in  <dig>  <cit> , because it allows calculation of the molecular mass of a protein independently of its shape, using a combination of the stokes radius  derived from gel filtration chromatography and the sedimentation coefficient  obtained from density gradient centrifugation.
the gel filtration chromatograms are presented in fig.
<dig>  showing in fig.
2a a major peak of oligomeric ttl and in fig.
2b superposed chromatograms of standard stokes radius markers.
using the equations described in methods, section on "determination of the protein oligomeric state", a and s were estimated to be  <dig>  nm and  <dig>  s, respectively, and with these values the native molecular mass for ttl was estimated to be  <dig>  kda.
since each ttl subunit has  <dig>  kda, we concluded that ttl is a tetramer, which is in agreement with the fact that ttr, as well as a.thaliana tlp, also have a tetrameric structure  <cit> .
moreover, the calculated frictional ratio,  <dig> , is consistent with a moderately elongated non-globular protein  <cit> .
thyroxin -binding assays
since ttr is a tetramer with two equivalent t4-binding sites, we decided to study the binding of t <dig> to ttl, using radiolabeled thyroxin and ttr as the sample control .
the ttr control has the typical behaviour of a specific t4-binding protein, in which the increase of cold t <dig> concentration causes the displacement of specifically bound radiolabeled t <dig> *.
on the contrary, t <dig> *-binding to ttl, besides being very low, was constant at different t <dig> concentrations, showing a non-specific binding.
this is a very different behaviour as compared to ttr, for which non-specific binding was only detected at the highest t <dig> concentration.
we concluded that the binding of t <dig> to ttl is not significant and clearly non-specific, since it is not altered in the presence of a competitor.
sequence alignment of human ttr, ttl and trps from salmonella dublin, b. subtilis and mouse are presented in fig.
<dig>  as mentioned previously, only the c-terminal domain of ttl has homology with ttr or trps.
several amino acids were reported as important for the binding of t <dig> to ttr, namely lys <dig>  glu <dig>  thr <dig> and thr <dig>  <cit> .
interestingly, they are not conserved in ttl, being replaced by his <dig>  arg <dig>  his <dig> and tyr <dig>  respectively , which are amino acids conserved in trps, and were reported to be important for 5-hiu hydrolase catalytic activity  <cit> .
according to our sequence alignment data  and the three-dimensional structure of several ttr and trp proteins  <cit> , these substitutions will probably have two main effects: i) create a narrower region at the inner part of the binding site due to the replacement of thr for tyr, which has a larger side chain and ii) alter the surface topography and introduce charge differences at the entrance of the channel, due to substitutions lys-his, glu-arg and thr-his.
these modifications will probably exclude the binding of t <dig> to trps and ttl.
ttl enzymatic activities
since trps are functionally associated to 5-hiu hydrolases, we decided to study the predicted 5-hiu hydrolase and ohcu decarboxylase activities for ttl in vitro.
the experiments were started by testing how ttl would affect the activity of uricase over uric acid by monitoring the differences in absorbance at  <dig> nm in the absence or presence of ttl .
although uricase alone was able to decompose uric acid, its activity was significantly accelerated in the presence of ttl.
by contrast, ttr did not influence this reaction.
this result is consistent with the predicted 5-hiu hydrolase and ohcu decarboxylase activities.
the rapid consumption of 5-hiu and ohcu should favour an equilibrium shift in the reaction catalysed by uricase.
5-hiu hydrolase activity was demonstrated in vitro for single-domain trps present in s. dublin, b. subtilis and mouse  <cit> .
the authors reported that uric acid was converted into 5-hiu by uricase and both the formation and subsequent degradation of this compound was monitored at  <dig> nm, since ohcu does not absorb at this wavelength  <cit> .
in our assay conditions, within  <dig> seconds incubation of uric acid with the candida sp.
uricase, the production of 5-hiu reached the maximum level .
at this point, ttl was added to the reaction solution resulting in a rapid decrease in absorbance at  <dig> nm .
as expected, addition of ttr, which was used as the negative control, failed to accelerate 5-hiu degradation.
this result shows that ttl is an enzyme that facilitates the hydrolysis of 5-hiu and also indicates that most probably four protein c-terminal domains associate forming a tetrameric structure, since the 5-hiu hydrolase catalytic site are located at the dimer-dimer interface in all structurally and functionally characterised trp members  <cit> .
although the crystal structure of the n-terminal ohcu domain of ttl from a. thaliana, which contains conserved residues critical for the ohcu decarboxylase activity, was recently reported  <cit> , its predicted enzyme activity was not experimentally demonstrated.
in fact, it was only observed in vitro in an isolated domain of a bifunctional enzyme from b. subtilis and also in zebrafish ohcu decarboxylase  <cit> .
monitoring the decarboxylase activity of ttl by spectrophotometry is not straightforward since ohcu absorbs below  <dig> nm and its absorption spectrum overlaps with that of 5-hiu  <cit> .
therefore, measurements at  <dig> nm are likely to be a sum of contributions by 5-hiu and ohcu.
however, the spontaneous degradation observed at this wavelength is clearly distinct from what is observed at  <dig> nm .
the spontaneous degradation followed at  <dig> nm shows that 5-hiu is completely degraded in  <dig> seconds , thus the spontaneous absorbance decay measured after this point at  <dig> nm  is exclusively due to the spontaneous degradation of ohcu.
therefore this degradation was used as control in the experiments testing the effect of ttl on degradation of ohcu.
in presence of ttl the degradation of 5-hiu followed at  <dig> nm was completed in  <dig> seconds  therefore after this point the absorbance decay measured at  <dig> nm  corresponds to the degradation of ohcu affected by ttl.
the rate of this degradation is excessively high as compared to the control spontaneous degradation of ohcu .
addition of ttl accelerated significantly the degradation of ohcu, supporting the decarboxylase activity of the protein.
taken together, our results demonstrated that ttl has 5-hiu hydrolase and ohcu decarboxylase activities in vitro, and they are both performed at the same time.
discussion
it seems that the ttr domain evolved from an enzyme present in bacteria, fungi, plants and animals to a hormone transporter present in vertebrates  <cit> .
its function changed completely, although there are only minor differences in sequence.
in the present study, we showed that ttl from a. thaliana is a tetramer with a moderately elongated shape.
the tetrameric structure is in agreement with the observation that the protein has 5-hiu hydrolase activity.
we expect that four c-terminal domains associate in a tetramer forming two active sites, as reported for trps, which contain only one domain.
moreover, the elongated shape is possibly due to the n-terminal ohcu decarboxylase domain associated to each of the four c-terminal sequences forming the tetramer.
the molecular model of this domain was determined in the presence of allantoin  <cit> ; it is an alpha-domain that associates as dimers in its crystal form.
however, a careful inspection of the available molecular models for trps and ohcu decarboxylase domain indicates that allantoin binding site is not in the region of the interaction between the two subunits that form the dimer  <cit>  and this kind of association most probably will not be present in ttl, since the ttl tetramer will be formed through contacts among the c-terminal domains.
we also demonstrated that ttl does not bind t <dig> in vitro, corroborating the observation that the ligand-binding site has characteristics which are different from those present in ttr.
these results are in agreement with previous observations, since the functionally characterized trps do not bind thyroid hormones  <cit>  and are 5-hiu hydrolases with active sites located in the regions corresponding to the t4-binding sites in ttr  <cit> .
the ohcu decarboxylase activity was also detected in the full-length ttl, indicating that the n-terminal domains do not inhibit the active sites formed by the c-terminal regions; the reverse is also valid, thus supporting the idea of a bifunctional enzyme.
ttl was initially characterized as a specific bri1-interactor that regulates br-mediated plant growth  <cit> .
here we show that the same protein is also enzymatically active.
several proteins with at least two totally different functions have been reported and named moonlighting proteins; the switch between functions is due to changes in cellular localization, cell type, oligomeric state or cellular concentration of a ligand  <cit> .
we think that ttl is a moonlighting protein that switches its function according to its subcellular location.
in eukaryotes, peroxisomes are the major organelles where oxidative reactions with molecular oxygen consumption are carried out.
in these organisms, uricase is located in peroxisomes, oxidizing uric acid to 5-hiu  <cit> .
as a consequence, 5-hiu accumulates in peroxisomes, providing the initial ttl substrate.
therefore, in peroxisomes, where uric acid degradation occurs, ttl, through its 324-residues isoform, functions as a bifunctional enzyme with 5-hiu hydrolase and ohcu decarboxylase activities.
in the cytoplasm, 5-hiu and ohcu are absent, since they are produced in peroxisomes.
consequently, the ttl 311-residues isoform most probably will not have these enzymatic functions.
moreover, it was shown to interact with bri <dig>  mediating br-regulated plant growth.
bri <dig> is an intrinsic membrane protein, located in the plasma membrane and containing a cytoplasmic kinase domain that interacts with ttl  <cit> .
the main difference between the 324- and 311-residues isoforms is the presence or absence of pts- <dig>  the subcellular location of three ttl isoforms is probably fundamental to define their role in vivo.
ttl 324-residues isoform location into peroxisomes was already shown by fluorescence microscopy, and provided conclusive results regarding its import  <cit> .
it was also observed that its import efficiency was low and it was hypothesized that could result from a low accessibility of the internal targeting signal to the peroxisomal import machinery.
internal pts- <dig> signal sequences are thought to be rare in nature and to occur preferentially in bifuctional enzymes resulting from gene fusion  <cit> .
the 311-residues isoform will probably remain in the cytoplasm in vivo, since it does not contain the pts- <dig> sequence  <cit> .
therefore in the absence of uric acid degradation intermediates, it interacts with the plasma membrane-located bri <dig>  mediating br-regulated plant growth  <cit> .
attempts for the three-dimensional structure determination of ttl are in progress and should represent a fundamental step in order to clarify ttl function, namely its signalling role in br-mediated responses.
CONCLUSIONS
the aminoacid sequence of ttl from a. thaliana reveals two domains similar to 5-hiu hydrolase and ohcu decarboxylase.
as a consequence, ttl was predicted to be a bifunctional enzyme.
here we reported the first experimental data showing that in fact the protein has both enzymatic activities in vitro.
moreover, we confirmed that ttl is in fact a bifunctional enzyme, by showing that it performs both activities simultaneously.

 the arabidopsis thaliana transthyretin-like protein was first identified as a potential substrate of brassinosteroid-insensitive, the principal brassinosteroid receptor, playing a negative role in br-mediated plant growth. sequence analysis shows that ttl displays an n-terminal domain corresponding to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, and a c-terminal domain that has approximately 42% sequence identity with transthyretin, a vertebrate-specific transport protein. ttr is a plasma protein which transports the thyroid hormones,3',5'-tetraiodo-l-thyronin and,3-triiodo-l-thyronin as well as retinol by association with the retinol-binding protein. ttrs are homotetrameric proteins, each monomer containing two four-stranded β-sheets and one short α-helix. they have a channel that runs through the dimer-dimer interface, where two identical thyroid hormone binding sites are located. proteins with sequence homology to ttr and with the characteristic c-terminal sequence motif tyr-arg-gly-ser, which is absent in ttrs, are named transthyretin-related proteins and are found in bacteria, fungi, animals and plants. trps are thought to be ttr ancestors. three ttl splice variants were reported; two of them are cytoplasmic and the other one is peroxisomal. in all three variants the n-terminal domain, ohcu decarboxylase, is composed of amino acids, whereas the c-terminal domain varies in length, having and amino acids for the 324-, 311- and 286-residues isoforms, respectively. the 324-residues isoform contains a type- peroxisomal targeting sequence, which is deleted in the other two isoforms. in most eukaryotes, 5-hiu hydrolases contain a pts- and ohcu decarboxylases contain a type- peroxisomal targeting sequence, except in plants. in a. thaliana these two enzymes are fused into a single polypeptide chain, containing a pts- sequence, between the two domains, in the 324-residues isoform. in a previous study, the c-terminal domain of ttl was expressed and studied separately. this single-domain was named transthyretin-like protein and is distinct from ttl, which contains both this c-terminal domain, with 5-hiu hydrolase activity, and a n-terminal domain, with ohcu decarboxylase activity, fused into the same polypeptide chain. tlp was described as a tetramer. the three-dimensional structure of trps from salmonella dublin, escherichia coli, zebrafish and the homologous domain from bacillus subtilis were determined. the overall structures of these trps and also the model predicted for tlp are very similar to those of ttrs, as expected due to sequence homology, although functionally they are different proteins. trps are hydrolases with a role in uric acid degradation and they do not bind thyroid hormones. ttl is the a. thaliana trp member, which contains an extra domain of amino acids as compared to other trps. until now, only trps from magnetospirillum magnetotacticum, bradyrhizobium japonicum,bacillus subtilis and plant species have been described as composed of two sequence domains, one related to ttr and the other with features common to ohcu decarboxylases. ttl was predicted by sequence homology as being a bifunctional enzyme with 5-hydroxyisourate hydrolase and ohcu decarboxylase activities, catalysing the two final steps in the uric acid degradation pathway. in most organisms, uric acid, the end product of purine degradation, is catabolised to allantoin. the process is initiated by uricase, which oxidizes uric acid into 5-hiu; this intermediate compound is then hydrolysed by 5-hiu hydrolase, leading to ohcu. finally, a third enzyme, ohcu decarboxylase, catalyses the decarboxylation of ohcu producing -allantoin. the spontaneous degradation of uric acid follows the same pathway, resulting into 5-hiu and ohcu intermediates, which can be further oxidized, producing potentially harmful reactive species. however, many organisms have an enzymatic pathway, possibly to convert these intermediates more rapidly into allantoin, a much less reactive species. the spontaneous pathway produces racemic allantoin, whereas the enzymatic pathway produces exclusively -allantoin. so far, two different functions have been assigned to ttl: i) a signalling role to the cytoplasmic isoforms and ii) an enzymatic role to the peroxisomal isoform. while the signalling role has already been studied, it is not known if the two enzymatic sequence domains that are in this protein fused into a long polypeptide chain do not compromise the role of each domain in an isolated form. in this work we studied the 311-residue cytoplasmic isoform, referred as ttl throughout the manuscript, to address the protein enzymatic role.  ttl oligomerization state recombinant ttl was purified and used to investigate the protein oligomerization state. we decided to use the method developed by siegel and monty in, because it allows calculation of the molecular mass of a protein independently of its shape, using a combination of the stokes radius derived from gel filtration chromatography and the sedimentation coefficient obtained from density gradient centrifugation. the gel filtration chromatograms are presented in fig. showing in fig. 2a a major peak of oligomeric ttl and in fig. 2b superposed chromatograms of standard stokes radius markers. using the equations described in methods, section on "determination of the protein oligomeric state", a and s were estimated to be nm and s, respectively, and with these values the native molecular mass for ttl was estimated to be kda. since each ttl subunit has kda, we concluded that ttl is a tetramer, which is in agreement with the fact that ttr, as well as a.thaliana tlp, also have a tetrameric structure. moreover, the calculated frictional ratio,, is consistent with a moderately elongated non-globular protein. thyroxin -binding assays since ttr is a tetramer with two equivalent t4-binding sites, we decided to study the binding of t to ttl, using radiolabeled thyroxin and ttr as the sample control. the ttr control has the typical behaviour of a specific t4-binding protein, in which the increase of cold t concentration causes the displacement of specifically bound radiolabeled t *. on the contrary, t *-binding to ttl, besides being very low, was constant at different t concentrations, showing a non-specific binding. this is a very different behaviour as compared to ttr, for which non-specific binding was only detected at the highest t concentration. we concluded that the binding of t to ttl is not significant and clearly non-specific, since it is not altered in the presence of a competitor. sequence alignment of human ttr, ttl and trps from salmonella dublin, b. subtilis and mouse are presented in fig. as mentioned previously, only the c-terminal domain of ttl has homology with ttr or trps. several amino acids were reported as important for the binding of t to ttr, namely lys glu thr and thr. interestingly, they are not conserved in ttl, being replaced by his arg his and tyr respectively, which are amino acids conserved in trps, and were reported to be important for 5-hiu hydrolase catalytic activity. according to our sequence alignment data and the three-dimensional structure of several ttr and trp proteins, these substitutions will probably have two main effects: i) create a narrower region at the inner part of the binding site due to the replacement of thr for tyr, which has a larger side chain and ii) alter the surface topography and introduce charge differences at the entrance of the channel, due to substitutions lys-his, glu-arg and thr-his. these modifications will probably exclude the binding of t to trps and ttl. ttl enzymatic activities since trps are functionally associated to 5-hiu hydrolases, we decided to study the predicted 5-hiu hydrolase and ohcu decarboxylase activities for ttl in vitro. the experiments were started by testing how ttl would affect the activity of uricase over uric acid by monitoring the differences in absorbance at nm in the absence or presence of ttl. although uricase alone was able to decompose uric acid, its activity was significantly accelerated in the presence of ttl. by contrast, ttr did not influence this reaction. this result is consistent with the predicted 5-hiu hydrolase and ohcu decarboxylase activities. the rapid consumption of 5-hiu and ohcu should favour an equilibrium shift in the reaction catalysed by uricase. 5-hiu hydrolase activity was demonstrated in vitro for single-domain trps present in s. dublin, b. subtilis and mouse. the authors reported that uric acid was converted into 5-hiu by uricase and both the formation and subsequent degradation of this compound was monitored at nm, since ohcu does not absorb at this wavelength. in our assay conditions, within seconds incubation of uric acid with the candida sp. uricase, the production of 5-hiu reached the maximum level. at this point, ttl was added to the reaction solution resulting in a rapid decrease in absorbance at nm. as expected, addition of ttr, which was used as the negative control, failed to accelerate 5-hiu degradation. this result shows that ttl is an enzyme that facilitates the hydrolysis of 5-hiu and also indicates that most probably four protein c-terminal domains associate forming a tetrameric structure, since the 5-hiu hydrolase catalytic site are located at the dimer-dimer interface in all structurally and functionally characterised trp members. although the crystal structure of the n-terminal ohcu domain of ttl from a. thaliana, which contains conserved residues critical for the ohcu decarboxylase activity, was recently reported, its predicted enzyme activity was not experimentally demonstrated. in fact, it was only observed in vitro in an isolated domain of a bifunctional enzyme from b. subtilis and also in zebrafish ohcu decarboxylase. monitoring the decarboxylase activity of ttl by spectrophotometry is not straightforward since ohcu absorbs below nm and its absorption spectrum overlaps with that of 5-hiu. therefore, measurements at nm are likely to be a sum of contributions by 5-hiu and ohcu. however, the spontaneous degradation observed at this wavelength is clearly distinct from what is observed at nm. the spontaneous degradation followed at nm shows that 5-hiu is completely degraded in seconds, thus the spontaneous absorbance decay measured after this point at nm is exclusively due to the spontaneous degradation of ohcu. therefore this degradation was used as control in the experiments testing the effect of ttl on degradation of ohcu. in presence of ttl the degradation of 5-hiu followed at nm was completed in seconds therefore after this point the absorbance decay measured at nm corresponds to the degradation of ohcu affected by ttl. the rate of this degradation is excessively high as compared to the control spontaneous degradation of ohcu. addition of ttl accelerated significantly the degradation of ohcu, supporting the decarboxylase activity of the protein. taken together, our results demonstrated that ttl has 5-hiu hydrolase and ohcu decarboxylase activities in vitro, and they are both performed at the same time. discussion it seems that the ttr domain evolved from an enzyme present in bacteria, fungi, plants and animals to a hormone transporter present in vertebrates. its function changed completely, although there are only minor differences in sequence. in the present study, we showed that ttl from a. thaliana is a tetramer with a moderately elongated shape. the tetrameric structure is in agreement with the observation that the protein has 5-hiu hydrolase activity. we expect that four c-terminal domains associate in a tetramer forming two active sites, as reported for trps, which contain only one domain. moreover, the elongated shape is possibly due to the n-terminal ohcu decarboxylase domain associated to each of the four c-terminal sequences forming the tetramer. the molecular model of this domain was determined in the presence of allantoin; it is an alpha-domain that associates as dimers in its crystal form. however, a careful inspection of the available molecular models for trps and ohcu decarboxylase domain indicates that allantoin binding site is not in the region of the interaction between the two subunits that form the dimer and this kind of association most probably will not be present in ttl, since the ttl tetramer will be formed through contacts among the c-terminal domains. we also demonstrated that ttl does not bind t in vitro, corroborating the observation that the ligand-binding site has characteristics which are different from those present in ttr. these results are in agreement with previous observations, since the functionally characterized trps do not bind thyroid hormones and are 5-hiu hydrolases with active sites located in the regions corresponding to the t4-binding sites in ttr. the ohcu decarboxylase activity was also detected in the full-length ttl, indicating that the n-terminal domains do not inhibit the active sites formed by the c-terminal regions; the reverse is also valid, thus supporting the idea of a bifunctional enzyme. ttl was initially characterized as a specific bri1-interactor that regulates br-mediated plant growth. here we show that the same protein is also enzymatically active. several proteins with at least two totally different functions have been reported and named moonlighting proteins; the switch between functions is due to changes in cellular localization, cell type, oligomeric state or cellular concentration of a ligand. we think that ttl is a moonlighting protein that switches its function according to its subcellular location. in eukaryotes, peroxisomes are the major organelles where oxidative reactions with molecular oxygen consumption are carried out. in these organisms, uricase is located in peroxisomes, oxidizing uric acid to 5-hiu. as a consequence, 5-hiu accumulates in peroxisomes, providing the initial ttl substrate. therefore, in peroxisomes, where uric acid degradation occurs, ttl, through its 324-residues isoform, functions as a bifunctional enzyme with 5-hiu hydrolase and ohcu decarboxylase activities. in the cytoplasm, 5-hiu and ohcu are absent, since they are produced in peroxisomes. consequently, the ttl 311-residues isoform most probably will not have these enzymatic functions. moreover, it was shown to interact with bri mediating br-regulated plant growth. bri is an intrinsic membrane protein, located in the plasma membrane and containing a cytoplasmic kinase domain that interacts with ttl. the main difference between the 324- and 311-residues isoforms is the presence or absence of pts- the subcellular location of three ttl isoforms is probably fundamental to define their role in vivo. ttl 324-residues isoform location into peroxisomes was already shown by fluorescence microscopy, and provided conclusive results regarding its import. it was also observed that its import efficiency was low and it was hypothesized that could result from a low accessibility of the internal targeting signal to the peroxisomal import machinery. internal pts- signal sequences are thought to be rare in nature and to occur preferentially in bifuctional enzymes resulting from gene fusion. the 311-residues isoform will probably remain in the cytoplasm in vivo, since it does not contain the pts- sequence. therefore in the absence of uric acid degradation intermediates, it interacts with the plasma membrane-located bri mediating br-regulated plant growth. attempts for the three-dimensional structure determination of ttl are in progress and should represent a fundamental step in order to clarify ttl function, namely its signalling role in br-mediated responses.  the aminoacid sequence of ttl from a. thaliana reveals two domains similar to 5-hiu hydrolase and ohcu decarboxylase. as a consequence, ttl was predicted to be a bifunctional enzyme. here we reported the first experimental data showing that in fact the protein has both enzymatic activities in vitro. moreover, we confirmed that ttl is in fact a bifunctional enzyme, by showing that it performs both activities simultaneously. 
we also found evidence for 5-hiu hydrolase and ohcu decarboxylase activities in vitro, in the full-length protein. the arabidopsis thaliana transthyretin-like protein is a tetrameric bifunctional enzyme, since it has 5-hiu hydrolase and ohcu decarboxylase activities, which were simultaneously observed in vitro. moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase and 5-hydroxyisourate hydrolase. experimental evidence is fundamental for knowing if ttl is a tetrameric protein, formed by the association of the 5-hiu hydrolase domains and, in this case, if the structural arrangement allows for ohcu decarboxylase activity. trps are single domain proteins that form tetrameric structures with 5-hiu hydrolase activity. the results show that ttl is composed of four subunits, with a moderately elongated shape. arabidopsis thaliana transthyretin-like protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. ttl is a member of the transthyretin-related protein family, which comprises a number of proteins with sequence homology to transthyretin and the characteristic c-terminal sequence motif tyr-arg-gly-ser.
 arabidopsis thaliana transthyretin-like protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase and 5-hydroxyisourate hydrolase. ttl is a member of the transthyretin-related protein family, which comprises a number of proteins with sequence homology to transthyretin and the characteristic c-terminal sequence motif tyr-arg-gly-ser. trps are single domain proteins that form tetrameric structures with 5-hiu hydrolase activity. experimental evidence is fundamental for knowing if ttl is a tetrameric protein, formed by the association of the 5-hiu hydrolase domains and, in this case, if the structural arrangement allows for ohcu decarboxylase activity. this work reports about the biochemical and functional characterization of ttl.  the ttl gene was cloned and the protein expressed and purified for biochemical and functional characterization. the results show that ttl is composed of four subunits, with a moderately elongated shape. we also found evidence for 5-hiu hydrolase and ohcu decarboxylase activities in vitro, in the full-length protein.  the arabidopsis thaliana transthyretin-like protein is a tetrameric bifunctional enzyme, since it has 5-hiu hydrolase and ohcu decarboxylase activities, which were simultaneously observed in vitro. 