BACKGROUND
uridine phosphorylase  is a ubiquitous enzyme involved in pyrimidine salvage and maintenance of uridine homeostasis  <cit> .
it catalyzes the reversible phosphorolysis of uracil ribosides and analogous compounds to their respective nucleobases and ribose-1-phosphate.
the structural mechanisms underlying the catalytic activity of this enzyme have been extensively studied through analysis of e. coli upp   <cit>  and more recently the s. typhimurium homologue  <cit> .
these structures have shown upp to belong to the nucleoside phosphorylase  super-family of proteins in the np-i subset of proteins possessing α/β folds and trimeric or hexameric  quaternary assemblies  <cit> .
additionally, based on conservation of sequence and ligand binding site architecture, it is probable that the general catalytic mechanism is retained between upp and related purine nucleoside phosphorylases .
humans possess two isoforms of upp  of which hupp <dig> is more widely distributed, more abundantly expressed, and better characterized.
hupp <dig> has been a subject of interest to cancer researchers due to its role in the activation of pyrimidine nucleoside analogues used in chemotherapy, such as 5-fluorouracil   <cit>  and its prodrug, capecitabine.
further, elevated levels of hupp <dig> activity in certain tumours may contribute positively to the selectivity of these cancer-killing reagents  <cit> .
other studies have explored the potential of hupp <dig> inhibitors as a means of raising endogenous uridine levels during the course of fluoropyrimidine nucleoside treatment, in order to protect normal tissues from the toxicity of these drugs  <cit> .
these inhibitors have been developed from a family of acyclouridine analogues and include 5-benzylacyclouridine   <cit> , a compound that has been investigated in clinical trials for its ability to increase the therapeutic index of 5-fu through induction of such uridine-mediated rescue  <cit> .
while structures of ecupp with bau and related molecular analogues have revealed the general mechanistic features of this competitive inhibitor which obstructs the enzyme's active site  <cit> , the structure of hupp <dig> and the details of its specific interactions with this potentially clinically-valuable drug have not been elucidated.
in the present study, we have determined the crystallographic structure of hupp <dig> at high resolution, both in ligand-free and bau-bound conformations .
the structures reveal significant global and local differences between the human enzyme and its microbial counterparts.
this knowledge will be valuable in the future discovery and design of more potent and specific inhibitors of hupp <dig> for development of improved chemotherapeutic regimens.
RESULTS
hupp <dig> is a dimeric enzyme
recombinantly expressed hupp <dig> was noted to possess a smaller than expected quaternary assembly during purification by gel filtration.
analysis by both calibrated size-exclusion chromatography and multi-angle static light scattering  confirmed that the enzyme was dimeric in solution, in contrast to the hexameric assembly predicted from sequence analysis and protein family relationships  <cit> .
consistent with this observation, both solved crystal forms also yielded dimeric enzymes, verifying that the human form of this enzyme has lost the higher order secondary trimerization of dimers seen in microbial homologues.
overall structure of hupp1
the overall global fold of hupp <dig> is highly similar to its microbial upp homologues and other members of the np-i family of proteins, with two notable exceptions.
first, there is substantial additional architecture at the n-terminus of the protein forming a strand-turn-strand structure bracketed by two short helices .
second, the third α-helix of the bacterial enzymes  has been replaced by an additional strand-turn-strand motif.
while not directly impacting the structure of the active site of the enzyme, both of these changes increase the buried surface area of the dimer interface .
quantitatively, these modifications together increase the interface area between subunits approximately 18%, from  <dig> sq.
Å in the e. coli enzyme to  <dig> sq.
Å in hupp <dig>
the alterations occurring around the region of microbial helix α <dig> also provide a molecular explanation for the loss of trimerization of dimers in the human enzyme.
the long loop preceding β* in hupp <dig> sterically interferes with the formation of the protein-protein interface for dimer oligomerization seen in previous structures.
additionally, the hydrophobic residues  that form the core of the trimer assembly surface in ecupp have been mutated to more polar, solvent-compatible residues in hupp <dig> .
another difference comparing the hupp <dig> structure to previously studied microbial counterparts is that the stabilizing k+ binding site observed at the dimer interface of those proteins  <cit>  is not occupied in either of the determined human structures .
from structure-based sequence alignment it becomes clear that the primary coordinating residue  is deleted from the human proteins with surprisingly little distortion in the surrounding architecture .
that k+ no longer plays a role in enzyme stabilization was unexpected, as high levels of salt needed to be maintained in all protein purification and crystallization buffers to prevent hupp <dig> aggregation and precipitation.
binding of the inhibitor bau to the hupp <dig> active site
analysis of the catalytic active site reveals that bau binds hupp <dig> in a manner consistent with that previously characterized using ecupp  <cit> .
in actuality, all residues interacting with the natural uridine and phosphate ligands are strictly conserved between the two enzymes .
this fact was unclear prior to this study, as regions of these proteins between β <dig> and β <dig> lack sufficient sequence similarity for accurate alignment in the absence of additional structural data.
despite strict identity retention in ligand-binding residues, the coordination of the phosphate ion deviates from that observed in previous microbial structures .
specifically, one of the three pocket arginine residues  is bent away from the anion, leaving only two coordinating guanidinium groups, one from each subunit of the dimer, to bind the substrate.
the reason for the unexpected conformation of this arginine is not immediately apparent, as the backbone cα of this residue is in an equivalent location with respect to the phosphate molecule as in other structurally analyzed upps.
however, in the ligand-free structure of hupp <dig> this residue is found to coordinate a sulfate ion from the crystallization liquor at a position  <dig>  Å removed from the catalytic phosphate binding pocket.
this finding may imply that in the human enzyme this residue has been adapted to ligand recruitment from the cytoplasm rather than ligand binding in the active site.
additional differences between the active sites of human and bacterial enzymes become apparent when the analysis is extended to regions of these proteins contacting chemical modifications of acyclouridine analogues such as bau.
the benzyl ring of this molecule is π-stacked on one side by tyr <dig>  a residue that is conserved as phenylalanine in microbes.
more significantly, the edge of this benzyl moiety interacts with a loop region that is structured very differently in hupp <dig> than in ecupp.
in structures of the e. coli enzyme, the equivalent loop appears highly flexible, to the point of frequently being too disordered for modelling.
these studies concluded that this loop undergoes an 'induced-fit' conformational change upon ligand or inhibitor binding   <cit> .
in contrast, the same loop in hupp <dig> is rigid with relatively lower thermal factors and remains structurally unaltered by bau binding.
this stability is likely the result of the insertion within this loop in hupp <dig> of two additional residues as compared to bacterial variants .
these structural differences have major implications toward understanding how various other existing or theoretical acyclouridine derivatives will interact with the human enzyme.
ligand induced motion between hupp <dig> domains
determination of both ligand-free and bau-bound forms of hupp <dig> allows direct analysis of the conformational changes induced in the enzyme upon substrate binding.
to this end, there are only subtle differences in the fold and residue orientation between overlaid monomers of hupp <dig>  however, there is a dramatic inter-domain motion between monomers leading to 3– <dig> Å changes in the relative positioning of the loops undergoing the greatest movement .
this hinge-type conformational change results in a closure of the active site around its substrates and appears to be driven by the formation of interactions between arg <dig> and phosphate, his <dig> and the ribose sugar group, and tyr <dig> and the nucleobase.
it is noteworthy that this domain motion has not been observed in any of the previously determined microbial structures despite multiple ligand-free structures, suggesting that the human enzyme is more mobile than its bacterial homologues.
discussion
human uridine phosphorylase  <dig> has been the molecular target for the design of specific inhibitors intended to boost endogenous uridine levels for the purpose of rescuing normal tissues from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents.
the structures reported here reveal significant differences between hupp <dig> and previously characterized microbial upps that have meaningful implications toward the rational design of novel reagents with improved potency.
in contrast to expectations, hupp <dig> is dimeric, having lost the higher order assembly  that results in hexameric rings in more primitive upps.
possibly as a direct consequence, hupp <dig> has two major architectural modifications, both of which serve to increase the contact surface area between domains.
these alterations likely increase the stability of dimer association, potentially as compensation for stability lost on dissolution of the larger ring complex.
another implication of the loss of hexameric structure appears to be that the human enzyme possesses greater inter-domain flexibility than its microbial counterparts.
while the ability of enzymes to 'breathe' to facilitate substrate and product exchange is a common phenomenon, it has not been observed in previous studies of upps.
whether this inter-subunit motion has a meaningful affect on the kinetics and catalytic turnover rates of hupp <dig>  in comparison to ecupp, remains to be investigated.
however, the 'open' conformation of hupp <dig> provides the opportunity to potentially develop a novel class of allosteric inhibitors of this enzyme that lock the protein in a functionally disabled form, with its catalytic residues too separated to lyse substrates.
CONCLUSIONS
in this report, we present the first structures of human uridine phosphorylase  <dig>  the specific biochemical features of the human version of this ubiquitous enzyme revealed by these studies offer improved understanding of how clinically-evaluated specific inhibitors, such as bau, bind to and inactivate this protein.
the molecular details regarding the residues of hupp <dig> lining the binding pocket for such acyclouridine derivatives offer clear approaches to improving the specificity of these compounds to the human enzyme.
previous studies have successfully increased the affinity of acyclouridine analogues to hupp <dig> through the creation of more hydrophobic variations of bau such as 5-m-benzyloxybenzylacyclouridine  <cit> .
given the recent revelation that bau may have cross-reactivity to other human enzymes such as aldehyde oxidase  <cit> , designing rational alterations in the moieties extending beyond the first benzyl ring may result in substantial improvements in both compound activity and selectivity.
extremely high affinity inhibitors of human pnp  have been obtained by using transition state mimetics derived from immucillins  <cit> .
with the conservation of active site structure and catalytic mechanism among all np-i family members, similarly potent antagonists of hupp <dig> should be creatable.
when combined with strategically selected structure-based modifications to optimize specificity for this enzyme, there is a strong potential to develop improved pharmaceuticals for incorporation into novel chemotherapeutic regimens with increased efficacy and reduced toxicity.

 uridine phosphorylase is a ubiquitous enzyme involved in pyrimidine salvage and maintenance of uridine homeostasis. it catalyzes the reversible phosphorolysis of uracil ribosides and analogous compounds to their respective nucleobases and ribose-1-phosphate. the structural mechanisms underlying the catalytic activity of this enzyme have been extensively studied through analysis of e. coli upp and more recently the s. typhimurium homologue. these structures have shown upp to belong to the nucleoside phosphorylase super-family of proteins in the np-i subset of proteins possessing α/β folds and trimeric or hexameric quaternary assemblies. additionally, based on conservation of sequence and ligand binding site architecture, it is probable that the general catalytic mechanism is retained between upp and related purine nucleoside phosphorylases. humans possess two isoforms of upp of which hupp is more widely distributed, more abundantly expressed, and better characterized. hupp has been a subject of interest to cancer researchers due to its role in the activation of pyrimidine nucleoside analogues used in chemotherapy, such as 5-fluorouracil and its prodrug, capecitabine. further, elevated levels of hupp activity in certain tumours may contribute positively to the selectivity of these cancer-killing reagents. other studies have explored the potential of hupp inhibitors as a means of raising endogenous uridine levels during the course of fluoropyrimidine nucleoside treatment, in order to protect normal tissues from the toxicity of these drugs. these inhibitors have been developed from a family of acyclouridine analogues and include 5-benzylacyclouridine, a compound that has been investigated in clinical trials for its ability to increase the therapeutic index of 5-fu through induction of such uridine-mediated rescue. while structures of ecupp with bau and related molecular analogues have revealed the general mechanistic features of this competitive inhibitor which obstructs the enzyme's active site, the structure of hupp and the details of its specific interactions with this potentially clinically-valuable drug have not been elucidated. in the present study, we have determined the crystallographic structure of hupp at high resolution, both in ligand-free and bau-bound conformations. the structures reveal significant global and local differences between the human enzyme and its microbial counterparts. this knowledge will be valuable in the future discovery and design of more potent and specific inhibitors of hupp for development of improved chemotherapeutic regimens.  hupp is a dimeric enzyme recombinantly expressed hupp was noted to possess a smaller than expected quaternary assembly during purification by gel filtration. analysis by both calibrated size-exclusion chromatography and multi-angle static light scattering confirmed that the enzyme was dimeric in solution, in contrast to the hexameric assembly predicted from sequence analysis and protein family relationships. consistent with this observation, both solved crystal forms also yielded dimeric enzymes, verifying that the human form of this enzyme has lost the higher order secondary trimerization of dimers seen in microbial homologues. overall structure of hupp1 the overall global fold of hupp is highly similar to its microbial upp homologues and other members of the np-i family of proteins, with two notable exceptions. first, there is substantial additional architecture at the n-terminus of the protein forming a strand-turn-strand structure bracketed by two short helices. second, the third α-helix of the bacterial enzymes has been replaced by an additional strand-turn-strand motif. while not directly impacting the structure of the active site of the enzyme, both of these changes increase the buried surface area of the dimer interface. quantitatively, these modifications together increase the interface area between subunits approximately 18%, from sq. Å in the e. coli enzyme to sq. Å in hupp the alterations occurring around the region of microbial helix α also provide a molecular explanation for the loss of trimerization of dimers in the human enzyme. the long loop preceding β* in hupp sterically interferes with the formation of the protein-protein interface for dimer oligomerization seen in previous structures. additionally, the hydrophobic residues that form the core of the trimer assembly surface in ecupp have been mutated to more polar, solvent-compatible residues in hupp. another difference comparing the hupp structure to previously studied microbial counterparts is that the stabilizing k+ binding site observed at the dimer interface of those proteins is not occupied in either of the determined human structures. from structure-based sequence alignment it becomes clear that the primary coordinating residue is deleted from the human proteins with surprisingly little distortion in the surrounding architecture. that k+ no longer plays a role in enzyme stabilization was unexpected, as high levels of salt needed to be maintained in all protein purification and crystallization buffers to prevent hupp aggregation and precipitation. binding of the inhibitor bau to the hupp active site analysis of the catalytic active site reveals that bau binds hupp in a manner consistent with that previously characterized using ecupp. in actuality, all residues interacting with the natural uridine and phosphate ligands are strictly conserved between the two enzymes. this fact was unclear prior to this study, as regions of these proteins between β and β lack sufficient sequence similarity for accurate alignment in the absence of additional structural data. despite strict identity retention in ligand-binding residues, the coordination of the phosphate ion deviates from that observed in previous microbial structures. specifically, one of the three pocket arginine residues is bent away from the anion, leaving only two coordinating guanidinium groups, one from each subunit of the dimer, to bind the substrate. the reason for the unexpected conformation of this arginine is not immediately apparent, as the backbone cα of this residue is in an equivalent location with respect to the phosphate molecule as in other structurally analyzed upps. however, in the ligand-free structure of hupp this residue is found to coordinate a sulfate ion from the crystallization liquor at a position Å removed from the catalytic phosphate binding pocket. this finding may imply that in the human enzyme this residue has been adapted to ligand recruitment from the cytoplasm rather than ligand binding in the active site. additional differences between the active sites of human and bacterial enzymes become apparent when the analysis is extended to regions of these proteins contacting chemical modifications of acyclouridine analogues such as bau. the benzyl ring of this molecule is π-stacked on one side by tyr a residue that is conserved as phenylalanine in microbes. more significantly, the edge of this benzyl moiety interacts with a loop region that is structured very differently in hupp than in ecupp. in structures of the e. coli enzyme, the equivalent loop appears highly flexible, to the point of frequently being too disordered for modelling. these studies concluded that this loop undergoes an'induced-fit' conformational change upon ligand or inhibitor binding. in contrast, the same loop in hupp is rigid with relatively lower thermal factors and remains structurally unaltered by bau binding. this stability is likely the result of the insertion within this loop in hupp of two additional residues as compared to bacterial variants. these structural differences have major implications toward understanding how various other existing or theoretical acyclouridine derivatives will interact with the human enzyme. ligand induced motion between hupp domains determination of both ligand-free and bau-bound forms of hupp allows direct analysis of the conformational changes induced in the enzyme upon substrate binding. to this end, there are only subtle differences in the fold and residue orientation between overlaid monomers of hupp however, there is a dramatic inter-domain motion between monomers leading to 3– Å changes in the relative positioning of the loops undergoing the greatest movement. this hinge-type conformational change results in a closure of the active site around its substrates and appears to be driven by the formation of interactions between arg and phosphate, his and the ribose sugar group, and tyr and the nucleobase. it is noteworthy that this domain motion has not been observed in any of the previously determined microbial structures despite multiple ligand-free structures, suggesting that the human enzyme is more mobile than its bacterial homologues. discussion human uridine phosphorylase has been the molecular target for the design of specific inhibitors intended to boost endogenous uridine levels for the purpose of rescuing normal tissues from the toxicity of fluoropyrimidine nucleoside chemotherapeutic agents. the structures reported here reveal significant differences between hupp and previously characterized microbial upps that have meaningful implications toward the rational design of novel reagents with improved potency. in contrast to expectations, hupp is dimeric, having lost the higher order assembly that results in hexameric rings in more primitive upps. possibly as a direct consequence, hupp has two major architectural modifications, both of which serve to increase the contact surface area between domains. these alterations likely increase the stability of dimer association, potentially as compensation for stability lost on dissolution of the larger ring complex. another implication of the loss of hexameric structure appears to be that the human enzyme possesses greater inter-domain flexibility than its microbial counterparts. while the ability of enzymes to'breathe' to facilitate substrate and product exchange is a common phenomenon, it has not been observed in previous studies of upps. whether this inter-subunit motion has a meaningful affect on the kinetics and catalytic turnover rates of hupp in comparison to ecupp, remains to be investigated. however, the'open' conformation of hupp provides the opportunity to potentially develop a novel class of allosteric inhibitors of this enzyme that lock the protein in a functionally disabled form, with its catalytic residues too separated to lyse substrates.  in this report, we present the first structures of human uridine phosphorylase the specific biochemical features of the human version of this ubiquitous enzyme revealed by these studies offer improved understanding of how clinically-evaluated specific inhibitors, such as bau, bind to and inactivate this protein. the molecular details regarding the residues of hupp lining the binding pocket for such acyclouridine derivatives offer clear approaches to improving the specificity of these compounds to the human enzyme. previous studies have successfully increased the affinity of acyclouridine analogues to hupp through the creation of more hydrophobic variations of bau such as 5-m-benzyloxybenzylacyclouridine. given the recent revelation that bau may have cross-reactivity to other human enzymes such as aldehyde oxidase, designing rational alterations in the moieties extending beyond the first benzyl ring may result in substantial improvements in both compound activity and selectivity. extremely high affinity inhibitors of human pnp have been obtained by using transition state mimetics derived from immucillins. with the conservation of active site structure and catalytic mechanism among all np-i family members, similarly potent antagonists of hupp should be creatable. when combined with strategically selected structure-based modifications to optimize specificity for this enzyme, there is a strong potential to develop improved pharmaceuticals for incorporation into novel chemotherapeutic regimens with increased efficacy and reduced toxicity. 
these distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower upp activity. they also reveal in detail the mechanism by which bau engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. the structural details underlying hupp1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of bau and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. the structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial upps. uridine phosphorylase is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hupp than in previously studied microbial homologues. in response to this limitation, specific inhibitors of upp, such as 5-benzylacyclouridine, have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-fu and its derivatives, so as to increase the therapeutic index of these agents. in this report we present the high resolution structures of human uridine phosphorylase in ligand-free and bau-inhibited conformations. additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. the observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous upp structures and may result from the simpler oligomeric organization.
 uridine phosphorylase is a key enzyme of pyrimidine salvage pathways, catalyzing the reversible phosphorolysis of ribosides of uracil to nucleobases and ribose 1-phosphate. it is also a critical enzyme in the activation of pyrimidine-based chemotherapeutic compounds such a 5-fluorouracil and its prodrug capecitabine. additionally, an elevated level of this enzyme in certain tumours is believed to contribute to the selectivity of such drugs. however, the clinical effectiveness of these fluoropyrimidine antimetabolites is hampered by their toxicity to normal tissue. in response to this limitation, specific inhibitors of upp, such as 5-benzylacyclouridine, have been developed and investigated for their ability to modulate the cytotoxic side effects of 5-fu and its derivatives, so as to increase the therapeutic index of these agents.  in this report we present the high resolution structures of human uridine phosphorylase in ligand-free and bau-inhibited conformations. the structures confirm the unexpected solution observation that the human enzyme is dimeric in contrast to the hexameric assembly present in microbial upps. they also reveal in detail the mechanism by which bau engages the active site of the protein and subsequently disables the enzyme by locking the protein in a closed conformation. the observed inter-domain motion of the dimeric human enzyme is much greater than that seen in previous upp structures and may result from the simpler oligomeric organization.  the structural details underlying hupp1's active site and additional surfaces beyond these catalytic residues, which coordinate binding of bau and other acyclouridine analogues, suggest avenues for future design of more potent inhibitors of this enzyme. notably, the loop forming the back wall of the substrate binding pocket is conformationally different and substantially less flexible in hupp than in previously studied microbial homologues. these distinctions can be utilized to discover novel inhibitory compounds specifically optimized for efficacy against the human enzyme as a step toward the development of more effective chemotherapeutic regimens that can selectively protect normal tissues with inherently lower upp activity. 