BACKGROUND
oligo- and polyfructosyl-sucrose polymers, collectively known as fructans, are synthesized by a significant number of bacteria and an estimated  <dig>  plant species  <cit>  either replacing or supplementing starch as a carbohydrate reserve.
fructans are synthesised from sucrose as the sole substrate, sharing a single sucrose starter unit βfru) to which fructofuranose units can become attached at various positions of the fructosyl or glucosyl ring, resulting in highly branched or linear polymers in a species-dependent fashion  <cit> .
the two prevailing forms of fructans are β -linked levan βfru n) and β -linked inulin βfru n), with the degree of polymerization of fructans varying between a few hundred and several thousands saccharide units.
in plants, fructans are thought to contribute to drought and frost tolerance by preventing rupture of cell membranes  <cit> , whereas in bacteria fructans are known to serve as food storage and to contribute to biofilm formation  <cit> .
while fructan synthesis in plants involves at least two enzymes with different fructosyl-donor and – acceptor specificities, levan or inulin synthesis in bacteria requires only a single enzyme, with sucrose initially acting as both fructosyl donor and acceptor substrate.
levansucrase , encoded in bacillus subtilis by the sacb gene, catalyses the fructosyl transfer reaction
sucrose + acceptor → glucose + fructosyl-acceptor
in vitro, levansucrase mediates invertase  or polymerase activity depending on the concentration of the fructosyl donor substrate: below  <dig> mm, sucrose is cleaved into glucose and fructose with water acting as fructosyl-acceptor, whereas above this concentration levan production occurs through successive transfer of fructosyl units from sucrose to the fructosyl 6'-hydroxyl -linkage) of the acceptor substrate  <cit> .
levansucrase belongs to family  <dig> of glycoside hydrolases  according to the classification of carbohydrate-active enzymes .
while structurally and functionally diverse, glycoside hydrolases share the requirement of two juxtaposed acidic side chains, acting as proton donor  and catalytic nucleophile or catalytic base, respectively.
hydrolysis of the glycosidic bond can result in either inversion or retention of the anomeric configuration in the substrate, corresponding to a single or double displacement reaction mechanism, respectively  <cit> .
kinetic studies of levansucrase established that sucrose hydrolysis follows a ping-pong kinetic reaction mechanism that retains the anomeric configuration and involves a covalently bound fructosyl-enzyme intermediate  <cit> .
we recently determined the crystal structures of b. subtilis levansucrase in the ligand-free form and bound to the fructosyl donor substrate sucrose  <cit> .
our structures established that the catalytic domain of gh family  <dig> enzymes folds into a 5-bladed β-propeller with the active site located in a deep axial pocket .
this fold is shared by the catalytic domain of gh family  <dig> of retaining enzymes  <cit> , as well as by the distantly related family  <dig> of inverting glycoside hydrolases  <cit>  .
in agreement with a rich body of biochemical data  <cit> , we proposed that the strictly conserved asp <dig>  and glu <dig>  represent the two canonical catalytic carboxylate groups, while a third invariant carboxylate, asp <dig>  may aid catalysis by stabilizing the transition state of the oxocarbenium ion by forming close hydrogen bond contacts with two of the fructosyl hydroxyls  <cit> .
while this assignment was confirmed in subsequent structural and biochemical studies of several ortho- and paralogs  <cit> , the characterization of the structural determinants of levan synthesis has remained incomplete.
in particular, the mode of and elements required for acceptor substrate binding remain unclear.
here, we report crystal structures of three single-site mutants, d86a, d247a and e342a, previously shown to be catalytically inactive  <cit> , in the ligand-free form, and that of the e342a mutant in complex with the fructosyl donor substrate raffinose.
comparisons between the ligand-free and substrate-bound structures shed light on a network of hydrogen bonds and ionic interactions surrounding the proton donor glu <dig>  this network reacts sensitively to presence or absence of the glu <dig> carboxylate, and to changes in the ligand-binding state.
we observed significant conformational flexibility of arg <dig>  a key residue in levan polymerisation, and propose that this role is linked to arg <dig> alternating between alternative rotamer states, facilitating participation in a transient docking site for the fructosyl acceptor.
RESULTS
apo structures of inactive mutants d86a, d247a and e342a
crystals of the inactive single site mutants d86a, d247a and e342a of b. subtilis levansucrase grew at similar solution conditions as the wild-type enzyme.
crystals were in space group p <dig>  diffracting to beyond  <dig>  Å. geometric constraints of the detector, rather than crystal quality limited data acquisition on the home source to a maximum resolution of  <dig>  Å .
the diffraction data of mutant-forms of levansucrase were highly isomorphous to those of crystal form i of wild-type levansucrase .
prior to structure building and refinement, structural differences were ascertained by way of difference fourier maps , and the structures were refined starting from the wild-type model, altering the mutation sites to alanine .
the backbone structures superimpose closely with that of the wild-type enzyme.
the root mean square deviation  for  <dig> backbone atoms between the mutant and wild type structures vary from  <dig>  Å  to  <dig>  Å .
the rmsd for  <dig> side chain atoms between mutants and wild type structures are  <dig>  Å or less , corresponding to ~ <dig> times the estimated coordinate error at  <dig>  Å. this indicates that overall the mutations induce only minimal structural changes.
the missing carboxylate groups in the d86a and d247a mutants are clearly marked by negative density in the difference fourier maps, but result in no other notable changes in the active site .
arsym = Σ/Σi
bhigh resolution shell in parentheses
arfree calculated using 5% of total reflections omitted from refinement
bramachandran statistics calculated using procheck
in marked contrast, mutation of the general acid glu <dig> to alanine has a profound impact on the rotamer state of functionally important side chains in the vicinity of the glu <dig> carboxylate .
in the apo structure of wild-type levansucrase, glu <dig> forms a tight salt bridge interaction with the guanido group of arg <dig>  and a strong hydrogen bond with the side chain hydroxyl of tyr <dig>  .
in e342a, the missing carboxylate prompts, firstly, the guanido group of arg <dig>  which is important for activity  <cit> , to swing about 90°  towards the axis of the β-propeller, overlapping in this configuration with the fructosyl binding site.
secondly, the e342a mutation eliminates the hydrogen bond to the tyr <dig> hydroxyl, causing a minor upward shift of the phenol ring .
thirdly, arg <dig> assumes an alternative rotamer state, involving an 80° rotation in χ <dig> and a near 90° rotation in χ <dig>  this drastic change in rotamer configuration of arg <dig>  which is required for polymerase activity  <cit> , is somewhat surprising: in wild-type levansucrase arg <dig> interacts through a  <dig> -Å hydrogen bond with tyr <dig>  but forms only a weak interaction with glu <dig> .
the alternative rotamer state of arg <dig> is ostensibly stabilised by a tight ionic interaction with glu <dig> , a residues involved in donor substrate binding .
in conclusion, the d247a and d86a mutations have little or no impact on the side chain configurations and interactions elsewhere in the active site, while the glu342a substitution has knock-on effects for the network of non-covalent interactions around the glu <dig> carboxylate.
we note however, that binding of the donor substrate to levansucrase-e342a largely restores the side chain configuration of apo wild-type levansucrase , prompting arg <dig> and arg <dig> to swing back into their original position.
the raffinose-bound complex of levansucrase-e342a
besides sucrose, levansucrase also accepts the trisaccharide d-raffinose αglcβfru) as fructosyl donor.
the products resulting from levansucrase-catalysed hydrolysis of raffinose are melibiose  and free fructose.
crystals of levansucrase-e342a were soaked in raffinose, followed by cryoprotection and flash-freezing in a  <dig> k nitrogen gas stream .
diffraction data to  <dig>  Å resolution were recorded in-house and difference electron density maps, comparing amplitudes of the raffinose-bound mutant and apo wild-type levansucrase, showed density that could be attributed unequivocally to the ligand .
with amplitudes on an  absolute scale, the occupancy of raffinose refined to a value near  <dig>  , suggesting full occupancy, while the average b factor of the raffinose atoms was  <dig>  Å <dig> compared to  <dig>  Å <dig> for the protein atoms .
in the refined structure, the raffinose ligand displays a configuration in which the planes of the three sugar rings are approximately orthogonal to each other: the fructose moiety lies almost 'flat' on the bottom of the active site, the glucose moiety rises up and the galactose ring is jutting out into the solvent .
the fructose and glucose rings superimpose closely with their counterparts in the sucrose-bound complex  <cit>  , as illustrated by an rmsd of  <dig>  Å for the atoms common between the two ligands.
following the terminology defined by davies et al.
<cit> , the active site of glycoside hydrolases can be divided into subsites with respect to the cleaved glycosidic bond.
applied to the present raffinose complex, subsite - <dig> coincides with the fructose, and subsites + <dig> and + <dig> with the glucose and galactose moieties, respectively .
it is apparent that, with the exception of arg <dig> and perhaps glu <dig>  side chains tend to form specificity-determining contacts with only one of the three subsites : the fructosyl moiety makes specificity-determining contacts with the side chains of trp <dig> , asp <dig> , arg <dig> , asp <dig> , and glu <dig> .
in subsite + <dig>  the 2-, 3- and 4-hydroxyls of the glucosyl moiety form tight h-bond contacts  with arg <dig> and glu <dig>  these specificity-determining contacts lock the fructosyl and glucosyl units into defined orientations, positioning the anomeric carbon of the fructosyl unit within  <dig>  Å of the nucleophile asp <dig>  and the glycosidic oxygen in close proximity  to the carboxylate of glu <dig>  in contrast, the galactosyl unit makes only few, water-mediated h-bonds, limited to the 6"-hydroxyl, with asn <dig> and tyr <dig>  whereas the 2"-, 3"- and 4"-hydroxyl groups point into solvent .
the configuration of active site residues in raffinose-bound e342a is very similar to wild-type levansucrase in the ligand-free form, but contrasts with the apo form the e342a mutant.
owing to steric overlap with the fructosyl moiety, arg <dig> swings back from the rotamer state in apo-e342a to the wild-type configuration.
similarly, arg <dig> resumes the wild-type configuration, stabilised by h-bond interactions  with the 2-, 3-hydroxyls of the glucosyl ring.
yet, in the donor substrate-bound state tyr <dig> tilts by ~20° towards the floor of the active site .
as a consequence, the h-bond between oh of tyr <dig> and nε of arg <dig> is not preserved in either of the raffinose- and sucrose-bound complexes .
the dip of tyr <dig> appears to result from van der waals interactions with the glucosyl ring  and with the guanido group of arg <dig> .
through the contacts to the ligand, arg <dig> inserts deeper into the active site than in the apo structure of wild-type levansucrase.
it remains unresolved whether the 20°-tilt of tyr <dig> would also occur in a donor substrate complex of the wild-type enzyme .
it is noteworthy, that the position of the missing carboxylate of glu <dig> is marked by two water molecules , a feature consistent with the sucrose-bound complex  <cit> .
among the protein side chains, there is very little change between sucrose- and raffinose-bound structures of e342a .
the rmsd of side chain atoms located within  <dig>  Å of the substrate  is  <dig>  Å. thus within the limits of the estimated coordinate error the two structures are identical with respect to the protein framework.
discussion
we present here the raffinose-bound complex of levansucrase-e342a in addition to the apo crystal structures of the three inactive point mutants d86a, d247a and e342a.
our previous study of the structure of b. subtilis levansucrase  <cit>  established, based largely on structural arguments, the function of these three strictly conserved carboxylate side chains in the active site.
the present raffinose complex reinforces a view that donor substrate recognition in b. subtilis levansucrase rests primarily on the common sucrosyl unit, whereas the galactosyl moiety, which protrudes out of the active site, makes only a few water-mediated h-bonds, pointing three unliganded hydroxyl groups to the bulk solvent.
this mode of binding is echoed by the raffinose-bound complex of thermotoga maritima invertase, which belongs to gh family  <dig> and which, like levansucrase, mediates hydrolysis of the glycosidic bond through a double displacement reaction mechanism  <cit> .
in the latter study, an inert complex was facilitated by mutating the proton donor  to aspartic acid.
superimposing the two complexes by matching the positions of  <dig> ligand atoms , reveals a very similar geometry of the ligand, and an almost perfect overlap of the catalytic residues.
while there is significant variation of structural elements mediating specificity-determining contacts with the ligand, specific recognition of the outermost saccharide unit is weak in both structures and does not involve direct h-bonds.
nevertheless, the t. maritima complex includes notable van der waals interactions between the galactose and trp <dig>  for which there is no counterpart in b. subtilis levansucrase .
the importance of glu <dig>  arg <dig> and arg <dig> in forming specificity-determining contacts with the donor substrate is illustrated by the mutagenesis data obtained for levansucrase from bacillus megaterium .
mutating these side chains was reported as nearly abolishing hydrolase activity  <cit> .
the functional assignment of the catalytic side chains in the active site of levansucrase raises the question of which structural features ensure that the requirement for differential protonation states of asp <dig>  and glu <dig>   <cit>  is met.
since the ph optimum of levansucrase lies in the range of ph  <dig>  to  <dig>   <cit> , the pka of glu <dig> must be raised to at least  <dig> –  <dig>  in order to serve as the general acid.
this could occur, for instance, through juxtaposition to hydrophobic or acidic side chains  <cit> .
yet, the shortest contacts of glu <dig>  are with tyr <dig>  and arg <dig> , neither of which is likely to result in the required effect.
still, a structure-based computational analysis using uhbd and scripts written by the wade group  <cit>  indicated a pka of glu <dig> at least  <dig> ph units above that of the free amino acid, arguing that a rise of the pka is the result of the cumulative effect of the ensemble of side chains and contacts surrounding glu <dig>  an environment that includes three acidic side chains, glu <dig> , glu <dig> , and asp <dig> .
moreover, from the sucrose- and raffinose-bound complexes of the e342a mutant one can infer that donor substrate binding results in additional contacts with hydrophobic moieties of the sugar, and it is conceivable that a pka shift only occurs upon substrate binding.
the distinct effects caused by the point mutation e342a on the configuration of adjacent side chains contrasts conspicuously with the minimal structural consequences of the d247a, d86a mutations.
this is despite the fact that all three mutants crystallised on isomorphous lattices .
the conformational flexibility observed for arg <dig> leads us to hypothesise that the interaction network, to which glu <dig> is central, helps to coordinate donor and acceptor substrate binding.
removal of the glu <dig> carboxylate not only eliminates the h-bond to the tyr <dig> hydroxyl , but also weakens the h-bond between the tyr <dig> hydroxyl and nε of arg <dig> , as the latter assumes an alternative rotamer state, which is stabilised by the tight salt bridge interaction with glu <dig> .
this observation suggests that, in the ligand-free state of wild-type levansucrase, the conformation of arg <dig> is stabilized, firstly, through the h-bond to tyr <dig>  and, secondly, through the interaction with glu <dig> .
this implies that in the ligand-free state  glu <dig> may be deprotonated.
it is conceivable that arg <dig>  in the absence of substrate, does not have a strong preference for the configuration seen in wild-type levansucrase, but may be free to assume the alternative conformation closer to glu <dig>  as both rotamer states of arg <dig> occur with about the same frequency in protein structures , and thus a switch between them is likely energy-neutral.
during the first reaction step of the double displacement mechanism, nucleophilic asp <dig> forms a covalent intermediate with the fructofuranosyl, while glu <dig> protonates the glucosyl leaving group .
upon binding of the donor substrate , the tyr <dig> phenol ring tilts towards the bottom of the active site, altering, and presumably weakening the interaction with arg <dig>  however, the conformation of arg <dig> in the donor-bound state is stabilized by h-bonds  to the 2-, 3-hydroxyls of the glucose moiety.
release of the leaving group deprives arg <dig> of these stabilising contacts, and it may be free to switch to the alternative rotamer state, engaging in the salt bridge with glu <dig>  which also has lost its contacts to the substrate .
in the second reaction step of the double displacement mechanism, the acceptor substrate binds  and, through nucleophilic attack of the terminal 6'-hydroxyl  linkage) on the anomeric carbon, the enzyme-bound fructosyl is added to the acceptor.
based on the structural requirements for catalysis and the geometry of the active site, one would predict that the terminal fructosyl of the acceptor substrate binds in a position that overlaps at least partially with the site of the glucosyl leaving group, such that the 6'-hydroxyl is positioned appropriately for activation by glu <dig> .
the precise mode of acceptor substrate binding is as yet unclear.
located at the rim of the active site pocket, asn <dig> has very recently emerged as a structural element required for polymerase activity  <cit> , in addition to arg <dig>  <cit> .
mutation of asn <dig> to aspartate in b. megaterium levansucrase  preserves polymerase activity, but removal of the side chain amide  abrogates polysaccharide synthesis without affecting hydrolysis activity  <cit> .
accordingly, homann et al.
suggested that asn252/asn <dig> contributes to the acceptor-substrate binding site, identifying asn252/asn <dig> as a part of the + <dig> subsite  .
in the present structure of apo-e342a, arg <dig> and asn <dig> are linked indirectly through h-bond/ionic interactions to glu <dig> , suggesting that all three side chains may form part of the fructosyl-acceptor binding site.
thus, we envisage a scenario where arg <dig> can alternate between two rotamer states, which contribute to the donor and acceptor substrate binding sites respectively.
given the variety of oligosaccharide products synthesised by b. megaterium levansucrase and levansucrases of other species it appears that acceptor binding occurs with low specificity  <cit> .
a flexible conformation of arg <dig>  acting as sort of a 'fishing hook', could contribute to accommodating acceptors in different orientations relative to the enzyme-bound fructosyl unit.
weak affinity of acceptor binding may also explain the donor substrate concentration-dependent switch between invertase  and polymerase  activity.
bearing in mind that the - <dig> subsite is occupied by the fructosyl-enzyme intermediate following the first step of the double displacement mechanism, the acceptor will find a binding surface that, compared to the deep central pocket of apo levansucrase, offers significantly less depth to bury solvent-accessible surface.
the raffinose complex, furthermore, illustrates that direct interactions between substrate and enzyme are limited to the - <dig> and + <dig> subsites of the donor substrate complex.
saccharide units beyond the + <dig> subsite might find it difficult to make specificity-determining contacts.
in our observation, an intact set of interactions at the - <dig> subsite seems to be a prerequisite for 'high' affinity binding of the donor: when soaking crystals of the inactive d247a and d86a mutants in  <dig> mm sucrose for  <dig> min, we observed binding to a secondary site at a crystal packing interface, but no ligand was detected in the active site , whereas lower concentrations  and shorter soaking times  were sufficient to obtain full occupancy complexes with e342a using the same approach.
this argues that productive binding of the donor depends on an intact set of interactions between the enzyme and the fructosyl moiety, and that the specific interactions of the glucose moiety, while conferring specificity, are less critical for achieving high affinity binding.
thus, in order to promote polymerisation, the acceptor substrate, which initially is sucrose, must be present at sufficiently high concentration to lead to productive binding and levan polymerisation.
CONCLUSIONS
the data presented here are consistent with a view that donor substrate recognition in sucrose- or raffinose-bound complexes of gh <dig> and gh68-family enzymes rests primarily on the sucrosyl unit, a view that is in agreement with the structure of raffinose-bound t. maritima invertase.
the recent activity data obtained for point mutants of b. megaterium levansucrase in conjunction with our structural data provide clues for the acceptor substrate binding site, a site to which asn <dig>  glu <dig> and arg <dig> appear to contribute.
the biochemical and structural data lend support to the hypothesis that the conformational flexibility of arg <dig> may play the role of a switch between donor and acceptor substrate binding modes.

 oligo- and polyfructosyl-sucrose polymers, collectively known as fructans, are synthesized by a significant number of bacteria and an estimated plant species either replacing or supplementing starch as a carbohydrate reserve. fructans are synthesised from sucrose as the sole substrate, sharing a single sucrose starter unit βfru) to which fructofuranose units can become attached at various positions of the fructosyl or glucosyl ring, resulting in highly branched or linear polymers in a species-dependent fashion. the two prevailing forms of fructans are β -linked levan βfru n) and β -linked inulin βfru n), with the degree of polymerization of fructans varying between a few hundred and several thousands saccharide units. in plants, fructans are thought to contribute to drought and frost tolerance by preventing rupture of cell membranes, whereas in bacteria fructans are known to serve as food storage and to contribute to biofilm formation. while fructan synthesis in plants involves at least two enzymes with different fructosyl-donor and – acceptor specificities, levan or inulin synthesis in bacteria requires only a single enzyme, with sucrose initially acting as both fructosyl donor and acceptor substrate. levansucrase, encoded in bacillus subtilis by the sacb gene, catalyses the fructosyl transfer reaction sucrose + acceptor → glucose + fructosyl-acceptor in vitro, levansucrase mediates invertase or polymerase activity depending on the concentration of the fructosyl donor substrate: below mm, sucrose is cleaved into glucose and fructose with water acting as fructosyl-acceptor, whereas above this concentration levan production occurs through successive transfer of fructosyl units from sucrose to the fructosyl 6'-hydroxyl -linkage) of the acceptor substrate. levansucrase belongs to family of glycoside hydrolases according to the classification of carbohydrate-active enzymes. while structurally and functionally diverse, glycoside hydrolases share the requirement of two juxtaposed acidic side chains, acting as proton donor and catalytic nucleophile or catalytic base, respectively. hydrolysis of the glycosidic bond can result in either inversion or retention of the anomeric configuration in the substrate, corresponding to a single or double displacement reaction mechanism, respectively. kinetic studies of levansucrase established that sucrose hydrolysis follows a ping-pong kinetic reaction mechanism that retains the anomeric configuration and involves a covalently bound fructosyl-enzyme intermediate. we recently determined the crystal structures of b. subtilis levansucrase in the ligand-free form and bound to the fructosyl donor substrate sucrose. our structures established that the catalytic domain of gh family enzymes folds into a 5-bladed β-propeller with the active site located in a deep axial pocket. this fold is shared by the catalytic domain of gh family of retaining enzymes, as well as by the distantly related family of inverting glycoside hydrolases. in agreement with a rich body of biochemical data, we proposed that the strictly conserved asp and glu represent the two canonical catalytic carboxylate groups, while a third invariant carboxylate, asp may aid catalysis by stabilizing the transition state of the oxocarbenium ion by forming close hydrogen bond contacts with two of the fructosyl hydroxyls. while this assignment was confirmed in subsequent structural and biochemical studies of several ortho- and paralogs, the characterization of the structural determinants of levan synthesis has remained incomplete. in particular, the mode of and elements required for acceptor substrate binding remain unclear. here, we report crystal structures of three single-site mutants, d86a, d247a and e342a, previously shown to be catalytically inactive, in the ligand-free form, and that of the e342a mutant in complex with the fructosyl donor substrate raffinose. comparisons between the ligand-free and substrate-bound structures shed light on a network of hydrogen bonds and ionic interactions surrounding the proton donor glu this network reacts sensitively to presence or absence of the glu carboxylate, and to changes in the ligand-binding state. we observed significant conformational flexibility of arg a key residue in levan polymerisation, and propose that this role is linked to arg alternating between alternative rotamer states, facilitating participation in a transient docking site for the fructosyl acceptor.  apo structures of inactive mutants d86a, d247a and e342a crystals of the inactive single site mutants d86a, d247a and e342a of b. subtilis levansucrase grew at similar solution conditions as the wild-type enzyme. crystals were in space group p diffracting to beyond Å. geometric constraints of the detector, rather than crystal quality limited data acquisition on the home source to a maximum resolution of Å. the diffraction data of mutant-forms of levansucrase were highly isomorphous to those of crystal form i of wild-type levansucrase. prior to structure building and refinement, structural differences were ascertained by way of difference fourier maps, and the structures were refined starting from the wild-type model, altering the mutation sites to alanine. the backbone structures superimpose closely with that of the wild-type enzyme. the root mean square deviation for backbone atoms between the mutant and wild type structures vary from Å to Å. the rmsd for side chain atoms between mutants and wild type structures are Å or less, corresponding to ~ times the estimated coordinate error at Å. this indicates that overall the mutations induce only minimal structural changes. the missing carboxylate groups in the d86a and d247a mutants are clearly marked by negative density in the difference fourier maps, but result in no other notable changes in the active site. arsym = Σ/Σi bhigh resolution shell in parentheses arfree calculated using 5% of total reflections omitted from refinement bramachandran statistics calculated using procheck in marked contrast, mutation of the general acid glu to alanine has a profound impact on the rotamer state of functionally important side chains in the vicinity of the glu carboxylate. in the apo structure of wild-type levansucrase, glu forms a tight salt bridge interaction with the guanido group of arg and a strong hydrogen bond with the side chain hydroxyl of tyr. in e342a, the missing carboxylate prompts, firstly, the guanido group of arg which is important for activity, to swing about 90° towards the axis of the β-propeller, overlapping in this configuration with the fructosyl binding site. secondly, the e342a mutation eliminates the hydrogen bond to the tyr hydroxyl, causing a minor upward shift of the phenol ring. thirdly, arg assumes an alternative rotamer state, involving an 80° rotation in χ and a near 90° rotation in χ this drastic change in rotamer configuration of arg which is required for polymerase activity, is somewhat surprising: in wild-type levansucrase arg interacts through a -Å hydrogen bond with tyr but forms only a weak interaction with glu. the alternative rotamer state of arg is ostensibly stabilised by a tight ionic interaction with glu, a residues involved in donor substrate binding. in conclusion, the d247a and d86a mutations have little or no impact on the side chain configurations and interactions elsewhere in the active site, while the glu342a substitution has knock-on effects for the network of non-covalent interactions around the glu carboxylate. we note however, that binding of the donor substrate to levansucrase-e342a largely restores the side chain configuration of apo wild-type levansucrase, prompting arg and arg to swing back into their original position. the raffinose-bound complex of levansucrase-e342a besides sucrose, levansucrase also accepts the trisaccharide d-raffinose αglcβfru) as fructosyl donor. the products resulting from levansucrase-catalysed hydrolysis of raffinose are melibiose and free fructose. crystals of levansucrase-e342a were soaked in raffinose, followed by cryoprotection and flash-freezing in a k nitrogen gas stream. diffraction data to Å resolution were recorded in-house and difference electron density maps, comparing amplitudes of the raffinose-bound mutant and apo wild-type levansucrase, showed density that could be attributed unequivocally to the ligand. with amplitudes on an absolute scale, the occupancy of raffinose refined to a value near, suggesting full occupancy, while the average b factor of the raffinose atoms was Å compared to Å for the protein atoms. in the refined structure, the raffinose ligand displays a configuration in which the planes of the three sugar rings are approximately orthogonal to each other: the fructose moiety lies almost'flat' on the bottom of the active site, the glucose moiety rises up and the galactose ring is jutting out into the solvent. the fructose and glucose rings superimpose closely with their counterparts in the sucrose-bound complex, as illustrated by an rmsd of Å for the atoms common between the two ligands. following the terminology defined by davies et al., the active site of glycoside hydrolases can be divided into subsites with respect to the cleaved glycosidic bond. applied to the present raffinose complex, subsite - coincides with the fructose, and subsites + and + with the glucose and galactose moieties, respectively. it is apparent that, with the exception of arg and perhaps glu side chains tend to form specificity-determining contacts with only one of the three subsites: the fructosyl moiety makes specificity-determining contacts with the side chains of trp, asp, arg, asp, and glu. in subsite + the 2-, 3- and 4-hydroxyls of the glucosyl moiety form tight h-bond contacts with arg and glu these specificity-determining contacts lock the fructosyl and glucosyl units into defined orientations, positioning the anomeric carbon of the fructosyl unit within Å of the nucleophile asp and the glycosidic oxygen in close proximity to the carboxylate of glu in contrast, the galactosyl unit makes only few, water-mediated h-bonds, limited to the 6"-hydroxyl, with asn and tyr whereas the 2"-, 3"- and 4"-hydroxyl groups point into solvent. the configuration of active site residues in raffinose-bound e342a is very similar to wild-type levansucrase in the ligand-free form, but contrasts with the apo form the e342a mutant. owing to steric overlap with the fructosyl moiety, arg swings back from the rotamer state in apo-e342a to the wild-type configuration. similarly, arg resumes the wild-type configuration, stabilised by h-bond interactions with the 2-, 3-hydroxyls of the glucosyl ring. yet, in the donor substrate-bound state tyr tilts by ~20° towards the floor of the active site. as a consequence, the h-bond between oh of tyr and nε of arg is not preserved in either of the raffinose- and sucrose-bound complexes. the dip of tyr appears to result from van der waals interactions with the glucosyl ring and with the guanido group of arg. through the contacts to the ligand, arg inserts deeper into the active site than in the apo structure of wild-type levansucrase. it remains unresolved whether the 20°-tilt of tyr would also occur in a donor substrate complex of the wild-type enzyme. it is noteworthy, that the position of the missing carboxylate of glu is marked by two water molecules, a feature consistent with the sucrose-bound complex. among the protein side chains, there is very little change between sucrose- and raffinose-bound structures of e342a. the rmsd of side chain atoms located within Å of the substrate is Å. thus within the limits of the estimated coordinate error the two structures are identical with respect to the protein framework. discussion we present here the raffinose-bound complex of levansucrase-e342a in addition to the apo crystal structures of the three inactive point mutants d86a, d247a and e342a. our previous study of the structure of b. subtilis levansucrase established, based largely on structural arguments, the function of these three strictly conserved carboxylate side chains in the active site. the present raffinose complex reinforces a view that donor substrate recognition in b. subtilis levansucrase rests primarily on the common sucrosyl unit, whereas the galactosyl moiety, which protrudes out of the active site, makes only a few water-mediated h-bonds, pointing three unliganded hydroxyl groups to the bulk solvent. this mode of binding is echoed by the raffinose-bound complex of thermotoga maritima invertase, which belongs to gh family and which, like levansucrase, mediates hydrolysis of the glycosidic bond through a double displacement reaction mechanism. in the latter study, an inert complex was facilitated by mutating the proton donor to aspartic acid. superimposing the two complexes by matching the positions of ligand atoms, reveals a very similar geometry of the ligand, and an almost perfect overlap of the catalytic residues. while there is significant variation of structural elements mediating specificity-determining contacts with the ligand, specific recognition of the outermost saccharide unit is weak in both structures and does not involve direct h-bonds. nevertheless, the t. maritima complex includes notable van der waals interactions between the galactose and trp for which there is no counterpart in b. subtilis levansucrase. the importance of glu arg and arg in forming specificity-determining contacts with the donor substrate is illustrated by the mutagenesis data obtained for levansucrase from bacillus megaterium. mutating these side chains was reported as nearly abolishing hydrolase activity. the functional assignment of the catalytic side chains in the active site of levansucrase raises the question of which structural features ensure that the requirement for differential protonation states of asp and glu is met. since the ph optimum of levansucrase lies in the range of ph to, the pka of glu must be raised to at least – in order to serve as the general acid. this could occur, for instance, through juxtaposition to hydrophobic or acidic side chains. yet, the shortest contacts of glu are with tyr and arg, neither of which is likely to result in the required effect. still, a structure-based computational analysis using uhbd and scripts written by the wade group indicated a pka of glu at least ph units above that of the free amino acid, arguing that a rise of the pka is the result of the cumulative effect of the ensemble of side chains and contacts surrounding glu an environment that includes three acidic side chains, glu, glu, and asp. moreover, from the sucrose- and raffinose-bound complexes of the e342a mutant one can infer that donor substrate binding results in additional contacts with hydrophobic moieties of the sugar, and it is conceivable that a pka shift only occurs upon substrate binding. the distinct effects caused by the point mutation e342a on the configuration of adjacent side chains contrasts conspicuously with the minimal structural consequences of the d247a, d86a mutations. this is despite the fact that all three mutants crystallised on isomorphous lattices. the conformational flexibility observed for arg leads us to hypothesise that the interaction network, to which glu is central, helps to coordinate donor and acceptor substrate binding. removal of the glu carboxylate not only eliminates the h-bond to the tyr hydroxyl, but also weakens the h-bond between the tyr hydroxyl and nε of arg, as the latter assumes an alternative rotamer state, which is stabilised by the tight salt bridge interaction with glu. this observation suggests that, in the ligand-free state of wild-type levansucrase, the conformation of arg is stabilized, firstly, through the h-bond to tyr and, secondly, through the interaction with glu. this implies that in the ligand-free state glu may be deprotonated. it is conceivable that arg in the absence of substrate, does not have a strong preference for the configuration seen in wild-type levansucrase, but may be free to assume the alternative conformation closer to glu as both rotamer states of arg occur with about the same frequency in protein structures, and thus a switch between them is likely energy-neutral. during the first reaction step of the double displacement mechanism, nucleophilic asp forms a covalent intermediate with the fructofuranosyl, while glu protonates the glucosyl leaving group. upon binding of the donor substrate, the tyr phenol ring tilts towards the bottom of the active site, altering, and presumably weakening the interaction with arg however, the conformation of arg in the donor-bound state is stabilized by h-bonds to the 2-, 3-hydroxyls of the glucose moiety. release of the leaving group deprives arg of these stabilising contacts, and it may be free to switch to the alternative rotamer state, engaging in the salt bridge with glu which also has lost its contacts to the substrate. in the second reaction step of the double displacement mechanism, the acceptor substrate binds and, through nucleophilic attack of the terminal 6'-hydroxyl linkage) on the anomeric carbon, the enzyme-bound fructosyl is added to the acceptor. based on the structural requirements for catalysis and the geometry of the active site, one would predict that the terminal fructosyl of the acceptor substrate binds in a position that overlaps at least partially with the site of the glucosyl leaving group, such that the 6'-hydroxyl is positioned appropriately for activation by glu. the precise mode of acceptor substrate binding is as yet unclear. located at the rim of the active site pocket, asn has very recently emerged as a structural element required for polymerase activity, in addition to arg. mutation of asn to aspartate in b. megaterium levansucrase preserves polymerase activity, but removal of the side chain amide abrogates polysaccharide synthesis without affecting hydrolysis activity. accordingly, homann et al. suggested that asn252/asn contributes to the acceptor-substrate binding site, identifying asn252/asn as a part of the + subsite. in the present structure of apo-e342a, arg and asn are linked indirectly through h-bond/ionic interactions to glu, suggesting that all three side chains may form part of the fructosyl-acceptor binding site. thus, we envisage a scenario where arg can alternate between two rotamer states, which contribute to the donor and acceptor substrate binding sites respectively. given the variety of oligosaccharide products synthesised by b. megaterium levansucrase and levansucrases of other species it appears that acceptor binding occurs with low specificity. a flexible conformation of arg acting as sort of a'fishing hook', could contribute to accommodating acceptors in different orientations relative to the enzyme-bound fructosyl unit. weak affinity of acceptor binding may also explain the donor substrate concentration-dependent switch between invertase and polymerase activity. bearing in mind that the - subsite is occupied by the fructosyl-enzyme intermediate following the first step of the double displacement mechanism, the acceptor will find a binding surface that, compared to the deep central pocket of apo levansucrase, offers significantly less depth to bury solvent-accessible surface. the raffinose complex, furthermore, illustrates that direct interactions between substrate and enzyme are limited to the - and + subsites of the donor substrate complex. saccharide units beyond the + subsite might find it difficult to make specificity-determining contacts. in our observation, an intact set of interactions at the - subsite seems to be a prerequisite for'high' affinity binding of the donor: when soaking crystals of the inactive d247a and d86a mutants in mm sucrose for min, we observed binding to a secondary site at a crystal packing interface, but no ligand was detected in the active site, whereas lower concentrations and shorter soaking times were sufficient to obtain full occupancy complexes with e342a using the same approach. this argues that productive binding of the donor depends on an intact set of interactions between the enzyme and the fructosyl moiety, and that the specific interactions of the glucose moiety, while conferring specificity, are less critical for achieving high affinity binding. thus, in order to promote polymerisation, the acceptor substrate, which initially is sucrose, must be present at sufficiently high concentration to lead to productive binding and levan polymerisation.  the data presented here are consistent with a view that donor substrate recognition in sucrose- or raffinose-bound complexes of gh and gh68-family enzymes rests primarily on the sucrosyl unit, a view that is in agreement with the structure of raffinose-bound t. maritima invertase. the recent activity data obtained for point mutants of b. megaterium levansucrase in conjunction with our structural data provide clues for the acceptor substrate binding site, a site to which asn glu and arg appear to contribute. the biochemical and structural data lend support to the hypothesis that the conformational flexibility of arg may play the role of a switch between donor and acceptor substrate binding modes. 
in marked contrast, the e342a mutant reveals conformational flexibility of functionally relevant side chains in the vicinity of the general acid glu including arg a residue required for levan polymerisation. the present structures, in conjunction with prior biochemical data, lead us to hypothesise that the conformational flexibility of arg is linked to it forming a transient docking site for the fructosyl-acceptor substrate, through an interaction network involving nearby glu and asn at the rim of a central pocket forming the active site. still, the structural determinants of the fructosyl transfer reaction thus far have been only partially defined. the d86a and d247a substitutions have little effect on the active site geometry. the raffinose-complex reveals a conserved mode of donor substrate binding, involving minimal contacts with the raffinose galactosyl unit, which protrudes out of the active site, and specificity-determining contacts essentially restricted to the sucrosyl moiety. biosynthesis of levan βfru n), an abundant form of bacterial fructan, is catalysed by levansucrase, utilizing sucrose as the sole substrate. previously, we described the tertiary structure of bacillus subtilis levansucrase in the ligand-free and sucrose-bound forms, establishing the mechanistic roles of three invariant carboxylate side chains, asp asp and glu which are central to the double displacement reaction mechanism of fructosyl transfer.
 fructans – β-d-fructofuranosyl polymers with a sucrose starter unit – constitute a carbohydrate reservoir synthesised by a considerable number of bacteria and plant species. biosynthesis of levan βfru n), an abundant form of bacterial fructan, is catalysed by levansucrase, utilizing sucrose as the sole substrate. previously, we described the tertiary structure of bacillus subtilis levansucrase in the ligand-free and sucrose-bound forms, establishing the mechanistic roles of three invariant carboxylate side chains, asp asp and glu which are central to the double displacement reaction mechanism of fructosyl transfer. still, the structural determinants of the fructosyl transfer reaction thus far have been only partially defined.  here, we report high-resolution structures of three levansucrase point mutants, d86a, d247a, and e342a, and that of raffinose-bound levansucrase-e342a. the d86a and d247a substitutions have little effect on the active site geometry. in marked contrast, the e342a mutant reveals conformational flexibility of functionally relevant side chains in the vicinity of the general acid glu including arg a residue required for levan polymerisation. the raffinose-complex reveals a conserved mode of donor substrate binding, involving minimal contacts with the raffinose galactosyl unit, which protrudes out of the active site, and specificity-determining contacts essentially restricted to the sucrosyl moiety.  the present structures, in conjunction with prior biochemical data, lead us to hypothesise that the conformational flexibility of arg is linked to it forming a transient docking site for the fructosyl-acceptor substrate, through an interaction network involving nearby glu and asn at the rim of a central pocket forming the active site. 