Donor substrate recognition in the raffinose-bound E342A mutant of fructosyltransferase Bacillus subtilis levansucrase

Oligo- and polyfructosyl-sucrose polymers, collectively known as fructans, are synthesized by a significant number of bacteria and an estimated 40,000 plant species [1] either replacing or supplementing starch as a carbohydrate reserve. Fructans are synthesised from sucrose as the sole substrate, sharing a single sucrose starter unit (αGlc(1–2)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 [2]. The two prevailing forms of fructans are β (2 → 6)-linked levan (αGlc(1–2)βFru [(2–6)βFru]_n) and β (2 → 1)-linked inulin (αGlc(1–2)βFru [(2-1)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 [2], whereas in bacteria fructans are known to serve as food storage and to contribute to biofilm formation [3].

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 (sucrose:2,6-β-D-fructan-6-β-D-fructosyl transferase, E.C.2.4.1.10), encoded in Bacillus subtilis by the sacB gene, catalyses the fructosyl transfer reactionsucrose + acceptor → glucose + fructosyl-acceptor

In vitro, levansucrase mediates invertase (hydrolase) or polymerase activity depending on the concentration of the fructosyl donor substrate: below 250 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 (assuming β (2 → 6)-linkage) of the acceptor substrate [4]. Levansucrase belongs to family 68 of glycoside hydrolases (GH) according to the classification of carbohydrate-active enzymes (CAZY, [5, 6]). While structurally and functionally diverse, glycoside hydrolases share the requirement of two juxtaposed acidic side chains, acting as proton donor (to the leaving group) 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 [7]. 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 [8–10].

We recently determined the crystal structures of B. subtilis levansucrase in the ligand-free form and bound to the fructosyl donor substrate sucrose [11]. Our structures established that the catalytic domain of GH family 68 enzymes folds into a 5-bladed β-propeller with the active site located in a deep axial pocket (Figure 1). This fold is shared by the catalytic domain of GH family 32 of retaining enzymes [12–15], as well as by the distantly related family 43 of inverting glycoside hydrolases [16, 17] (see also reference [18] for a review of structure-function relationships in levansucrases). In agreement with a rich body of biochemical data [8, 19–22], we proposed that the strictly conserved Asp86 (nucleophile) and Glu342 (proton donor) represent the two canonical catalytic carboxylate groups, while a third invariant carboxylate, Asp247, may aid catalysis by stabilizing the transition state of the oxocarbenium ion by forming close hydrogen bond contacts with two of the fructosyl hydroxyls [11]. While this assignment was confirmed in subsequent structural and biochemical studies of several ortho- and paralogs [12–15, 23–25], 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 [11], 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 Glu342. This network reacts sensitively to presence or absence of the Glu342 carboxylate, and to changes in the ligand-binding state. We observed significant conformational flexibility of Arg360, a key residue in levan polymerisation, and propose that this role is linked to Arg360 alternating between alternative rotamer states, facilitating participation in a transient docking site for the fructosyl acceptor.

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 [11] 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 32 and which, like levansucrase, mediates hydrolysis of the glycosidic bond through a double displacement reaction mechanism [23]. In the latter study, an inert complex was facilitated by mutating the proton donor (Glu190) to aspartic acid. Superimposing the two complexes by matching the positions of 3 ligand atoms (Figure 5), 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 Trp41, for which there is no counterpart in B. subtilis levansucrase (Figure 5). The importance of Glu340, Arg246 and Arg360 in forming specificity-determining contacts with the donor substrate is illustrated by the mutagenesis data obtained for levansucrase from Bacillus megaterium (74% identity on amino acid level). Mutating these side chains was reported as nearly abolishing hydrolase activity [24].

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 Asp86 (nucleophile) and Glu342 (general acid) [28] is met. Since the pH optimum of levansucrase lies in the range of pH 6.0 to 6.5 [4, 24], the pKa of Glu342 must be raised to at least 6 – 6.5 in order to serve as the general acid. This could occur, for instance, through juxtaposition to hydrophobic or acidic side chains [29, 30]. Yet, the shortest contacts of Glu342 (in the apo wild-type structure) are with Tyr411 (H-bond, 2.6 Å) and Arg246 (2.9 Å), 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 [31–33] indicated a pKa of Glu342 at least 2 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 Glu342, an environment that includes three acidic side chains, Glu340 (4.1 Å), Glu262 (3.9 Å), and Asp247 (5.0 Å). 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 (Table 1). The conformational flexibility observed for Arg360 leads us to hypothesise that the interaction network, to which Glu342 is central, helps to coordinate donor and acceptor substrate binding.

Removal of the Glu342 carboxylate not only eliminates the H-bond to the Tyr411 hydroxyl (2.6 Å), but also weakens the H-bond between the Tyr411 hydroxyl and Nε of Arg360 (from 2.7 to 3.2 Å), as the latter assumes an alternative rotamer state, which is stabilised by the tight salt bridge interaction with Glu340 (2.9 Å). This observation suggests that, in the ligand-free state of wild-type levansucrase, the conformation of Arg360 is stabilized, firstly, through the H-bond to Tyr411, and, secondly, through the interaction with Glu342 (distance of 4.9 Å). This implies that in the ligand-free state (and at the condition of crystal growth, pH = 6.3) Glu342 may be deprotonated. It is conceivable that Arg360, 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 Glu340, as both rotamer states of Arg360 occur with about the same frequency in protein structures (6.5% vs. 6%), and thus a switch between them is likely energy-neutral.

During the first reaction step of the double displacement mechanism, nucleophilic Asp86 forms a covalent intermediate with the fructofuranosyl, while Glu342 protonates the glucosyl leaving group [see panels A and B in Additional File 1]. Upon binding of the donor substrate (sucrose or raffinose), the Tyr411 phenol ring tilts towards the bottom of the active site, altering, and presumably weakening the interaction with Arg360. However, the conformation of Arg360 in the donor-bound state is stabilized by H-bonds (3.1 Å) to the 2-, 3-hydroxyls of the glucose moiety. Release of the leaving group deprives Arg360 of these stabilising contacts, and it may be free to switch to the alternative rotamer state, engaging in the salt bridge with Glu340, which also has lost its contacts to the substrate [panel B in Additional file 1].

In the second reaction step of the double displacement mechanism, the acceptor substrate binds [panel C in Additional file 1] and, through nucleophilic attack of the terminal 6'-hydroxyl (assuming a polymer with β (2 → 6) 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 Glu342 (now acting as general base).

The precise mode of acceptor substrate binding is as yet unclear. Located at the rim of the active site pocket, Asn242 has very recently emerged as a structural element required for polymerase activity [24], in addition to Arg360 [20, 22]. Mutation of Asn252 to aspartate in B. megaterium levansucrase (corresponding to Asn242 in B. subtilis) preserves polymerase activity, but removal of the side chain amide (N252A, N252G) abrogates polysaccharide synthesis without affecting hydrolysis activity [24]. Accordingly, Homann et al. suggested that Asn252/Asn242 contributes to the acceptor-substrate binding site, identifying Asn252/Asn242 as a part of the +2 subsite (relative to the positioning of the fructosyl donor) [see panels C and D in Additional file 1]. In the present structure of apo-E342A, Arg360 and Asn242 are linked indirectly through H-bond/ionic interactions to Glu340 (Figures 2C and 3A), suggesting that all three side chains may form part of the fructosyl-acceptor binding site. Thus, we envisage a scenario where Arg360 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 [24]. A flexible conformation of Arg360, 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 (< 250 mM) and polymerase (> 250 mM) activity. Bearing in mind that the -1 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 -1 and +1 subsites of the donor substrate complex. Saccharide units beyond the +1 subsite might find it difficult to make specificity-determining contacts. In our observation, an intact set of interactions at the -1 subsite seems to be a prerequisite for 'high' affinity binding of the donor: when soaking crystals of the inactive D247A and D86A mutants in 500 mM sucrose for 30 min, we observed binding to a secondary site at a crystal packing interface, but no ligand was detected in the active site (data not shown), whereas lower concentrations (150 mM) and shorter soaking times (10 min) 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 GH32 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 Asn242, Glu340 and Arg360 appear to contribute. The biochemical and structural data lend support to the hypothesis that the conformational flexibility of Arg360 may play the role of a switch between donor and acceptor substrate binding modes.