General comments about search models and description of the monomer structure
N-terminally His-tagged Cest-2923, when overexpressed using the pURI3-TEV vector as in our previous structural studies , was revealed to be marginally stable in solution and therefore not suitable for crystallization experiments. By contrast, the C-terminally His-tagged variant behaved well in solution and was suitable for crystallization. The enzyme crystallized in the hexagonal space group P6322 (unit-cell dimensions a = b = 141.65 Å, c = 165.74 Å, α = β = 90º, γ = 120º) with two independent protein molecules per asymmetric unit. Diffraction data up to 2.1 Å resolution were measured at beamline ID29 at the European Synchrotron Radiation Facility (ESRF) (Grenoble, France) on a Pilatus 6M detector (Dectris Ltd, Baden, Switzerland) and the structure of the esterase was solved by molecular replacement. At that time, there were two sets of atomic coordinates for Cest-2923 in the PDB, one deposited by the NESG (PDB code: 3d3n) and the other one by the JCSG (PDB code: 3bxp). Previous inspection of these models revealed two aspects: first, the two independent molecules of the corresponding asymmetric units were incomplete, lacking a catalytically relevant region around residues Gly231 to Tyr250 and, second, most remarkably, they exhibited structural features incompatible with the presence of the catalytic triad typical of an α/β hydrolase fold, which is the basic architecture of this enzyme. These latter anomalous features are considered in detail below. Only the shortest deposited model for Cest-2923 (molecule A from the PDB entry 3d3n) is devoid of these anomalous structural features and therefore was used as search model. This model lacks the 25-residue sequence stretch between residues Gly231 and Ala255, in conflict with the classic definition of the catalytic triad because it contains the catalytic residue His233 (see below), plus a three-residue loop (Phe28-Thr30).
In this regard, the residues forming the catalytic triad of Cest-2923 (Ser116, His233 and Asp201) were predicted in a straightforward manner from a simple blast search against the PDB, which in turn revealed the putative sugar hydrolase YeeB from Lactococcus lactis (PDB entry 3hxk) and the carboxylesterase lp_1002 (PDB entry 3bjr) as the closest homologues (33% and 32% sequence identity, respectively) to be structurally characterized (Fig. 1).
Figure 1. Amino acid sequence alignment of Cest-2923 with its closest homologuess. Cest-2923 shares 33% and 32% sequence identity with the putative sugar hydrolase YeeB from L. lactis and the carboxylesterase lp_1002 from L. plantarum, respectively. Colour code: blue, conserved amino acid residues; yellow, similar residues; red, residues forming the catalytic triad in YeeB and lp_1002.
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The two Cest-2923 independent molecules making up the asymmetric unit of our hexagonal crystal are essentially identical (0.36 Å rmsd for 275 Cα atoms). We arbitrarily chose chain A as reference for description of the protein. As indicated above, the structure of the Cest-2923 subunit is based on a canonical α/β hydrolase fold [6-8] consisting of a central eight-stranded β-sheet, with strand β2 being the only antiparallel strand (Fig. 2). The β-sheet shows a marked left-handed twist with an approximate angle of 90º between strands β1 and β8. This core is surrounded by five helices, with α1 (Ala54-Met61) and α7 (Trp258-Glu268) lying on one side of the sheet and α2 (Trp84-His103), α3 (Ala117-Val128) and α6 (Ile207-Gln218) on the opposite side. Although Cest-2923 is currently classified as member of the HSL subfamily of α/βhydrolases  by the ESTHER database, the enzyme does not exhibit a distinct cap domain over the active site. Curiously, the loop between strand β8 (Thr223-Phe228) and helix α7 is highly flexible as deduced from the above mentioned models from the JCSG and NESG consortia for the enzyme where this region cannot be modelled, and may perhaps function as a pseudo-flap.
Figure 2. Overall fold of the Cest-2923 subunit from L. plantarum WCFS1. The tertiary structure of the Cest-2923 subunit fits the canonical α/β hydrolase fold where a central eight-stranded β-sheet (shown in green) is surrounded by five α-helices (orange). Two orthogonal views of the protein subunit are shown.
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Comparison with other esterases
A dali search  revealed numerous structures with high structural similarity, as otherwise expected as a result of the high level of conservation of the α/β hydrolase fold [6-8]. Sugar hydrolase YeeB from L. lactis (PDB entry: 3hxk) and carboxylesterase lp_1002 from L. plantarum WCFS1 (PDB entry: 3bjr) have the highest structural similarity (Z-score = 34.7 and 28.8; rmsd = 1.5 and 2.0 Å for 250 and 244 Cα atoms, respectively). Other esterases from the HSL subfamily such as Est1 from environmental samples , Sto-Est from Sulfolobus tokodaii , PestE from Pyrobaculum calidifontis  and acetyl esterase from Salmonella typhimurium (PDB entry: 3ga7) are also structurally similar (Z-score = 25.4, 24.8, 24.6 and 24.5; rmsd = 2.5, 2.6, 2.5 and 2.6 Å for 236, 236, 234 and 240 Cα atoms, respectively). In these latter cases, the structural similarity is limited to the α/β hydrolase fold (core β-sheet plus the two surrounding layers of α-helices) because, as indicated above, Cest-2923 lacks a cap domain characteristic of some HSL enzymes.
Definition of the active site of Cest-2923: the catalytic triad
As a member of the α/β hydrolase superfamily, the catalytic machinery of Cest-2923 is based on a catalytic triad formed by Ser, His and Asp/Glu residues (Ser116, His233 and Asp201), which are located at canonical sites along the protein sequence. The nucleophile Ser116 of Cest-2923 is situated in the so-called nucleophilic elbow, within a highly conserved sequence (Gly-X-Ser-X-Gly) (with X denoting any amino acid) , with its backbone angles residing in an unfavourable region of the Ramachandran plot (φ = 52º, ψ = −118º). This constrained conformation is stabilized by a dense network of hydrogen bonds affecting both the polypeptide chain (two 2.9 Å hydrogen bonds between the carbonyl oxygen atom and the amide nitrogen atoms of Gly119 and Val156, respectively, and a 3.0 Å hydrogen bond between the amide nitrogen atom of Ser116 and the carbonyl oxygen of Gly153) and the hydroxyl side chain (a hydrogen bond (2.8 Å) between the Oγ atom and the Nε2 atom of His233) (Fig. 3A).
Figure 3. Catalytic triad machinery of the Cest-2923 subunit from L. plantarum WCFS1. (A) Stereo view of the catalytic triad environment of Cest-2923. Residues of the catalytic triad (Ser116, His233, Asp201) are shown as orange sticks and those participating in hydrogen bonding interactions as grey sticks; potential H-bonds are indicated as black, broken lines. A conserved water molecule that interacts with Asp201, which is conserved within the HSL family of enzymes, is shown as a blue sphere. (B) Stereo view of the surroundings of the sulfate molecule found in the active site of Cest-2923. The electron density map (2Fo – Fc in blue; contoured at 1-σ level) for the sulfate is shown. Residues of the catalytic triad are shown as light yellow sticks. Distances are given in Å.
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The proton carrier residues His233 and Asp201 of the charge-relay system locate downstream the strands β8 and β7, respectively. Particularly, the amino acid His233 is located within the 29-residue long loop connecting strand β8 to helix α7. The Nδ1 atom of His233 is hydrogen-bonded to the Oδ1 atom (2.7 Å) and to the Oδ2 atom (3.1 Å) of Asp201. The conformation of this last side chain is further stabilized by a hydrogen bond between the Oδ2 atom and the NH of Val204 (2.9 Å) and is also at hydrogen bond distance to the CO of this latter residue (3.1 Å). In addition, the Oδ1 atom is hydrogen-bonded to the Nε2 atom of Gln197 (3.0 Å) and also to a highly ordered water molecule (2.7 Å). This solvent molecule together with its close interacting neighbour (2.9 Å distance between them) are present in all structural homologues of Cest-2923 found with dali and have been proposed to play a structural role in this family of enzymes . In particular, in Cest-2923 these solvent molecules may contribute to maintain the proper conformation of this part of the active site by bridging the loops where His233 and Asp201 are situated. Thus, molecule HOH45 hydrogen bonds to the NH atom (3.0 Å) and to the Oγ1 atom (2.8 Å) of Thr198 from the loop connecting strand β7 to helix α6, and HOH4 makes hydrogen bonds to the NH atom (3.1 Å) of Leu235 and to the CO atom (2.8 Å) of Ile232 from the loop between strand β8 and helix α7.
Interestingly, a spherical blob of electron density appeared in close contact to the hydroxyl group of the nucleophile Ser116 of both independent molecules that has been assigned to a sulfate molecule coming from the crystallization solution (Fig. 3B). This molecule may be claimed to mimic the negatively-charged tetrahedral intermediates of the catalytic reaction, which are stabilized by the so-called oxyanion hole . In this case, the sulfate molecule is stabilized by polar interactions with amide nitrogen atoms of Gly43, Gly44 and Ala117, which would suggest the existence of a tridentate oxyanion hole similar to those from heroin esterase , esterase EstE1  or carboxylesterase EST2 from Alicyclobacillus acidocaldarius , in contrast to the most commonly observed bidentate counterparts .
As indicated above, the atomic models of Cest-2923 deposited by the JCSG and NESG consortia (PDB code: 3d3n and 3bxp, respectively), exhibited structural features that are incompatible with the presence of a catalytic triad (Fig. S1). Specifically, in molecule A from PDB entry 3d3n, which is the shortest model, the Asp201 side chain location overlaps with that of His233 (in our model), which, in this model, is not defined (Fig. S1A). Conversely, the trace of the loop that contains the catalytic residue His233 in molecule B places this residue at a distance of 15 Å from the nucleophile Ser116 (distance between Cα atoms), a geometry incompatible with the built-up of a catalytic triad (Fig. S1B). The conformation of the connecting loop between strand β7 and helix α6 almost coincides with our model, placing the catalytic Asp201 in a similar position. Similar anomalous structural features can be detected in molecules A and B from PDB entry 3bxp (Fig. S1B): in this case, the trace of the connecting loop between strand β7 and helix α6 from molecule A places Asp233 in a wrong position and orientation, preventing the catalytic His233 residue from being located in its proper, canonical position, close to the nucleophile Ser116 (Fig. S1C). Finally, similar features can be identified in molecule B (Fig. S1D).
Oligomeric states of Cest_Lp2923: a case of pH-dependent pleomorphism
The two molecules in the asymmetric unit of the Cest-2923 hexagonal crystal tightly associate, forming a dimer. The association mode is similar to the one observed for other dimeric esterases from the α/β hydrolase superfamily [16, 17] and involves the antiparallel association between β8 strands and interactions between helices preceding (α6) and following (α7) this latter β strand (Fig. 4a). We refer to these dimers as canonical ones. Analysis of the interfaces within the Cest-2923 crystal with pisa  and pic  web servers reveals that the contact region between monomers has an interface area of ~ 980 Å2. The intermolecular contacts identified in this area are 26 hydrogen bonds, 20 hydrophobic and aromatic–aromatic interactions and two salt bridges. Among the set of hydrogen bonds detected, there are four between main chain atoms, which are those directly involved in the association between β8 strands (particularly from residues Tyr225 and Leu227).
Figure 4. Dimeric and tetrameric assemblies of Cest-2923 from L. plantarum WCFS1. (A) Canonical dimer of Cest-2923. The association of the subunits involves interactions between strands β8 and α-helices α6 and α7. The two subunits are shown in different colours. (B) Noncanonical tetramer of Cest-2923. The contacting region between canonical dimers is mainly formed by α-helix α1, the contacting loops between strands β2 and β3 and between strand β8 and α-helix α7 and the C-terminal end. The four subunits are shown in different colours. (C) Canonical tetramer of HSL esterases. As an example, it is shown the tetramer of the hyperthermophilic esterase EstE1 (PDB code: 2c7b) . Further details on the comparison between canonical and noncanonical tetramers are provided in Fig. S3. (D) Three-dimensional superposition between the noncanonical tetramers of Cest-2923 (in transparent, grey cartoons) and those found for the putative sugar hydrolase YeeB from L. lactis (PDB code: 3hxk).
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In addition, this same analysis revealed the existence of a second contact region between symmetry-related molecules (molecules A-A and B-B) involving an even larger interface area (1460 Å2). The assembly of Cest_Lp2923 monomers through the joint combination of these two contact regions renders a tetrameric structure made up of two canonical dimers, which is stable according to pisa (total buried area: 9780 Å2; Gibb′s free energy of dissociation, ΔGdiss: 11.5 kcal·mol−1) (Fig. 4B). In this regard, it is worth noting that canonical dimers of close relatives of Cest-2923 such as EstE1 , Sto-Est , PestE  and Brefeldin A esterase  also form tetramers within the crystals (‘canonical tetramers’), which, nevertheless, are not comparable to the one observed for Cest-2923 (Fig. 4C). It is obvious that these observations raise the question of whether the Cest-2923 tetramers are crystallographic (i.e. by-products of the crystallization process). In this regard, results from different experimental approaches reveal a complex behaviour of Cest-2923: first, the crystal forms analysed by the structural genomics consortia (PDB code: 3d3n and PDB 3bxp) reveal canonical dimers as the highest order oligomeric form within the crystals, which would support the crystallographic nature of the observed tetramers. By contrast, analyses of the oligomeric state of Cest-2923 in solution by analytical ultracentrifugation techniques (Fig. 5) reveals a complex scenario that is consistent with an associative system involving monomeric, dimeric and tetrameric species. Thus, sedimentation velocity studies carried out in Tris buffer (20 mm Tris, pH 8.0, with 0.1 m NaCl) show that Cest-2923 behaves as (at least) two molecular species in solution, with sedimentation coefficients of 3.2 ± 0.2 S (n = 3) and 6.6 ± 0.1 S (n = 3), and estimated mean molecular masses of 32 kDa and 96 kDa, respectively, which are values consistent with monomeric and trimeric Cest-2923 species (Fig. 5a). Considering the above crystallographic results, where dimers and tetramers have been observed for this protein, this scenario can be easily explained in terms of two association equilibria: a fast equilibrium (within the time scale of the sedimentation velocity experiment, namely 6 h) between dimers and tetramers, therefore explaining the mean molecular mass of the 6.8 S species (intermediate between dimers and tetramers), and a second, slow equilibrium between monomers and dimers, therefore explaining the peak assignable to the monomer.
Figure 5. Analytical ultracentrifugation analysis of Cest-2923 at neutral and acidic conditions. (A) Sedimentation coefficient c(s) distributions for Cest-2923 (13 μm) in Tris buffer (20 mm Tris-HCl, pH 8.0, with 0.1 m NaCl). Raw sedimentation velocity profiles for this analysis were acquired using A280, 148 288 g, 20 °C and different times (not shown). Calculations were conducted using sedfit . (B) Sedimentation equilibrium analysis of Cest-2923 (13 μm) in Tris buffer (20 mm Tris-HCl, pH 8.0, with 0.1 m NaCl) at 10 545 g (open circles) and 21 916 g (open squares). A280 is plotted against the radial position from the centre of the rotor. The fit to the data set (solid line curves) corresponds to a monomer–tetramer association equilibrium. Residuals from this fit are shown in the inset. (C) As in (B) but where the fit corresponds to an ideal species. The best-fit weight mean molecular mass is 69.8 ± 5.4 kDa. (D) Sedimentation equilibrium analysis of Cest-2923 (13 μm) in acetate buffer (20 nm sodium acetate, pH 5.5, with 0.1 m NaCl) at 13 149 g (open circles) and 27 845 g (open squares). Other experimental conditions are identical to those at neutral conditions. The fit to the data set (solid line curves) corresponds to a monomer–tetramer association equilibrium. Residuals from this fit are shown in the inset. (E) As shown in (D) but where the fit corresponds to an ideal species. The best-fit weight mean molecular mass is 98.6 ± 0.6 kDa.
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As expected from these latter ultracentrifugation results, sedimentation equilibrium experiments carried out under identical experimental conditions not only fitted well to a monomer to tetramer association scheme, but also to an ideal model considering a unique species with an estimated mean molecular mass of 69.8 ± 5.4 kDa (Fig. 5B and 5C), which would indicate that the system is mainly shifted to the dimeric species under neutral conditions. In this regard, it is worth noting that the two crystal forms prepared by the structural genomics consortia, where only canonical dimers were observed, were both obtained at pH 7.5 (PDB code: 3bxp: 30% 1,2-propanediol, 20% poly(ethylene glycol) 400, 0.1 m Hepes, pH 7.5; PDB code: 3d3n: 5% poly(ethylene glycol) 8000, 0.1 m calcium acetate, 0.1 m HEPES, pH 7.5).
Conversely, because the Cest-2923 crystals described in the present study, which revealed the presence of tetramers, were obtained in 1.7 m ammonium sulfate and 0.15 m sodium acetate (pH 4.6), we also analysed the behaviour of the enzyme in solution at acidic conditions. Unexpectedly, we found that Cest-2923 precipitates in sodium acetate buffer (20 mm sodium acetate, pH 4.6 with 0.1 m NaCl) even at a low protein concentration (0.2 mg·mL−1), although it was stable at pH 5.5. In this regard, both the far-UV CD spectrum at 25 °C and the thermal denaturation curve measured in acetate buffer, pH 5.5, are indistinguishable from those registered at pH 8.0, indicating that there are no significant structural differences and changes in stability (Fig. S2). Under these acidic conditions, sedimentation velocity analyses revealed a profile similar to those observed at pH 8.0, although with a relative lower contribution of the monomeric species for samples with the same protein concentration in comparable experiments (not shown). As expected, sedimentation equilibrium experiments fitted well to a monomer to tetramer associative model, and also to an ideal one with a unique species with an mean molecular mass of 98.6 ± 0.6 kDa (Fig. 5D and 5E), indicating a displacement of the equilibria towards the tetrameric form relative to the neutral conditions.
Combining the results derived from these two distinct experimental approaches (crystallographic and analytical ultracentrifugation), we can deduce that Cest-2923 behaves in solution as an associative system well described by two equilibria; namely, a fast equilibrium between species with molecular masses consistent with monomers and dimers and another much faster one between dimeric and tetrameric species and, second, that the crystallization process is an active player in selecting a pre-existing oligomeric form: dimers at neutral conditions and tetramers at acidic ones. Hence, we claim that the oligomeric forms determined by protein crystallography are not a mere crystallographic by-product but reveal intrinsic, associative properties of Cest-2923. Nevertheless, determination of the structural basis of the pH-dependence of the complex associative behaviour of Cest-2923 is not straightforward. Analysis of the interactions between Cest-2923 subunits with pisa  and pic  revealed that the hydrophobic and hydrogen-bond interactions are the main driving force for protein association not only for dimer formation, but also for tetramer formation. If this is the case, a displacement of the association equilibria towards tetramer formation can be predicted both under acidic or basic conditions with respect to the Cest-2923 isoelectric point (theoretical value of 6.5). In this sense, we carried out analytical ultracentrifugation studies of Cest-2923 at pH 9.0 (20 mm Tris, pH 9.0, 0.1 m NaCl) essentially as described above (both sedimentation velocity and equilibrium assays). The results obtained (Fig. S3) are similar to those obtained under acidic conditions, in agreement with an association process mainly guided by hydrophobic interactions.
As indicated above, EstE1 , Sto-Est , PestE  and Brefeldin A esterase  form canonical tetramers within the crystals (Fig. 4C). These tetramers have not been considered as a crystallographic by-product because they were observed in different crystal lattices with crystals prepared in disparate conditions. It is worth noting that this conclusion was derived despite these proteins being dimers in solution at much lower concentrations . It is obvious that this behaviour resembles the one described for Cest-2923 and therefore similar association equilibria for these proteins cannot be dismissed. From a structural viewpoint, Cest-2923 tetramers are not canonical ones, and are almost perfectly superimposable on those of the putative sugar hydrolase YeeB from L. lactis (PDB code: 3hxk), one of its closest structural relatives (Fig. 4D). When compared, it can be seen that both canonical and noncanonical tetramers result from a head-to-head association because the same region from both canonical dimers is the one involved in the association, although this region is different in both types of tetramers: thus, defining the cis face of the dimers as the one in which the C-terminal α-helix of the participating monomers is situated, noncanonical tetramers would result from a cis-to-cis association, whereas canonical ones would result from a trans-to-trans association. In both cases, the final oligomer exhibits a 222 point group, although it is evident that the relative orientation of the dimers within tetramers is also different: if one dimer of each tetramer is fixed as a reference and is equally oriented for comparison, the other dimers are rotated with respect to each other around 60º (Fig. S4).
In sum, these results indicate that Cest-2923 displays a pleomorphic behaviour because it can form different oligomeric assemblies depending on pH, protein concentration and probably other environmental conditions that can be derived from crystallization experiments. We consider that this behaviour reflects an intrinsic property of the enzyme, although, on the other hand, we have no experimental basis to claim that this property has functional consequences in vivo. Undoubtedly, this needs to be investigated further.
The most relevant enzymatic properties of Cest-2923 have been examined (Fig. 6). The optimum pH for hydrolytic activity against p-nitrophenyl acetate (pNPA) (see below) is 7.0 (Fig. 6A), which is a value typically observed for esterases, in contrast to the higher pH values (~ 8.0) displayed by lipases . With respect to temperature, the esterase presented the highest activity at ~ 30 °C, although, at 45 °C, it exhibited a high level of activity (~ 65%) (Fig. 6B). These values for optimum pH and temperature are commonly found in other esterases from Lactobacilli [24-26]. On the other hand, temperature stability measurements show a drastic reduction in Cest-2923 hydrolytic activity upon incubation of the esterase at 55 °C (Fig. 6C). This result agrees well with the analysis of protein thermostability carried out by far-UV CD spectroscopy, which revealed an apparent t1/2 value at ~ 60 °C at neutral and acidic pH values (Fig. S2).
Figure 6. Biochemical characterization of Cest-2923 from L. plantarum WCFS1. (A) Dependence on pH of hydrolytic activity of Cest-2923 against pNPA. Open squares indicate measurements in Tris buffer (pH 7.0) and phosphate buffer (pH 8.0) carried out to discard buffer-specific side effects. (B) Dependence on temperature of hydrolytic activity of Cest-2923 against pNPA. The optimum temperature for esterase activity was ~ 30 °C. (C) Analysis of the temperature stability of Cest-2923. Recombinant esterase was incubated in 50 mm sodium phosphate buffer pH 7.0 at 20 (open circles), 30 °C (closed circles), 37 °C (open squares), 45 °C (closed squares), 55 °C (open triangles) and 65 °C (closed triangles) for 15 min, 30 min, and 1, 2, 3, 4, 6 and 20 h. In all cases, the values shown are the mean of three independent experiments. (D) Dependence of the esterase activity of Cest-2923 on the aliphatic chain length of p-nitrophenyl (p-NP): p-NP acetate (C2); p-NP butyrate (C4); p-NP caprylate (C8); p-NP laurate (C12); and p-NP myristate (C14).
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The acyl-length selectivity against p-nitrophenyl ester substrates follows the order: C2 > C4 > C8 > C12 > C14, indicating a preference for short acyl-length esters (Fig. 6D). The kinetic parameters for C2 and C4 substrates were determined spectrophotometrically. In both cases, Cest-2923 exhibited a hyperbolic Michaelis–Menten kinetics (not shown). The kinetic parameters are shown in Table 1. From the values of these parameters, it can be deduced that the catalytic efficiency (kcat/Km) for pNPA hydrolysis is ~ 75-fold greater than that observed for p-nitrophenyl benzoate hydrolysis (pNPB). Also, substrate specificity has been analysed with the use of a library of esters, as previously described . This study reveals maximum hydrolysis against phenyl acetate, which is considered as reference (100% activity), and also significant activity (> 10% of the activity observed for phenyl acetate) against methyl bromoacetate and isopropenyl acetate. Within the limitations of the ester library employed, this result suggests a broader specificity for the alcohol part of the substrate and a preference for small moieties for the acid part.
Table 1. Kinetic parameters for pNPA and pNPB hydrolysis by Cest-2923. Enzyme activities were determined at 30 °C in 50 mm sodium phosphate buffer (pH 7.0). Results are the mean ± SD of three independent experiments.
|Substrate||Vmax (μmol·min−1·mg−1)||Km (mm)||kcat (s−1)||kcat/Vmax (s−1·mm−1)|
|pNPA||660 ± 50||1.7 ± 0.2||343.6 ± 26||202 ± 28|
|pNPB||40 ± 8||7.6 ± 1.2||20.8 ± 4||2.7 ± 0.7|
These three substrates were subsequently used for crystallographic studies aiming to determine the structure of the corresponding complexes. Accordingly, native crystals were incubated for ~ 60 s with a cryosolution containing the substrate at a concentration of 10 mm. Diffraction data recorded at beamline ID23-1 from the ESRF (Grenoble, France) with two types of these crystals (those prepared with methyl bromoacetate rapidly cracked) allowed the opportunity to solve their structure, which unfortunately did not result in the structure of the complexes but unexpectedly provided evidence of catalysis occurring within the crystals. Thus, we observed that, upon incubation with the corresponding cryosolution, the space group of the native crystals (P6322) changed: those crystals incubated with phenyl acetate became monoclinic (C2 space group), whereas those incubated with isopropenyl acetate changed to the P622 hexagonal space group. Interestingly, in both cases, the hexagonal packing was preserved and, indeed, indexing of the diffraction data identified the native unit cell and 622 point group, which suggests that these changes in crystal symmetry result from the ordered incorporation of new molecules within the crystal lattice (Table 2). One interesting observation is the presence in both types of crystals of an acetate molecule in the vicinity of the nucleophile Ser116, instead of a sulfate molecule, which appeared in native crystals (Fig. S5). We consider this to be remarkable because acetate by itself cannot replace sulfate in the active site as deduced from the native structure. We interpret this result as indicating the substitution of sulfate by the corresponding substrate, which is further hydrolysed. After this step, the alcohol is released in contrast to the acetate molecule, which remains in the active site.
Table 2. Data collection and refinement statistics.
|PDB accession code|| 4BZW || 4C01 || 4BZZ |
|Beamline||ID29 (ESRF)||ID23-1 (ESRF)||ID23-1 (ESRF)|
|Space group||P 6322||C 2||P 622|
|Unit-cell parameters (Å)|| || || |
|Resolution range (Å)||46.37–2.15||49.20–2.30||48.97–2.99|
|Number of measured reflections||1051 757||509 575||74 713|
|Number of unique reflectionsa||53 876 (5215)||124 688 (12 482)||9994 (1306)|
|Mean I/σ(I)a||17.2 (6.2)||143.2 (46.5)||13.7 (4.1)|
|Completeness (%)a||99.9 (98.8)||99.9 (100)||99.6 (98.9)|
|Redundancya||19.5 (18.0)||4.1 (4.3)||7.5 (7.3)|
|Rmerge (%)a; Rpim (%)a||13.3 (51.4); 3.1 (12.4)||14.3 (66.1); 13.6 (60.9)||11.9 (47.1); 4.6 (18.5)|
|Wilson B-factor (Å2)||21.2||21.2||41.98|
|Mean B-factors (Å2); protein||27.0||41.0||49.7|
|Mean B-factors (Å2); water||39.1||38.7||45.2|
|Mean B-factors (Å2); other||76.0||64.8||94.2|
|rmsd bond length (Å)||0.007||0.008||0.008|
|rmsd angles (º)||1.000||1.160||1.060|
|Ramachandran favoured (%)||97.0||92.0||95.0|
|Ramachandran outliers (%)||0.0||0.3||0.0|