Crystal Structure of Exo-Inulinase from Aspergillus Awamori: the Enzyme Fold and Structural Determinants of Substrate Recognition

doi:10.1016/j.jmb.2004.09.024 J. Mol. Biol. (2004) 344, 471–480

Crystal Structure of Exo-inulinase from Aspergillus awamori: The Enzyme Fold and Structural Determinants of Substrate Recognition

R. A. P. Nagem1†, A. L. Rojas1†, A. M. Golubev2, O. S. Korneeva3 E. V. Eneyskaya2, A. A. Kulminskaya2, K. N. Neustroev2 and I. Polikarpov1*

1Instituto de Fı´sica de Saõ Exo-inulinases hydrolyze terminal, non-reducing 2,1-linked and 2,6-linked Carlos, Universidade de Saõ b-D-fructofuranose residues in inulin, levan and sucrose releasing Paulo, Av. Trabalhador b-D-fructose. We present the X-ray structure at 1.55 A˚ resolution of Saõ-carlense 400, CEP exo-inulinase from Aspergillus awamori, a member of glycoside hydrolase 13560-970, Saõ Carlos, SP family 32, solved by single isomorphous replacement with the anomalous Brazil scattering method using the heavy-atom sites derived from a quick cryo- soaking technique. The tertiary structure of this enzyme folds into 2Petersburg Nuclear Physics two domains: the N-terminal catalytic domain of an unusual five-bladed Institute, Gatchina, St b-propeller fold and the C-terminal domain folded into a b-sandwich-like Petersburg, 188300, Russia structure. Its structural architecture is very similar to that of another 3Voronezh State Technological member of glycoside hydrolase family 32, invertase (b-fructosidase) from Academy, pr. Revolutsii 19 Thermotoga maritima, determined recently by X-ray crystallography The Voronezh, 394017, Russia exo-inulinase is a glycoprotein containing five N-linked oligosaccharides. Two crystal forms obtained under similar crystallization conditions differ by the degree of protein glycosylation. The X-ray structure of the enzyme:fructose complex, at a resolution of 1.87 A˚ , reveals two catalytically important residues: Asp41 and Glu241, a nucleophile and a catalytic acid/base, respectively. The distance between the side-chains of these residues is consistent with a double displacement mechanism of reaction. Asp189, which is part of the Arg-Asp-Pro motif, provides hydrogen bonds important for substrate recognition. q 2004 Elsevier Ltd. All rights reserved. Keywords: exo-inulinase; Aspergillus awamori; glycoside hydrolase; crystal- *Corresponding author lographic structure; X-ray structure

Introduction Jerusalem artichoke (Helianthus tuberosus)and chicory (Cichorium endivia).3 Levan, a similar poly- Most plants store starch or sucrose as reserve fructan, which consists of linear b-(2,6)-linked carbohydrates; however, about 15% of ﬂowering fructofuranosyl units, is produced in dicotyledo- plant species store fructans,1 which are branched nous species, and is found in several types of grass, polymers of fructose. Inulin is a widespread plant e.g. Dactylis glomerata.4 Mixed levan, consisting of polyfructan that has linear chains of b-(2,1)-linked both 2/1 and 2/6 linked b-D-fructosyl residues, is fructose residues attached to a terminal sucrose found in most plant species belonging to the Poales residue.2 Inulin serves as a storage polysaccharide family, including wheat and barley.4,5 in the Compositae and Gramineae, and is accumu- Inulin, due to its role as a relatively inexpensive and lated in the underground parts of several plants abundant substrate for production of rich fructose of the Asteracea, including Vernonia herbacea, syrups, is of considerable industrial interest.3,6 This type of syrup may be obtained by acid hydrolysis 8 † R.A.P.N. and A.L.R. contributed equally to this work. of inulin at 80–100 C; however, under these conditions fructose easily degrades, producing Abbreviations used: EI, exo-inulinase; GHF, glycoside 7 hydrolase family; Bsl, Bacillus subtilis levansucrase. colored products, such as difructose anhydrides. E-mail address of the corresponding author: The enzymatic hydrolysis with inulinases and [email protected] exo-inulinases (EI) offers an alternative approach to

produce fructose free of undesirable by-products. form and was refined to the final Rwork and Rfree of Successful biotechnological applications of inulinases 17.0% and 19.8%, respectively. The model has good have brought significant attention to these enzymes.8 stereochemical parameters, with root-mean-square Inulinases expressed by Penicillium,9–11 Kluyvero- deviations (rmsd) of 0.007 A˚ for bond distances, myces,12 and Aspergillus13,14 are the most intensively and 1.178 for bond angles. The crystallographic studied fungal enzymes of this class. Exo-inulinases model was also refined in the orthorhombic crystal ˚ (EC 3.2.1.80, also known as fructan b-fructosidase or form at 1.89 A resolution, with a final Rwork and exo-b-D-fructosidase) hydrolyze terminal, non- Rfree of 16.5% and 19.9%, respectively (rmsd of reducing 2,1-linked and 2,6-linked b-D- 0.011 A˚ for bond distances and 1.318 for bond fructofuranose residues in fructans, with concomi- angles). These structures were found to be essen- tant release of b-D-fructose. The natural substrates tially the same, with the exception of the degree of of exo-inulinases include inulin, levan and glycosylation. The crystallographic structure of sucrose. Nucleotide and amino acid sequences EI:fructose complex was refined at 1.87 A˚ resolution 15 were reported for exo-inulinases from yeast, with final Rwork and Rfree of 14.5% and 16.9%, 16 14 chicory, Aspergillus awamori and Geobacillus respectively. All residues but one fall in the allowed 17 stearothermophilus. regions of the Ramachandran plot, as determined Based on amino acid sequence comparisons, it by PROCHECK.22 Ser348 lies outside of the allowed has been demonstrated that the exo-inulinase (EI) regions, probably because its Og forms hydrogen from A. awamori belongs to the glycoside hydrolase bonds with both Asn333 Nd2 (2.80 A˚ ) and Trp331 18 family (GHF) 32. This family includes invertase, N31 (3.15 A˚ ), which results in energetically unfavor- inulinase, levanase and two types of 1-fructosyl able conformation of this residue. transferase. The catalytic activity of the enzymes The enzyme folds into two domains (Figure 1). from GHF 32 is based on a similar mechanism that The N-terminal domain, containing the catalytic involves overall retention of the configuration at site, comprises 353 amino acid residues (Phe20 to the anomeric carbon atom of the substrate. The Gln372). It belongs to the b-propeller fold with five similarities of the amino acid sequences and activity b-sheets arranged like blades of a propeller, radially toward the fructose containing poly- and oligo- and pseudosymmetrically around a central axis. saccharides possibly reflect an evolutionary Each b-sheet contains four antiparallel b-strands. relationship among GHF 32 members. Strands 1, 2, 3, and 4 are connected by hairpin turns, Here, we describe the first crystal structure of and strand 4 of one sheet is connected to strand 1 of exo-inulinase in a ligand-free and fructose-bound the next. Strand 1, in each “blade” of the b-propeller, form, and discuss a structural basis of the catalytic is the closest to the central axis and runs parallel mechanism of the enzyme. with it. The overall shape of the domain can be described as cylindrical, with strand 4 of each blade being on the outside of the cylinder. Results and Discussion The five-bladed b-propeller fold is rare, and is exemplified by tachylectin-2,23 Cellvibrio japonicus 24 Quality of the model and a three-dimensional a-L-arabinanase A43 and Bacillus subtilis levansu- structure of exo-inulinase crase.25 Remarkably, the latter two enzymes are members of GHF 43 and 68, respectively. Common Two crystal forms of the native exo-inulinase sequence motifs have been identified between GHF were obtained under the same crystallization 32 and 68,26 as well as between GHF 68 and 4327 conditions. Crystallization of the enzyme in the despite low (w15%) sequence identity between primitive orthorhombic P212121 space group has these families. been reported.14 The new crystal form belongs to The EI C-terminal domain, comprising 156 amino the monoclinic P21 space group. The crystallo- acid residues (Arg382 to Asn537), consists of graphic structure of the enzyme was solved by the two b-sheets, each composed of six b-strands, SIRAS method, using a quick cryo-soaking tech- assembled into a sandwich-like structure. The first nique,19–21 at 1.55 A˚ resolution in the monoclinic and second domains are connected by a short

Figure 1. (a) Ribbon representation of the secondary structure elements of exo-inulinase. The N- terminal domain belongs to the five-bladed b-propeller fold. Each blade is shown in a different color (blade 1 is yellow, blade 2 is marine, blade 3 is red, blade 4 is lime and blade 5 is orange). The second domain, colored in slate, consists of 12 b-strands arranged in a b-sandwich fold. The short polypeptide chain connecting the two domains is depicted in pink. The five N-linked oligosaccharides present in the orthorhombic crystal are represented as stick. (b) Representation of exo-inulinase surface with the fructose molecule in the activity site. A. awamori Exo-inulinase 3D Structure 473 polypeptide linker, with relative positions and although the amino acid sequences of these orientations of the domains stabilized by multiple enzymes hardly bear any similarity (amino acid hydrogen bonds and hydrophobic interactions. sequence identity is only 12%), the fold of both Comparison of the present EI model with the protein domains is very similar with rmsd between structures deposited with the Protein Data Bank, 105 Ca atom pairs of 1.97 A˚ (Figure 2(b)). Overall, carried out using the DALI server,28 reveals a the fold of EI is essentially the same as that of significant structural similarity between the EI Thermotoga maritima invertase, another member of N-terminal b-propeller domain and the N-terminal GHF 32, which has been determined very recently domain of levansucrase from B. subtilis (PDB code by protein crystallography (PDB code 1UYP).30 1OYG),25 a member of GHF 68. The rmsd values for Their amino acid sequence is 29% identical; the 189 Ca atom pairs is 1.98 A˚ , while the sequence structural similarity extends over the entire core of identity between these two enzymes is only 13% two enzymes and the rmsd values between 392 (Figure 2(a)). An additional search shows structural correspondent Ca positions is only 1.6 A˚ . The close similarity of the inulinase C-terminal domain with structurally homology of two distinct members of carbohydrate recognition domain of rat calcium- the GHF 32 enables us to assert that the observed dependent lectin p58 (PDB code 1GV9).29 Strikingly, fold represents a template for other members of

Figure 2. (a) Stereoview of a superposition of the EI N-terminal domain (blue) with levansucrase from Bacillus subtilis (red). (b) Stereoview of the EI C-terminal domain, depicted in blue, superimposed with the carbohydrate recognition domain of rat lectin p58, painted in red. 474 A. awamori Exo-inulinase 3D Structure this family. Figure 3 shows primary structure sucrose:sucrose 1-fructosyl transferase (GenBanke alignments of representative members of GHF 32 CAA04131.1), Gluconacetobacter diazotrophicus leva- together with the secondary structure elements nase (GenBanke AAF16405.1), Aspergillus ﬁcuum of exo-inulinase from A. awamori. Alignment of inulinase (GenBanke CAA07345.1) and T. maritima amino acid sequences of A. awamori exo-inulinase invertase (GenBanke AAD36485.1) was performed (GenBanke CAC44220.1), Aspergillus foetidus with program CLUSTAL W.31

Figure 3. Sequence alignment of representative members of glycoside hydrolase family 32. The following sequences were used in the alignment: Exo-inulinase, Aspergillus awamori exo-inulinase; Suc:suc 1-fruct., Aspergillus foetidus sucrose:sucrose 1-fructosyl transferase; Levanase, Gluconacetobacter diazotrophicus levanase; Inulinase, Aspergillus ﬁcuum inulinase; Invertase, Thermotoga maritima invertase. The amino acid similarity between primary structures as calculated by the program ALSCRIPT55 is shown in three different shades of blue. The darker color corresponds to higher levels of sequence similarity. Secondary structure elements for EI as suggested by the program PROCHECK are shown below the amino acid sequences (the same color codes as those used in Figure 1 were applied). Catalytic residues and the conserved RDP motif are highlighted by green and red boxes, respectively. A. awamori Exo-inulinase 3D Structure 475

Fructose-binding site and catalytic residues Table 1. Close contacts between protein and fructose non- carbon atoms

Superposition of the native EI structure with the Fructose hydroxyl crystallographic model of the fructose-containing groups Direct hydrogen bond complex of enzyme enables 3D analysis of the Protein atom Distance (A˚ ) fructose-binding site. A single fructose molecule, clearly deﬁned by the difference Fourier electron OH-1 Asp41 OD1 2.49 Trp335 NE1 2.95 density map, is bound between the blades of OH-2 Glu241 OE2 2.70 the b-propeller almost at the central axis of the Water 2.72 N-terminal domain (Figure 1(b)). The angle OH-3 Asp189 OD2 2.58 between the plane of the sugar ring and the central Arg188 NE 2.99 8 Glu241 OE2 3.05 axis is approximately 70 . Figure 4(a) shows the OH-4 Ser103 N 3.16 fructose molecule bound to EI, together with the Asp189 OD1 2.61 amino acid residues of the active site. Direct OH-5 Gln57 NE2 2.59 hydrogen bond distances between the protein, Trp65 ND2 2.96 water molecules and OH groups of fructose, Asn40 ND2 2.97 depicted in Figure 4(b), are given in Table 1. A distance cut-off of 3.3 A˚ was used. The enzymatic hydrolysis catalyzed by glycoside hydrolases takes place via a general acid catalysis that requires two catalytic residues: a proton donor and a nucleophile.32 The hydrolysis occurs via two major mechanisms giving rise to either an overall

Figure 4. (a) Stereoview of fructose:enzyme interactions in the catalytic site of exo-inulinase. The fructose and the K neighboring residues are shown. An omit electron density map (mFobs DFcalc, fcalc), contoured at 3s, is displayed around the catalytic site of EI. (b) Schematic design of the interactions between the active-site residues and the fructose molecule (in pink). Hydrogen bonds are shown as cyan dotted lines. The Figure was drawn using the program LIGPLOT.56 476 A. awamori Exo-inulinase 3D Structure retention or inversion of the anomeric configuration positions and conformations of the active-site of the substrate.33 In both, retaining and inverting residues are conserved between EI and T. maritima glycosidases, the proton donor is found within a invertase. The putative reaction mechanism hydrogen bond distance from the glycosidic oxygen involving Asp41 and Glu241 in hydrolysis of atom. In retaining enzymes, the nucleophile/base is fructose containing oligossacharides is depicted in in close vicinity of the sugar anomeric carbon Figure 5. atom. However, the nucleophile is more distant in Asp41 and Glu241 are located, respectively, at the inverting enzymes that must accommodate a water b-1 strand of the first blade of the b-propeller and molecule between the catalytic base and sugar. the b-1 strand of the fourth blade. Although EI Consequently, the average distances of approxi- belongs to the group of enzymes that retain the mately 5.5 A˚ and 10 A˚ are commonly observed overall anomeric configuration of the substrate, between the two catalytic residues in the retaining the distances from Asp41 Od2 and Glu241 O32 to C1 and inverting enzymes, respectively.34 On the basis of fructose are slightly larger (3.19 A˚ and 3.26 A˚ , of the 3D superposition of the EI structure and its respectively) than those found normally in the complex with fructose (Figure 4(a)), we identified retaining carbohydrases (approximately 3.0 A˚ ). The Asp41 and Glu241 as two putative catalytic residues. possible reason for this observation is related to The former is presumably a nucleophile, whereas the fact that the position and orientation of the the latter is a proton donor. This structure-based fructose, the inhibitor and a product of the assignment of the catalytic residues is in agreement enzymatic reaction, may be slightly different from with the site-directed mutagenesis studies of those of the natural substrate, polyfructan. another member of GHF 32, yeast invertase.35,36 Amino acid sequence alignment reveals 36% The conserved RDP motif identity and 52% similarity of these two enzymes. EI Asp41, the residue of the conserved sequence The multiple sequence alignment of fructosyl motif 38-WMNDPNG-44, and Glu241 of the con- transferases, invertases, levanases, inulinases served motif 241-ECPGL-245 (EI amino acid and sucrose-6-phosphate hydolases revealed the numbering), are equivalent, respectively, to Asp23 presence of a conserved Arg-Asp-Pro (RDP) motif, and Glu204 of yeast invertase. Mutation of Asp23 to which is presumed to have a common functional Asn rendered the enzyme basically inactive,35 role.37–39 The amino acid sequence of EI contains the whereas mutagenizing Glu204 to Ala resulted in a conserved RDP motif (Arg188-Asp189-Pro190). 3000-fold reduction in the kcat of invertase, indicat- Residues Arg188 and Asp189 of this motif partici- ing that these two residues play a major role in pate in substrate binding, and are involved in catalysis.36 This conclusion is strengthened further hydrogen bond interactions with the hydroxyl by the recent results of site-directed mutagenesis groups 3 and 4 of fructose. It seems plausible, and structural analysis of B. subtilis levansucrase therefore, that these interactions are important for (Bsl).25 Mutation of Asp86 (nucleophile) or Glu342 recognition of the sugar ring and might be (general acid), that are structurally analogous to responsible for specificity of the enzyme toward Asp41 and Glu241 in EI, to alanine completely fructopyranosyl residues (Figure 4). abrogates Bsl enzymatic activity. Moreover, Recently, the role of Asp of the RDP motif of

Figure 5. A representation of a double displacement mechanism of reaction with an overall retention of the configuration at the anomeric carbon atom of the substrate, catalysed by exo-inulinase from Aspergillus awamori. The R group may represent fructose units of inulin. A. awamori Exo-inulinase 3D Structure 477 levansucrase from Acetobacter diazotrophicus SRT4 a-mannosidases during cultivation of the fungi. A (LsdA) was studied by site-directed mutagenesis.40 similar process has been observed for Trichoderma The D309N LsdA mutant showed considerable reesei.41 Further selection of the less glycosylated reduction in catalytic activity. The kcat of the molecules could occur during crystallization as a D309N LsdA was decreased 75-fold, while Km consequence of variation of protein solubility with value was similar to those of the wild-type enzyme, different content of glycosyl moieties. It is known indicating that RDP motif Asp plays an essential that mannose oligosaccharides attached to role in the catalytic mechanism.40 Along the same other glycoside hydrolases in the process of post- line, mutation of Asp274 to alanine resulted in translational modification aid in self-association inactivation of Bsl.25 Asp274 makes part of the RDP into tetramers, hexamers and other oligomeric conserved sequence motif found also in GHF 68. assemblies.36,42,43 It is, therefore, not surprising In order to elucidate the result of a similar that protein glycosylation interferes strongly in its mutation within the RDP motif on catalytic activity assembly during nucleation and crystal growth. of EI, we replaced Asp189 by Asn in the crystallographic model of the enzyme (D189N mutation). This mutation does not significantly affect overall Conclusions positions or orientations of the active-site residues, but it opens a possibility for a new hydrogen bond The high-resolution X-ray model of the exo- between catalytic Asp41 and Asn189 that does not inulinase from A. awamori presented here reveals existinthenativeEI.Suchhydrogenbond that the protein is composed of two separated interaction would change the nucleophilicity of domains, the larger N-terminal catalytic domain of Asp41, causing the electron density of the hydroxyl the rare five-bladed b-propeller fold and the smaller groups of the Asp41 side-chain to shift towards the C-terminal domain, folded as a b-sandwich. Asn189 amide group. This would render Asp41 less Structural comparison of the native enzyme with nucleophilic in character, affecting the efficiency of the enzyme:fructose complex enables the identifi- sucrose hydrolysis, but not the enzyme specificity. cation of the active site, and residues Glu241 as the This hypothesis is in agreement with the site- proton donor and Asp41 as the nucleophile. Asp41 directed mutagenic studies of levansucrase and Glu241 are located, respectively, at the blades 1 from A. diazotrophicus,40 and provides a structural and 4 of the N-terminal b-propeller domain. The basis for the importance of the conserved RDP motif average distance of 6.1 A˚ between the four pairs of for glycosyl transferase specificity toward side-chain oxygen atoms of the catalytic residues is fructosylsaccharides. similar to that (5.5 A˚ ) found in enzymes with the overall retention of the anomeric configuration of Carbohydrate moiety and its effect on the substrate. The EI structures shed light on the crystallization putative role of the Asp189 residue that is part of the conserved RDP motif. This residue is found in The inulinase is a glycoprotein containing five the active site of the enzyme and is important sites of N-glycosylation. During the inulinase for substrate recognition. We speculate that the crystallization, two crystal forms, primitive mono- D189N mutation might result in a new hydrogen bond between Asn189 and catalytic nucleophile clinic, P21, and primitive orthorhombic, P212121, were obtained in the same crystallization drops. Asp41, affecting its nucleophilic properties, but X-ray diffraction data for both crystal forms were not changing its conformation. This could decrease collected and the difference in glycosylation the catalytic efficiency significantly, but not the between EI structures determined in the two specificity, of the enzyme toward sucrose hydro- forms was analyzed. Five glycosylation sites in lysis, which is in agreement with biochemical both crystal forms are located at asparagine studies of structurally similar enzymes. residues; Asn67, Asn111, Asn300, Asn398, and Asn430. In the monoclinic form, only N-acetyl- galactosamine residues were located in the electron Materials and Methods density map. In the orthorhombic form, a branched tetramannosaccharide attached to two N-acetyl- Protein purification, crystallization and data galactopyranoside residues was found at Asn67. It collection is tempting to conclude that this antenna-like sugar structure prevented crystallization in the mono- Extracellular A. awamori EI was purified and prelimi- clinic space group. Indeed, a superposition of nary X-ray data were collected from an orthorhombic crystal form as described.14 A new monoclinic form of the X-ray structure refined in space group P212121 onto the 3D model determined in the monoclinic crystals was found under similar crystallization con- crystal form proves that the tetramannosaccharide ditions. X-ray diffraction studies revealed that EI crystal- lizes in the monoclinic space group P21, with the unit cell would cause a clash with the symmetry-related parameters aZ49.95 A˚ , bZ94.03 A˚ , cZ67.63 A˚ , bZ107.048 molecules. In nature, all five N-glycosylation and the crystals diffracted beyond 1.55 A˚ resolution. sites could contain antenna-like mannosaccharides. Calculation of the Matthews coefficient suggested the Z ˚ 3 K1 Their absence from the crystal may be explained by presence of a monomer (VM 2.6 A Da ) in the asym- presuming that they were cleaved with A. awamori metric unit with a solvent content of approximately 52% 478 A. awamori Exo-inulinase 3D Structure

(v/v). Native EI crystals (monoclinic) were used to modification with solvent flattening was performed prepare an iodine derivative according to the quick with the program SOLOMON.49 In the orthorhombic 19–21 cryo-soaking approach for derivatization by immer- case (P212121), initial phases for native structure were sing a crystal in the cryogenic solution containing 0.5 M obtained by the molecular replacement (MR) method 50 sodium iodide for a few minutes and then flash-cooled to with program AMoRe using the refined P21 structure as 100 K in a stream of cold nitrogen. Similarly, the search model. fructose:enzyme complex was obtained by soaking native EI crystals in 20% (w/v) fructose solution. X-ray Initial model building in monoclinic form and diffraction data were collected on a MAR345 image plate structure refinement using osmic mirror-focused Cu Ka X-rays, generated by a RIGAKU ultra X 18 rotating anode operating at 80 mA and 50 kV. Solvent-flattened, iodine-derived electron density map In total, three complete data sets (monoclinic form) and structure factor amplitudes from the native monoclinic data set were used by the program ARP/wARP51 were collected: native, iodine derivative and fructose 52 complex. Diffraction images were processed and scaled combined with cycles of REFMAC for an automatic 44 45 build of the EI 3D structure. Electron density maps with with the programs MOSFLM and SCALA, respect- K ively. Data collection statistics of each data set, including coefficients 2mFobs DFcalc and model-derived phases the previously collected orthorhombic form, are shown in enabled the identification of the side-chains of the protein. Table 2. The manual model construction and the initial side-chain assignment were performed using the programs O53 and XtalView.54 Phasing procedure After careful identification of the amino acid sequence, the 3D model went through several cycles of refinement Due to the strong incorporation of iodine anions into with REFMAC and water molecules insertion with the P21 crystallographic structure, the phase problem in ARP-WATERS from the CCP4 program package. The the monoclinic form could be solved either by single N-linked oligosaccharides were added using the Xfit anomalous dispersion (SAD) or single isomorphous program from the XtalView suite. replacement with anomalous scattering (SIRAS) methods. Analysis of the electron density maps of the EI:fructose Nevertheless, only more robust SIRAS phases were used complex revealed clear electron density for the fructose for structure determination. The majority of the iodine bound to the active site of the enzyme. The structures of positions were determined by direct methods with the the fructose:enzyme complex and the iodine derivative programs DREAR46 and Shake-and-Bake.47 This heavy- were refined using the same protocols as those used for atom substructure was then set as an input into the the native structure. The structure in the orthorhombic SHARP program48 for phase calculations. Density form was refined in a very similar way, after identification

Table 2. Data collection and reﬁnement statistics of exo-inulinase (EI) crystals

Data set Native protein Iodine derivative EI:fructose

A. Crystal preparation Cryoprotectant solution Mother liquora 20% glycerol Mother liquor 20% Mother liquor 20% glycerol 0.5 M NaI fructose Soaking time A few seconds 15 minutes 2 minutes B. Data collection Wavelength (A˚ ) 1.54 1.42 1.54 1.54 Space group P21 P212121 P21 P21 Unit cell parameters a (A˚ ) 49.95 64.82 49.97 49.83 b (A˚ ) 94.03 82.13 93.67 93.56 c (A˚ ) 67.63 136.22 83.36 67.58 b (deg.) 107.04 106.92 107.29 Resolution (A˚ ) 17.8–1.55 (1.63–1.55) 19.9–1.89 (1.99–1.89) 26.6–1.87 (1.97–1.87) 26.6–1.87 (1.97–1.87) No. reflections 487.330 310.283 834.739 365.375 hI/(s)i 10.3 (4.5) 8.0 (3.8) 12.8 (5.9) 11.7 (4.0) Multiplicity 2.9 (2.9) 5.5 (5.0) 7.9 (7.4) 3.5 (3.3) Completeness (%) 91.3 (85.7) 99.8 (99.6) 92.1(84.2) 99.6 (97.6) b Rmerge (%) 4.5 (15.0) 5.4 (29.7) 4.2 (12.8) 5.7 (19.0) C. Structure refinement R-factor (%) 17.0 16.5 17.8 14.5 R-free (%) 19.8 19.9 21.6 16.9 No. water molecules 921 639 549 755 No. carbohydrate residues 5 11 3 6 No. iodine ionsc – – 25 – No. glycerol molecules 2413 Rmsd values Bond lengths (A˚ ) 0.007 0.011 0.012 0.013 Bond angles (deg.) 1.17 1.31 1.34 1.39 Statistical values for the highest-resolution shells are shown in parentheses. a Mother liquorP consists ofP 15% (w/v) PEG 3350, 1 mM sodium cacodylate, 20–100 mM sodium acetate (pH 4.0–5.0). b Z K = Rmerge hkl jIhkl hIhklij hkl Ihkl. c Iodine sites were not fully occupied. A. awamori Exo-inulinase 3D Structure 479 of the correct molecular replacement solution. The final 17. Tsujimoto, Y., Watanabe, A., Nakano, K., Watanabe, refinement statistics for each data set are given in Table 2. K., Matsui, H., Tsuji, K. et al. (2003). Gene cloning, expression, and crystallization of a thermostable exo- inulinase from Geobacillus stearothermophilus KP1289. Appl. Microbiol. 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Edited by I. Wilson

(Received 8 June 2004; received in revised form 26 August 2004; accepted 14 September 2004)