Structural basis for substrate recognition by Erwinia chrysanthemi GH30 glucuronoxylanase L ˇ ubica Urba ´ nikova ´ 1 ,Ma ´ ria Vrs ˇ anska ´ 2 , Kristian B. R. Mørkeberg Krogh 3 , Tine Hoff 3 and Peter Biely 2 1 Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia 2 Institute of Chemistry, Center of Glycomics, Slovak Academy of Sciences, Bratislava, Slovakia 3 Novozymes A ⁄ S, Bagsvaerd, Denmark Introduction The important industrial enzyme endo-b-1,4-xylanase ( EC 3.2.1.8) has been placed into several glycoside hydrolase (GH) families on the basis of hydrophobic cluster analysis, 3D, and mode of action [1] (carbohy- drate-active enzymes server at http://www.cazy.org). The best-characterized xylanases belong to GH fami- lies 10 and 11. These enzymes do not seem to be spe- cialized for hydrolysis of a particular xylan, because they are capable of degrading hardwood acetyl glucu- ronoxylans, cereal arabinoxylans, and even algal b-1,4-b-1,3-xylan (rhodymenan) [2–4]. The activity of xylanases belonging to these two families does not appear to be dependent on the type of side chain dec- orations of the xylan main chain, but is strongly dependent on the density of substituents [2,5]. The cleavage of the xylan main chain by GH10 xylanases Keywords crystal structure with ligand; Erwinia chrysanthemi; GH30; glucuronoxylan-specific xylanase; substrate recognition Correspondence P. Biely, Institute of Chemistry, Center of Glycomics, Slovak Academy of Sciences, Du ´ bravska ´ cesta 9, SK-845 38 Bratislava, Slovakia Fax: +421 2 5941 0222 Tel: +421 2 5941 0275 E-mail: chempbsa@savba.sk (Received 17 December 2010, revised 10 April 2011, accepted 13 April 2011) doi:10.1111/j.1742-4658.2011.08127.x Xylanase A from the phytopathogenic bacterium Erwinia chrysanthemi is classified as a glycoside hydrolase family 30 enzyme (previously in family 5) and is specialized for degradation of glucuronoxylan. The recombinant enzyme was crystallized with the aldotetraouronic acid b- D-xylopyranosyl- (1 fi 4)-[4-O-methyl-a- D-glucuronosyl-(1 fi 2)]-b-D-xylopyranosyl-(1 fi 4)- D-xylose as a ligand. The crystal structure of the enzyme–ligand complex was solved at 1.39 A ˚ resolution. The ligand xylotriose moiety occupies sub- sites )1, )2 and )3, whereas the methyl glucuronic acid residue attached to the middle xylopyranosyl residue of xylotriose is bound to the enzyme through hydrogen bonds to five amino acids and by the ionic interaction of the methyl glucuronic acid carboxylate with the positively charged guan- idinium group of Arg293. The interaction of the enzyme with the methyl glucuronic acid residue appears to be indispensable for proper distortion of the xylan chain and its effective hydrolysis. Such a distortion does not occur with linear b-1,4-xylooligosaccharides, which are hydrolyzed by the enzyme at a negligible rate. Database Structural and experimental data are available in the Protein Data Bank database under accession number 2y24 [45]. Abbreviations GH, glycoside hydrolase; GlcA, D-glucuronic acid; MeGlcA, 4-O-methyl-D-glucuronic acid; MeGlcA 2 Xyl 2 ,4-O-methyl-a-D-glucuronosyl-(1 fi 2)- b- D-xylopyranosyl-(1 fi 4)-D-xylose; MeGlcA 2 Xyl 3 , b-D-xylopyranosyl-(1 fi 4)-[4-O-methyl-a-D-glucuronosyl-(1 fi 2)]-b-D-xylopyranosyl-(1 fi 4)-D- xylose; MeGlcA 3 Xyl 3 ,4-O-methyl-a-D-glucuronosyl-(1 fi 2)-b-D-xylopyranosyl-(1 fi 4)-b-D-xylopyranosyl-(1 fi 4)-D-xylose; MeGlcA 3 Xyl 4 , b- D-xylopyranosyl-(1 fi 4)-[4-O-methyl-a-D-glucuronosyl-(1 fi 2)]-b-D-xylopyranosyl-(1 fi 4)-b-D-xylopyranosyl-(1 fi 4)-D-xylose; MeXyl 3 Xyl 3 , 4-O-methyl-a- D-glucuronosyl-(1 fi 2)-b-D-xylopyranosyl-(1 fi 4)-b-D-xylopyranosyl-(1 fi 4)-D-xylose; MeXyl 3 Xyl 4 , b-D-xylopyranosyl-(1 fi 4)-[4-O- methyl-a- D-glucuronosyl-(1 fi 2)]-b-D-xylopyranosyl-(1 fi 4)-b-D-xylopyranosyl-(1 fi 4)-D-xylose; VS, virtual screening; Xyl, xylose; XynA, Erwinia chrysanthemi GH30 xylanase. FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works 2105 requires at least two consecutive unsubstituted xylo- pyranosyl residues, whereas hydrolysis by GH11 xylanases requires three consecutive unsubstituted xylopyranosyl residues [2–6]. Heavily substituted xylan, such as corn fiber xylan [7], is completely resis- tant to the action of members of these two xylanase families (P. Biely, unpublished results). An interesting endoxylanase, classified in GH family 8, was found to be produced by an Antarctic bacterium, Pseudoaltero- monas haloplanktis [8]. This enzyme showed the high- est activity on rhodymenan [8], which indicates that the enzyme might be specialized for hydrolysis of the linear xylan present in algae. Unique xylanases are found in GH family 30 [1,9]. These enzymes were originally classified in GH fam- ily 5. Some bacterial GH30 xylanases are specialized for the hydrolysis of xylans that contain d-glucuronic acid (GlcA) or 4-O-methyl-d-glucuronic acid (MeG- lcA) side residues. However, not all GH30 xylanases show this specificity. The recently described GH30 xy- lanase from the fungus Bispora sp. does not show such a requirement for these side residues [10]. With Bacil- lus subtilis GH30 xylanase and Erwinia chrysanthemi GH30 xylanase (XynA), it was clearly demonstrated that cleavage of the xylan main chain is dependent on the presence of MeGlcA side residues [11–14]. A simi- lar enzyme from another Bacillus species was recently described [15]. The cleavage of the main xylan chain takes place at the second glycosidic linkage from the MeGlcA side group towards the reducing end of the xylan chain. The elucidation of the three-dimensional structure of the E. chrysanthemi GH30 enzyme [16], together with its established mode of action [14], allowed us to present a hypothesis for the basis of sub- strate recognition in this group of so-called ‘append- age-dependent xylanases’ [11]. Examination of the structure of XynA [14] for the presence of aromatic amino acids and positively charged amino acid groups in the vicinity of the identified catalytic glutamic acids (Glu253, nucleophile; Glu165, acid ⁄ base) indicated that the substituted xylopyranosyl residue should be accommodated at the hypothetical subsite )2. Tyr290 and Trp289 near subsite )2 were considered to consti- tute a suitable place for binding of MeGlcA. However, the space between the two aromatic amino acids was too narrow to accommodate the uronic acid. An ionic interaction between the negatively charged MeGlcA carboxylate and the positively charged Arg293 (pK a > 12) occurring in the vicinity of the Tyr290 ⁄ Trp289 sandwich was also proposed to play an important role in uronic acid binding [14]. It became clear that definite understanding of the recognition of uronic acid by GH30 xylanases would require X-ray crystallographic studies of the enzyme–ligand complex. Preliminary data on the crystallization of the B. subtilis GH30 xylanase have also been released, but as yet without a proper ligand [17]. Here we report an X-ray structure of the complex of XynA with the aldotetraouronic acid b-d-xylopyr- anosyl-(1 fi 4)-[4-O-met hyl-a-d-glucuronosyl-(1 fi 2)]-b-d- xylopyranosyl-(1 fi 4)-d-xylose (MeGlcA 2 Xyl 3 )(Fig.S1). The ligand filled three of the hypothetical subsites on the glycone (subsites with negative designation) side of the substrate-binding site [18,19]. Subsite )2 accommo- dates the xylopyranosyl residue substituted with MeG- lcA. A detailed analysis of the enzyme–ligand complex confirmed the ionic interaction of the substrate carbox- ylate group with the enzyme. Furthermore, it pointed to a number of hydrogen bonds formed between the enzyme and its substrate. Results Crystallization and data collection Electrophoretically homogeneous recombinant XynA was subjected to dynamic light scattering analysis before crystallization. This method gives information on the homogeneity and size distribution of particles in solution [20]. Despite a relatively high measured polydispersity (Fig. S2), the protein crystallized rela- tively easily and produced high-quality crystals. Attempts were made to obtain crystals of the pro- tein–MeGlcA 2 Xyl 3 complex by diffusion of the ligand into pregrown crystals of the ligand-free enzyme or by cocrystallization. Crystals of ligand-free XynA were obtained under several conditions, which were further optimized to give diffraction-quality crystals. Crystals of two distinct habits were obtained (Fig. S3A,B); however, they were found to belong to the same P3 2 21 space group. Crystals of the XynA–MeGlcA 2 Xyl 3 complex were obtained by both methods tested; however, the best diffraction data were recorded with the crystal of the complex obtained by cocrystallization (Fig. S3C). These data are reported here. The crystals of the complex belonged to the P3 2 21 space group, with dimensions a = b = 59.578 A ˚ and c = 168.296 A ˚ , c = 120°. Crystal symmetry, unit cell dimensions and the molecular mass of the protein gave a Matthews coefficient of 2.05 and a 40% solvent con- tent in the crystal for one protein molecule in the asymmetric unit [21]. Diffraction data statistics are shown in Table 1. For a comparison, the first structure of XynA, crys- tallized without any ligand, belonged to the monoclinic X-ray structure of xylanase A–ligand complex L ˇ . Urba ´ nikova ´ et al. 2106 FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works P2 1 space group [16,22]. The authors reported multiple crystal forms, including hexagonal crystals with a P6 n space group and unit cell dimensions of a = b = 60.32 A ˚ and c = 165.78 A ˚ , which are very close to the unit cell dimensions reported here. In all cases, only one protein molecule was found in the asymmetric unit. As expected, the crystal contacts in the monoclinic and trigonal forms were different. Structure description The structure of XynA in complex with MeGlcA 2 Xyl 3 was solved by molecular replacement at resolution 1.39 A ˚ , with the original XynA crystal structure as a search model (1NOF) [16]. The final R-factor and R free - factor were 12.2% and 16.9%, respectively. The refine- ment statistics are shown in Table 1. The model consists of 383 amino acids (numbered 31–413 in the sequence), a single MeGlcA 2 Xyl 3 ligand, an imidazole, three mole- cules of poly(ethylene glycol), and 571 water molecules. MeGlcA 2 Xyl 3 , imidazole and poly(ethylene glycol) mol- ecules were modeled at later stages of refinement, when the electron density was unambiguous (Fig. 1) [the poly(ethylene glycol) molecules are not shown]. The overall structure of XynA in the complex with MeGlcA 2 Xyl 3 (Fig. 2A,B) is nearly identical to the 1NOF structure described previously by Larson et al. [16]. The enzyme consists of a (b ⁄ a) 8 -barrel catalytic domain and a b-sheet immunoglobulin-like C-terminal domain (a potential xylan-binding module) connected by amino acids 45 and 317–322. One cis-peptide bond has been found between Val200 and Ala201. Superposition of the structure of the ligand-free enzyme with the structure of the enzyme in the complex using 378 CA atoms (CA atoms with two alternative conformations were omitted) resulted in root mean square, average and maximum xyz displacements of 0.275 A ˚ , 0.241 A ˚ , and 0.849 A ˚ , respectively. It is Table 1. Data collection and refinement statistics. R merge = P hkl P i |I i (hkl)–ÆI(hkl)æ| ⁄ P hkl P i I i (hkl), where I i (hkl) is the intensity measure- ment for the ith observation of reflection hkl and ÆI(hkl)æ is the average intensity for multiple measurements for this reflection. R = P ||F obs | ) |F calc || ⁄ P |F obs |, where F obs and F calc are observed and calculated structure factor amplitudes. A random subset (5%) of data excluded from the refinement was used for R free factor calculation. XynA–MeGlcA 2 Xyl 3 Data collection Beamline X13 EMBL Hamburg Wavelength (nm) 0.831 Space group P3 2 21 (No. 154) Unit cell dimensions a, b, c (A ˚ ) 59.578, 59.578, 168.296 a, b, c (°) 90, 90, 120 Resolution range, overall ⁄ outer shell (A ˚ ) 1.39–20.0 ⁄ 1.388–1.395 No. of observed reflections, overall ⁄ outer shell 468 272 ⁄ 3188 No. of unique reflections, overall ⁄ outer shell 70 207 Completeness, overall ⁄ outer shell (%) 98.6 ⁄ 98.6 Mean I ⁄ r (I ), overall ⁄ outer shell 8.5 ⁄ 1.3 Wilson B-factor (A ˚ ) 18.89 R merge , overall ⁄ outer shell (%) 8.0 ⁄ 41.8 Refinement R overall ⁄ R working ⁄ R free (%) 12.20 ⁄ 11.96 ⁄ 16.93 Asymmetric unit content (No. of molecules) Protein ⁄ MeGlcA 2 Xyl 3 ⁄ imidazole ⁄ poly(ethylene glycol) ⁄ water 1 ⁄ 1 ⁄ 1 ⁄ 3 ⁄ 571 B average (A ˚ 2 ) Main chain ⁄ side chain ⁄ ligands ⁄ water 11.14 ⁄ 13.24 ⁄ 22.96 ⁄ 29.74 Model quality Ramachandran plot Preferred region [% (number of residues)] 90.1 (301 ⁄ 334 a ) Allowed region [% (number of residues)] 9.6 (32 ⁄ 334 a ) Generously allowed region [% (number of residues)] 0.3 (1 ⁄ 334 a ) Geometry Rmsd bond distances (A ˚ ) 0.024 Rmsd bond ⁄ torsion angles (°) 1.973 ⁄ 6.841 Estimated standard uncertainties based on R-value ⁄ R free (A ˚ ) 0.056 ⁄ 0.056 a Number of nonglycine and nonproline residues. L ˇ . Urba ´ nikova ´ et al. X-ray structure of xylanase A–ligand complex FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works 2107 interesting that the side chain conformations of amino acids interacting with MeGlcA 2 Xyl 3 did not change as a result of binding. The superposition of both structures also showed that the acetate anion observed in the first published structure (1NOF) [16] interacts with the posi- tively charged guanidinium group of Arg293 in a man- ner similar to the carboxylate group of MeGlcA 2 Xyl 3 . The protein substrate-binding site has a total area of 321.9 A ˚ 2 , and is composed of 17 amino acids; 44.4% of the binding site surface is hydrophobic, i.e. covered by carbon atoms. The remaining 55.6% is polar, cov- ered by nitrogen and oxygen atoms (Table S1; Fig. 3A). Thirteen of the 17 amino acids form 174 van der Waals contacts and 10 hydrogen bonds with MeG- lcA 2 Xyl 3 (Table 2; Fig. 3B). The three xylose (Xyl) units of the ligand take part in the stacking interac- tions with the aromatic rings of Trp289, Tyr172 and Trp55 in subsites ) 1, )2, and )3, as shown in detail in Fig. 4A,B. The Xyl in subsite )1 is also coordinated with Trp113, Asn164, and the catalytic Glu165 and Glu253 (Fig. 4B). The MeGlcA moiety interacts with the edges of the aromatic rings of the Trp289 and Tyr290 side chains, and also forms one hydrogen bond with the Trp289 amide nitrogen, NE1. The most important interaction for substrate recognition appears to be an ionic interaction between the positively charged Arg293 guanidinium group and the negatively charged carboxylate of MeGlcA (Fig. 4C). An electron density found in the proximity of the catalytic amino acids Glu165 and Glu253 was ascribed to imidazole, a component of the crystallization buffer. Imidazole interacts with Tyr168 and Trp232, and is also electrostatically bound to the catalytic Glu165 (Fig. 4D). Thus, imidazole appears to occupy sub- site +1, interacting with the Xyl or xylosyl residues of the enzyme-cleaved substrates. Depending on the char- acter of the substrate, this Xyl becomes the product of hydrolysis or the nonreducing end of the leaving group. A stereo view of the mode of binding of MeGlcA 2 Xyl 3 is shown in Fig. 5A. The interactions of the enzyme with MeGlcA 2 Xyl 3 and imidazole are sum- marized in Table 2. Binding energy calculations and ligand-docking studies The energy of ligand binding was estimated with lead- finder [24]. The scoring functions of leadfinder are based on a semiempirical molecular mechanical approach that explicitly accounts for various types of molecular interaction. The DG-scoring is a measure of binding energy, and the virtual screening (VS) scoring corresponds to the ligand-binding potency. The experimentally determined structure of the pro- tein–MeGlcA 2 Xyl 3 complex was used for calculating the binding energy at pH 5.5, which is the pH MeGlcA 2 Fig. 1. MeGlcA 2 Xyl 3 and imidazole in the 2F o ) F c electron density map (gray mesh), contoured at the 1.0 r level. Atoms are shown as sticks and colored as follows: C, green; O, red; N, blue. Three Xyl resi- dues with the MeGlcA moiety are bound in subsites )1, )2, and )3. A B Fig. 2. The arrangement of protein and ligand molecules in the XynA–MeGlcA 2 Xyl 3 crystal asymmetric unit. (A) A direct view of the structure and (B) a view of the structure rotated 90° around the y-axes, showing the active site of XynA. The catalytic (b ⁄ a) 8 -barrel domain is in red, the C-terminal b 9 -barrel domain is in blue, the con- necting region is in orange, the catalytic amino acids Glu165 and Glu253 are in ball-and-stick representations, and MeGlcA 2 Xyl 3 , imid- azole and three poly(ethylene glycol) molecules are in ball-and-stick representations with bonds in bold green, yellow, and turquoise, respectively. X-ray structure of xylanase A–ligand complex L ˇ . Urba ´ nikova ´ et al. 2108 FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works optimum of the enzyme [14]. Similar calculations were performed for its three analogs. For the first one, b-d- xylopyranosyl-(1 fi 4)-[4-O-methyl-a-d-xy lopyranosyl- (1 fi 2)]-b-d-xylopyranosyl-(1 fi 4)- d-xylose (MeXyl 2 Xyl 3 ), the carboxyl group of MeGlcA was replaced by hydrogen; that is, MeGlcA was converted to 4-O-methyl-d-xylose. For the second one, 4-O-methyl- a-d-glucuronosyl-(1 fi 2)-b-d-xylopyranosyl-(1 fi 4)- d-xylose (MeGlcA 2 Xyl 2 ), the nonreducing xylosyl residue of the ligand was replaced by hydrogen. The third compound examined in this regard was Xyl 3 , the core xylooligosaccharide. In addition to the above calculations, the Xyl mono- mer was docked into the hypothetical subsite +1, which is occupied by imidazole in the crystal structure. The program offered several different positions for Xyl bound in subsite +1. The position displayed in Fig. 5B corresponds to the lowest DG and VS scores, and is also optimal from the structural point of view. The Xyl O4 atom appears to be hydrogen bonded to the catalytic Glu165 and positioned in a relatively short distance (2.83 A ˚ ) from the glycosidic oxygen (O1) of the reduc- ing-end xylosyl residue bound in subsite )1 (Fig. 5B). The results of the binding energy calculations and molecular docking are summarized in Table 3. The dif- ference between the binding energies of MeGlcA 2 Xyl 3 and its virtual analog MeXyl 2 Xyl 3 indicates that the ionic interaction of the ligand carboxyl group with Arg293 corresponds to about 36% of the total binding energy of MeGlcA 2 Xyl 3 ()2.29 kcalÆmol )1 versus Table 2. Protein–MeGlcA 2 Xyl 3 and protein–imidazole interactions. The numbers in parentheses correspond to Xyl-binding subsites. Protein atom Ligand atom ⁄ group Distance (A ˚ ) Type of interaction MeGlcA 2 Xyl 3 Arg293 NH1 MeGlcA COOH group 2.86 Salt bridge Arg293 NE MeGlcA COOH group 2.93 Salt bridge Tyr295 OH MeGlcA O6 2.65 Hydrogen bond Ser258 OG MeGlcA O3 3.38 Hydrogen bond Tyr255 OH MeGlcA O2 2.69 Hydrogen bond Trp289 NE1 MeGlcA O5 3.36 Hydrogen bond Tyr295 OH MeGlcA O5 3.14 Hydrogen bond Trp113 NE1 Xyl (–1) O3 2.86 Hydrogen bond Glu253 OE2 Xyl (–1) O2 2.76 Hydrogen bond Asn164 ND2 Xyl (–1) O2 2.95 Hydrogen bond Glu165 OE1 Xyl (–1) O1 2.60 Hydrogen bond Glu165 OE2 Xyl (–1) O1 2.64 Hydrogen bond Trp289 aromatic ring Xyl (–1) 3.86–6.05 Stacking Tyr172 aromatic ring Xyl (–2) 4.07–4.59 Stacking Trp55 aromatic ring Xyl (–3) 3.56–4.78 Stacking Imidazole Glu165 OE1 Imidazole N1 2.70 Hydrogen bond Glu165 OE2 Imidazole N1 3.47 Hydrogen bond Trp168 aromatic ring Imidazole ring 3.7–4.7 Stacking Tyr232 aromatic ring Imidazole ring 3.5–4.6 Stacking A B Fig. 3. Details of the interactions of XynA with MeGlcA 2 Xyl 3 and imidazole. (A) Stick representation of MeGlcA 2 Xyl 3 and imidaz- ole (atoms: green, C; red, O; blue, N). Amino acids involved into substrate binding are in ball-and-stick representations (atoms: gray, C; red, O; blue, N). Hydrogen bonds are marked by dashed lines. (B) The van der Waals surface representation of the enzyme, showing the active site cleft filled by MeGlcA 2 Xyl 3 and imidazole. L ˇ . Urba ´ nikova ´ et al. X-ray structure of xylanase A–ligand complex FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works 2109 )6.22 kcalÆmol )1 ). The sum of both ionic and nonionic enzyme–MeGlcA interactions corresponds to about 55% of the total binding energy ()3.47 kcalÆmol )1 ver- sus )6.22 kcalÆmol )1 ). The binding energy of the non- reducing xylosyl residue in subsite )3 is only 9% of the total DG ()0.55 kcalÆmol )1 versus )6.22 kcalÆ- mol )1 ). The calculated binding energies for the ligand, its two virtual analogs and Xyl 3 (Table 3) correspond to specific enzyme activities on different substrates (see below). Specific activity on aldouronic acids and linear xylooligosaccharides Two aldotetraouronic acids differing in the presence of the nonreducing xylopyranosyl residue filling subsite )3 were available: 4-O-methyl-a-d-glucuronosyl-(1 fi 2)-b- d-xylopyranosyl-(1 fi 4)-b-d-xylopyranosyl-(1 fi 4)-d- xylose (MeGlcA 3 Xyl 3 ), the shortest acidic oligosac charide liberated from glucuronoxylan by endoxylanas- es of GH10 [4], and aldopentaouronic acid, b-d-xylo pyranosyl-(1 fi 4)-[4-O-methyl- a-d-glucuronosyl-(1 fi 2)]- b-d-xylopyranosyl-(1 fi 4)-b-d-xylopyranosyl-(1 fi 4)- d-xylose (MeGlcA 3 Xyl 4 ), the shortest acidic oligosaccha ride liberated from glucuronoxylan by endoxylanases of GH family 11 [4] (Fig. S1). XynA hydrolyzed the ald- opentaouronic acid more efficiently. Specific activities at 4mm substrate were 42 mmolÆmin )1 Æmg )1 for the pent- amer and 13 mmolÆmin )1 Æmg )1 for the tetramer. These data suggest that subsite )3 also contributes to sub- strate binding. In view of the recent information that a Bacillus GH30 xylanase shows activity on linear b-1,4-xylooligosaccharides [15], we also examined the rate of hydrolysis of xylotetraose and xylopentaose. AB C D Fig. 4. Detailed view of the interaction of the enzyme with individual carbohydrate residues of MeGlcA 2 Xyl 3 derived from the enzyme–ligand complex with imidazole bound in aglycone subsite +1. Amino acids and ligands are in ball-and-stick representations, with sticks colored gold and green, respectively. The atoms are colored as follows: red, O; blue, N; gold and green, C. Hydrogen bonds are marked by dashed lines. Stacking interactions are also highlighted as dashed lines connecting the centers of interacting groups marked by asterisks. The distances are in A ˚ . (A) Stacking interactions of Tyr172 and Trp55 with xylosyl residues in subsites )2 and )3. (B) Hydrogen bonds between the enzyme and xylosyl residue in subsite )1. The stacking interaction with Trp289 is also indicated. (C) Coordination of the MeGlcA residue of the ligand with Tyr255, Ser258, Trp289, Arg293, and Tyr295. There is no stacking interaction of MeGlcA with the sandwich of Trp289 ⁄ Tyr290. (D) Stacking interactions of imidazole in subsite +1 with Trp168 and Tyr232, and its hydrogen bond with the catalytic Glu165. The six-membered aromatic ring of Trp168 and Leu204 might be involved in binding of Xyl in subsite +2. X-ray structure of xylanase A–ligand complex L ˇ . Urba ´ nikova ´ et al. 2110 FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works At 4 mm, both oligomers served as enzyme substrates, but with specific activities three orders of magnitude lower than those on aldouronic acids. These observa- tions point again to a crucial role for the MeGlcA carboxylate in enzyme substrate recognition and the role of MeGlcA as an essential specificity determinant. We should mention that xylopentaose was hydrolyzed about three times faster than xylotetraose, which is also in accord with the results of the docking experiments and calculated binding energies (Table 3). Discussion The xylanase investigated in this work is one of the appendage-dependent endoxylanases, which are of bacterial origin and can be found in the GH30 A B Fig. 5. Stereoview of the interactions of XynA with MeGlcA 2 Xyl 3 and imidazole (IMD). (A) Enzyme–MeGlcA 2 Xyl 3 interactions (for clarity, Ser258, forming a hydrogen bond to MeGlcA, is not shown). Ligands and amino acids involved in ligand binding are in ball-and-stick represen- tations (atoms: black, C; red, O; blue, N) with sticks colored green and light gray, respectively. Hydrogen bonds and ionic interactions are marked by dashed lines. Glu253 is marked by an asterisk. (B) Interactions of the enzyme with Xyl docked at subsite +1. For comparison, Xyl (derived from the MeGlcA 2 Xyl 3 structure) in subsite )1 and imidazole are also shown. The length of the hydrogen bonds is indicated in A ˚ . Table 3. Summary of molecular modeling experiments and binding energy calculation. Ligand Calculation based on Binding energy, DG (kcalÆmol )1 ) VS score Difference in binding energies, DG 1 ) DG 2 Ligands kcalÆmol )1 Functional group of ligand MeGlcA 2 Xyl 3 Crystal structure )6.22 )10.10 – – – MeXyl 2 Xyl 3 a Crystal structure )3.93 )8.15 MeGlcA 2 Xyl 3 – MeXyl 2 Xyl 3 )2.29 COOH group MeGlcA 2 Xyl 2 b Crystal structure )5.67 )9.18 MeGlcA 2 Xyl 3 – MeGlcA 2 Xyl 2 )0.55 Xyl at subsite )3 Xyl 3 Crystal structure )2.75 )6.03 MeGlcA 2 Xyl 3 – Xyl 3 )3.65 MeGlcA Xyl 1 at subsite +1 Docking )3.51 )5.06 – – – a COOH group of MeGlcA was replaced by hydrogen. b Nonreducing Xyl was replaced by hydrogen. L ˇ . Urba ´ nikova ´ et al. X-ray structure of xylanase A–ligand complex FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works 2111 (formerly GH5) family [1,8,13–15]. These enzymes are specialized for depolymerization of xylans that contain GlcA or MeGlcA side substituents. They exhibit a unique mode of action. The cleavage of the glucuron- oxylan main chain takes place exclusively at the second glycosidic linkage from the branch towards the reduc- ing end of the polysaccharide chain. In other words, the cleavage occurs during the formation of the pro- ductive enzyme–substrate complex, in which the substi- tuted xylopyranosyl residue is bound in the hypothetical subsite )2. In this way, the MeGlcA or GlcA residues determine the site of substrate cleavage, and the content of these uronic acids determines the xylan chain cleavage frequency. In this work, we con- firm the hydrolysis of linear xylooligosaccharides by a GH30 xylanase [15]; however, the rate of their hydro- lysis with XynA was negligible in comparison with the rate of hydrolysis of aldouronic acids. After the 3D structure of the enzyme became known [16] and the mode of GH30 xylanase action had been elucidated [13,14], a question emerged con- cerning the basis for the recognition of the MeGlcA and GlcA residues by the enzyme. We have postu- lated an ionic interaction between the uronic acid car- boxylate and the positively charged Arg293 occurring in the vicinity of a sandwich of two aromatic amino acids, Tyr290 ⁄ Trp289, that could interact with the uronic acid [14]. However, because the space between Tyr290 and Trp289 in the published crystal structure was too narrow to accommodate the uronic acid, it became clear that the enzyme should be crystallized in a complex with a suitable ligand and that the structure of the complex could provide the required information. We have succeeded obtaining crystals of XynA with the aldotetraouronic acid MeGlcA 2 Xyl 3 , which is a product of the cleavage of MeGlcA 3 Xyl 4 by the same enzyme. In the crystal structure, the ligand was found to be bound in a manner similar to the one that we have predicted [14]. The xylopyranosyl residue substi- tuted by MeGlcA was bound in subsite )2, and MeG- lcA was in a position that clearly indicates an ionic interaction between its carboxyl group and the posi- tively charged Arg293. However, MeGlcA was not sandwiched between Tyr290 and Trp289, as proposed earlier [14]. Instead, in addition to the ionic interaction with Arg293, it interacts with the side chains of the aromatic amino acids Tyr255, Trp289, and Tyr295, and with Ser258, through several hydrogen bonds, which are listed in Table 2 and shown in Figs 4C and 5A. It is interesting that the ligand occurs in the complex with XynA in the form of its a-anomer. Such a config- uration corresponds to the enzyme a-glycosyl ester intermediate with the catalytic glutamate Glu165. This is interesting in light of the fact that the enzyme is a retaining GH [1]. At this stage of our work, we do not have any explanation for this observation. An important question to be answered in connec- tion with the mode of action of GH30 xylanases is why the enzymes do not efficiently attack linear b- 1,4-linked xylooligosaccharides. The MeGlcA carbox- ylate is involved in binding by Arg293. According to the calculations of the binding energies of the ligand and its virtual analogs (Table 3), the interaction of the enzyme with MeGlcA is stronger than with the xylopyranosyl residues in the negatively numbered subsites. The ionic interaction could also be impor- tant for the first contact of the enzyme with sub- strates, and also indispensable for creating a stable enzyme–substrate complex. In the next steps, the enzyme–substrate complex formation could be based on stacking interactions between aromatic amino acids covering the enzyme binding site and Xyl resi- dues of the xylan main chain. The final step could be the locking of the substrate, namely MeGlcA and a xylosyl or xylobiosyl moiety, at subsites +1 and +2, in a proper position for cleavage. The importance of Xyl binding at subsite +1 is supported by calcula- tions of the binding energy of free Xyl in subsite +1 (Table 3; Fig. 5B). One can envisage strong bending of the xylan chain as a consequence of both ionic and stacking interactions. This apparently cannot occur with linear oligosaccharides or a xylan main chain that is either unsubstituted or carries uncharged side substituents such as l-arabinose. The strong bending could be the reason why the enzyme hardly recognizes linear xylooligosaccharides as substrates and does not attack arabinoxylan [14]. To learn more about the enzyme–substrate interactions, complexes of the enzyme with larger, nonhydrolyzable ligands should be crystallized and their structure elucidated. An alternative approach could include the preparation of inactive enzyme mutants and crystallization of these mutants with natural substrates. Conclusions The crystal structure of XynA with MeGlcA 2 Xyl 3 shows that the unique substrate specificity and mode of action of bacterial GH30 xylanases on xylans with MeGlcA and GlcA side substituents is achieved mainly by recognition of the uronic acid side residue. A crucial role in this recognition is ascribed to ionic interaction of the enzyme with the uronic acid carboxylate. Lack of the uronic acid renders the X-ray structure of xylanase A–ligand complex L ˇ . Urba ´ nikova ´ et al. 2112 FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works xylan main chain virtually resistant to the enzyme’s action. The specific activities on unsubstituted b-1,4- xylooligosaccharides are three orders of magnitude lower than those on similar substrates containing MeGlcA. Experimental procedures Cloning and expression of XynA Recombinant XynA was obtained by expressing its synthetic gene in B. subtilis A164delta5 [25]. The synthetic gene, based on the published gene sequence (Swiss-Prot: Q46961), was generated by the company DNA2.0 (Menlo Park, CA, USA) and delivered as a cloned fragment in their standard cloning vector (kanamycin-resistant). The synthetic gene sequence (Fig. S3A) was codon-opti- mized for expression in B. subtilis following the recommen- dations of Gustafsson et al. [26]. The expressed DNA sequence can be found in Fig. S3B. The xylanase gene was cloned with the signal peptide from Savinase [26] (included in the vector), replacing the native secretion signal. The coding region without the native signal was amplified by PCR from the plasmid containing the synthetic gene, and cloned into the expression vector pDG268neo [25]). The PCR primers contained an N-terminal ClaI site and a C-terminal Mlu I site. The PCR fragment and vector were digested with ClaI and MluI. The vector and fragment were ligated and transformed into Escherichia coli. Several rec- ombinants were obtained. A plasmid containing the correct gene sequence was transformed into B. subtilis, following the methods in Widner et al. [27]. A recombinant B. subtilis clone containing the integrated expression construct was grown in PS-1 liquid culture medium [27]. The enzyme was purified from the culture supernatant. Purification of recombinant XynA The culture supernatant, collected by centrifugation (17 700 g for 30 min), was filtered (0.22 lm), and the filtrate was adjusted to pH 8.5 and subsequently loaded onto an MEP HyperCel (Pall, East Hills, NY, USA) XK 26 ⁄ 20 col- umn (GE Healthcare Bio-Sciences, Piscataway, NJ, USA). The column (60 mL) was equilibrated in 50 mm Tris ⁄ HCl buffer (pH 8.5) (buffer A). Unbound protein was washed off with 300 mL of buffer A. The proteins were eluted with 50 mm sodium acetate buffer (pH 4.5) (buffer B). Fractions were analyzed by SDS ⁄ PAGE, and fractions containing the enzyme were combined and their pH was adjusted to pH 6.0. The combined fractions were diluted five times in 25 mm Mes buffer (pH 6.0) (buffer C) and applied to a cation exchange SP Sepharose Fast Flow (GE Healthcare Biosciences, Uppsala, Sweden) XK 26 ⁄ 20 column (GE Healthcare Bio- Sciences, Piscataway, NJ). The cation exchanger (20 mL) was equilibrated in buffer C. Unbound protein was washed off with 100 mL of buffer C. The XynA was eluted with a linear gradient of NaCl (0–0.5 m) in buf- fer C, using five column volumes. Fractions were analyzed by SDS ⁄ PAGE, and those containing XynA were combined. Other enzymes GH3 b-xylosidase was a product of a recombinant Saccharomyces cerevisiae strain expressing a plasmid-borne Aspergillus niger XlnD gene [28], GH67 a-glucuronidase was obtained from R. P. deVries and J. Visser (Agricultural University of Wageningen, The Netherlands), and GH115 a-glucuronidase was a product of Pichia stipitis [29]. Substrates and oligosaccharide ligand The ligand used for cocrystallization with XynA was MeGlcA 2 Xyl 3 . This aldotetraouronic acid was prepared from MeGlcA 2 Xyl 4 , the shortest acidic product generated from hardwood glucuronoxylan by a family 11 endo-b- 1,4-xylanase [27] by the action of recombinant XynA. The enzyme catalyzed the reaction MeGlcA 3 Xyl 4 fi MeGlcA 2 Xyl 3 + Xyl [14]. MeGlcA 3 Xyl 4 (20 mg), isolated from glucuronoxylan-spent medium of Thermomyces la- nuginosus [30], was incubated in 2 mL of water with 0.3 mg of purified recombinant XynA at 30 °C. After the hydrolysis was completed (examined by TLC), the prod- uct was isolated from the reaction mixture by preparative paper chromatography on Whatman No. 3 (prewashed with deionized water) in the solvent system ethyl ace- tate ⁄ acetic acid ⁄ water (18 : 7 : 8, v ⁄ v ⁄ v) for 17 h. The sugars on guide strips were localized with the silver nitrate reagent. The water eluate of the desired product was filtered and freeze-dried. The structure of the product as MeGlcA 2 Xyl 3 was confirmed enzymatically (Fig. S1). The compound was resistant to GH67 a-glucuronidase but served as a substrate for GH115 a-glucuronidase to yield MeGlcA and xylotriose. It was hydrolyzed by the GH3 b-xylosidase [28] to Xyl and MeGlcA 2 Xyl 2 , giving MeGlcA and xylobiose with both types of a-glucuroni- dase. MeGlcA 3 Xyl 3 was isolated from glucuronoxylan hydrolysate by endoxylanase of GH10 as the shortest acidic oligosaccharide [4]. Xylotetraose and xylopentaose were from Megazyme (Ireland). Crystallization An enzyme solution was prepared by concentrating the pro- tein in 25 mm MES buffer (pH 6.0), containing150 mm NaCl, to a concentration of 20 mgÆmL )1 , using an Amicon stirred cell and a Biomax membrane with cutoff 5 kDa. Fifty-microliter aliquots of the concentrated solution were L ˇ . Urba ´ nikova ´ et al. X-ray structure of xylanase A–ligand complex FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works 2113 stored at )20 °C until use. Crystals were prepared by the vapor diffusion method in a hanging drop, with XRL plates and plastic coverslips (Molecular Dimensions, Suffolk, UK). The drops were composed of the protein stock solution and precipitant solution at a 1 : 1 ratio in a final volume of 2 lL, and equilibrated against 500 lLof precipitant solution. In the case of the cocrystallization, the 3-lL drops were prepared by mixing the protein, ligand and precipitant solutions at a 1 : 1 : 1 ratio. An aqueous solution of MeGlcA 2 Xyl 3 (20 mm) was used as the ligand solution. Pact Premier I and II and Crystal Clear I crystalli- zation kits (Molecular Dimensions) were used for prelimin- ary crystallization screening. Clusters of thin and fragile needle crystals were obtained under 19 conditions, which were further optimized. Diffraction-quality crystals were prepared by crystal seeding. Data were collected from the crystal of the XynA–MeGlcA 2 Xyl 3 complex obtained by cocrystallization with 0.1 m imidazole ⁄ d,l-malic acid buffer (pH 7.5) and 20% (w ⁄ v) poly(ethylene glycol) 1500 as a precipitant solution. Data collection and structure determination The crystals were tested and data were collected at the X13 beamline at EMBL c ⁄ o DESY, Hamburg, Germany. The crystals were mounted on the loops, soaked in a cryopro- tectant solution, and flash cooled in a stream of cold nitro- gen gas (100 K) directly at the goniometer head. As cryoprotectants, Paratone-N, perfluoropolyether, paraf- fin oil and precipitant solution enriched with glycerol to a final concentration of 20% were tested. The best results were obtained with paraffin oil. Data were collected at 100 K, according to the strategy proposed by best [31], and processed by xds [32], and scala [33], reindex and com- bat from ccp4 suite 6.1.3 (Collaborative Computational Project Number 4, 1994 [23]), using ccp4i Interface 2.0.6 [34] running under Windows. The structure was solved by the molecular replacement method with molrep [35] and xylanase A (Protein Data Bank code 1NOF [16]) as a model structure. The structure was refined with ref- mac 5.5.01 [36] in combination with coot-findwaters, and the model was visualized and rebuilt with coot [37]. All electron density maps were calculated by FFT [38]. For structure validation, procheck was used [39,40], and for structure analyses, areaimol, contact and other programs of the ccp4 suite were used with the default parameters. Figures were prepared with molscript [41] and pymol [42]. Molecular modeling Docking experiments and binding energy calculations were performed with leadfinder [23]. This program was also used for the preparation of the protein structure for dock- ing by addition of hydrogen atoms according to optimal ionization states of protein residues at a given pH. The ligand structures were prepared for molecular modeling in their optimal protonation state with ChemAxon marvin suite [43]. Specific activity on aldouronic acids and linear xylooligosaccharides Four-millimolar solutions of the compounds in 50 mm sodium acetate buffer (pH 5.5) were incubated at 40 °C with XynA at various dilutions. At time intervals, aliquots were taken to determine the reducing sugars by the Somo- gyi–Nelson procedure [44]. The high background of the substrates reduced the accuracy of the measurements, particularly at early stages of hydrolysis. Acknowledgements The authors are grateful to M. Czisza ´ rova ´ for excellent technical assistance. This work was supported by VEGA grants 2 ⁄ 0001 ⁄ 10 and 2 ⁄ 0165 ⁄ 08 from the Slo- vak Academy of Sciences. We acknowledge the EMBL X13 beamline at the DORIS storage ring, DESY, Hamburg for providing us with synchrotron source facilities. We thank M. Groves (EMBL Hamburg) for his help with data processing, and O. Stroganov (BioMolTech) for technical help with leadfinder. Note added in proof During processing of this article for publication we have learned about the appearence of the paper describing similar substrate recognition mechanism by a GH30 xylanase from Bacillus subtilis using a crystal structure of the complex of the enzyme with different ligand (St John FJ, Hurlbert JC, Rice JD, Preston JF & Pozharski E (2011) Ligand bound structures of a glycosyl hydrolase family 30 glucuronoxylan xylanohy- drolase. J Mol Biol 407, 92–109). References 1 Henrissat B & Davies GJ (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7, 637–644. 2 Biely P (2003) Xylanolytic enzymes. In Handbook of Food Enzymology (Whitaker JR, Voragen AGJ & Wond DWS eds), pp. 879–915. Marcel Dekker, New York. 3 Collins T, Gerday C & Feller G (2005) Xylanases, xylanase families and extremophilic xylanases. FEMS Microbiol Rev 29, 3–23. 4 Biely P, Vrs ˇ anska ´ M, Tenkanen M & Kluepfel D (1997) Endo-b-1,4-xylanase families: differences in catalytic properties. J Biotechnol 57, 151–166. X-ray structure of xylanase A–ligand complex L ˇ . Urba ´ nikova ´ et al. 2114 FEBS Journal 278 (2011) 2105–2116 Journal compilation ª 2011 FEBS. No claim to original Slovakian government works [...]... information The following supplementary material is available: Fig S1 Conversion of aldopentaouronic acid generated from glucuronoxylan by GH11 endoxylanases to aldotetraouronic acid used as the ligand (framed structure) for cocrystallization of E chrysanthemi GH30 xylanase Fig S2 Dynamic light scattering profile of E chrysanthemi GH30 xylanase Fig S3 Examples of distinct habits of E chrysanthemi GH30. .. protein sequences of E chrysanthemi GH30 xylanase Table S1 Characterization of the binding site surface and the list of the interactions between E chrysanthemi GH30 xylanase and MeGlcA2Xyl3 ligand This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such... 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Rosa AP, Day J, Larson SB, Keen NT & McPherson A (1997) Crystallization of xylanase from Erwinia chrysanthemi: influence of heat and polymeric substrate Acta Crystallogr D53, 256–261 23 Collaborative Computational Project Number 4 (1994) The CCP4 suite: programs for protein crystallography Acta Crystallogr D50, 760–763 24 Stroganov VO, Novikov FN, Stroylov VS, Kulkov V & Chilov GG (2008) LeadFinder: an . Structural basis for substrate recognition by Erwinia chrysanthemi GH30 glucuronoxylanase L ˇ ubica Urba ´ nikova ´ 1 ,Ma ´ ria. main chain by GH10 xylanases Keywords crystal structure with ligand; Erwinia chrysanthemi; GH30; glucuronoxylan-specific xylanase; substrate recognition Correspondence P.