Crystal Structure of Bacillus sp. GL1 Xanthan Lyase, Which Acts on the Side Chains of Xanthan*

Xanthan lyase, a member of polysaccharide lyase family 8, is a key enzyme for complete depolymerization of a bacterial heteropolysaccharide, xanthan, in Bacillus sp. GL1. The enzyme acts exolytically on the side chains of the polysaccharide. The x-ray crystallographic structure of xanthan lyase was determined by the multiple isomorphous replacement method. The crystal structures of xanthan lyase and its complex with the product (pyruvylated mannose) were refined at 2.3 and 2.4 Å resolution with finalR-factors of 17.5 and 16.9%, respectively. The refined structure of the product-free enzyme comprises 752 amino acid residues, 248 water molecules, and one calcium ion. The enzyme consists of N-terminal α-helical and C-terminal β-sheet domains, which constitute incomplete α5/α5-barrel and anti-parallel β-sheet structures, respectively. A deep cleft is located in the N-terminal α-helical domain facing the interface between the two domains. Although the overall structure of the enzyme is basically the same as that of the family 8 lyases for hyaluronate and chondroitin AC, significant differences were observed in the loop structure over the cleft. The crystal structure of the xanthan lyase complexed with pyruvylated mannose indicates that the sugar-binding site is located in the deep cleft, where aromatic and positively charged amino acid residues are involved in the binding. The Arg313 and Tyr315 residues in the loop from the N-terminal domain and the Arg612 residue in the loop from the C-terminal domain directly bind to the pyruvate moiety of the product through the formation of hydrogen bonds, thus determining the substrate specificity of the enzyme.

There is a large number of polysaccharide-degrading enzymes. 1 Generally, they can be classified into two groups, hydrolases and lyases. The former catalyze the hydrolysis reaction responsible for breaking glycosidic bonds in polysaccha-rides. The properties of glycosyl hydrolases that act on polyand oligosaccharides have been well documented, and the three-dimensional structures of many polysaccharide hydrolases, such as amylases, chitinases, and cellulases, have already been reviewed (1,2).
To determine the structural and functional relationships exhibited by polysaccharide lyases, we have recently been focusing on bacterial heteropolysaccharide lyases (lyases for alginate (14), gellan (15), and xanthan (16)) with either an endotypic or exotypic reaction mode and with either a backbone or side chain type of cleavage site. We have already determined the crystal structure of the endotype alginate lyase from Sphingomonas sp. A1 (9).
Xanthan is an exopolysaccharide produced by the plant pathogenic bacterium Xanthomonas campestris (17). This exopolysaccharide consists of a main cellulosic chain with trisaccharide side chains composed of one glucuronyl and two mannosyl residues attached at the C-3 position of alternate glucosyl residues (18) (Fig. 1A). The internal and terminal mannosyl residues of the side chains have an O-acetyl group at the C-6 position and a pyruvate ketal at the C-4 and C-6 positions, respectively, although the extents of acetylation and pyruvation vary with the growth conditions and bacterial strain (19). Because the polymer has the peculiar rheological properties of pseudoplasticity (reversible decrease in viscosity with increase in shear rate), high viscosity at low concentrations, and tolerance to a wide range of pH and temperatures, it is widely utilized as a gelling and stabilizing agent in the food, pharmaceutical, and oil industries (20).
Xanthan lyase produced by Bacillus sp. GL1 acts exolytically on the side chains of xanthan and liberates pyruvylated mannose (PyrMan) 2 through the ␤-elimination reaction (Fig. 1A) (16). The enzyme is synthesized as a precursor form (99 kDa) and is then converted into the mature form (ϳ75 kDa) through posttranslational excision of the signal peptide (2 kDa) and C-terminal polypeptide (ϳ22 kDa) (21). On the basis of amino acid (aa) sequence similarity, the enzyme is classified into polysaccharide lyase family 8, 1 which contains lyases for hyaluronate and chondroitin AC in addition to xanthan lyase, although xanthan lyase does not act on hyaluronate and chondroitin (21).
Moreover, xanthan lyase is peculiar in that it acts on the side chains of a polysaccharide and releases the nonreducing terminal saccharides of the side chains, because almost all polysaccharide lyases (including those for pectate, alginate, hyaluronate, chondroitin, and heparin) endolytically cleave the glycosidic bonds in the main chains of polysaccharides. Therefore, it is thought that the structural analysis of xanthan lyase will contribute to clarification of the structural features that determine the uronate recognition site, the ␤-elimination reaction, the reaction mode (endo/exo type), and the cleavage site (main/side chain type).
In this study, the three-dimensional structures of xanthan lyase and its complex with the product were determined by x-ray crystallography at 2.3 and 2.4 Å resolution, respectively. We also identified the active cleft of the enzyme and aa residues responsible for both the recognition of the substrate and the catalytic reaction.
Assays for Enzyme and Protein-Xanthan lyase was assayed as described previously (16). Briefly, the enzyme was incubated in 1 ml of a reaction mixture containing 0.05% xanthan and 50 mM sodium acetate buffer, pH 5.5, and then the activity was determined by monitoring the increase in absorbance at 235 nm. One unit of the enzyme activity was defined as the amount of enzyme required to produce an increase of 1.0 in absorbance at 235 nm/min. Protein was determined by the method of Lowry et al. (22) with bovine serum albumin as a standard or by measuring the absorbance at 280 nm, assuming that E280 ϭ 2.06 corresponded to 1 mg/ml, as calculated from the aa sequence using ProtParam (www.expasy.org/tools/protparam.html).
Purification of Xanthan Lyase-Unless otherwise specified, all operations were carried out at 0 -4°C. Cells of Escherichia coli strain BL21(DE3)pLysS harboring a plasmid (pET17b-XL4) (21) were grown in 6 liters of LB medium (1.5 liters/flask), collected by centrifugation at 6000 ϫ g and 4°C for 5 min, washed with 20 mM potassium phosphate buffer (KPB), pH 7.0, and then resuspended in the same buffer. The cells were disrupted ultrasonically (Insonator model 201M, Kubota, Tokyo, Japan) at 0°C and 9 kHz for 20 min, and the clear solution obtained upon centrifugation at 15,000 ϫ g and 4°C for 20 min was used as the cell extract containing the precursor form (97 kDa) of the enzyme. The cell extract, after supplementation with 1 mM phenylmethylsulfonyl fluoride and 0.1 M pepstatin A, was fractionated with ammonium sulfate. The precipitate (0 -30% saturation) was collected by centrifugation at 15,000 ϫ g and 4°C for 20 min, dissolved in 20 mM KPB, pH 7.0, and then applied to a DEAE-Toyopearl 650 M column (2.6 ϫ 15 cm) equilibrated with 20 mM KPB, pH 7.0. The enzyme was eluted with a linear gradient of NaCl (0 -0.7 M) in 20 mM KPB, pH 7.0 (200 ml), with 2 ml fractions collected every 2 min. The active fractions, which were eluted with 0.4 M NaCl, were combined and dialyzed against 20 mM KPB, pH 7.0. The dialysate was used as the purified precursor form (97 kDa) of the enzyme. To convert the precursor (97 kDa) autocatalytically to the mature form (ϳ75 kDa), the purified precursor was kept at 4°C for 1 week. After confirmation of the conversion by SDS-PAGE (23), the enzyme solution was applied to a Super Q-Toyopearl 650C column (1.7 ϫ 5 cm) equilibrated with 20 mM KPB, pH 7.0, and eluted with a linear gradient of NaCl (0 to 0.5 M) in 20 mM KPB, pH 7.0 (50 ml). 1-ml fractions were collected every minute. The active fractions, which were eluted with about 0.2 M NaCl, were combined and dialyzed against 20 mM Tris-HCl, pH 7.5, and the dialysate was used as the purified mature form (ϳ75 kDa) of the enzyme.
Preparation of PyrMan-Xanthan (0.5%) dissolved in 50 mM sodium acetate (pH 5.5) (200 ml) was treated with the purified xanthan lyase (10 mg). The solution was mixed with ethanol (400 ml) and then centrifuged at 15,000 ϫ g and 4°C for 20 min. The supernatant was concentrated to 1.5 ml through evaporation and then applied to a Bio-Gel P2 column (0.9 by 122 cm) equilibrated with distilled water. The sugar eluted was determined to be PyrMan by confirming the release of mannose and pyruvate on hydrolysis with trifluoroacetic acid, as described previously (16). The fractions containing PyrMan were collected, freeze-dried, and dissolved in distilled water. The purity and content of PyrMan were determined by TLC analysis as described previously (16).
Crystallization and X-ray Diffraction-The mature form (ϳ75 kDa) of xanthan lyase was crystallized by the hanging-drop vapor diffusion method. The solution for a crystallization drop was prepared on a siliconized coverslip by mixing 3 l of protein solution (7.18 mg of protein/ml) with 3 l of mother liquor comprising 23% polyethylene glycol 4000, 0.2 M ammonium formate, and 0.1 M sodium Bicine buffer, pH 9.0. The crystals were soaked in several heavy atom derivative solutions comprising 2 mM NaAuCl 4 , 0.2 mM AgNO 3 , 1 mM Ac 2 UO 2 , 2 mM SmCl 3 , 1 mM GdCl 3 , 1 mM HoCl 3 , and 1 mM CdCl 2 for 1 or 2 h at 20°C. Crystals were also soaked in a sugar solution containing 75 mM PyrMan. All heavy atom and sugar solutions were prepared with a modified mother liquor consisting of 23% polyethylene glycol 4000, 0.2 M ammonium formate, and 0.1 M Tris-HCl buffer, pH 7.4. Diffraction data for the native and derivative crystals were collected with a Bruker Hi-Star multiwire area detector at 20°C, using CuK␣ radiation generated by a MAC Science M18XHF rotating anode generator, and were processed with the SADIE and SAINT software packages (Bruker, Karlsruhe, Germany) ( Table I).
Structure Determination and Refinement-The crystal structure of xanthan lyase was solved by the multiple isomorphous replacement (m.i.r.) method. The major sites of heavy atoms were determined by interpretation of the peaks in difference Patterson maps obtained at 3.0 Å resolution. Additional heavy atom sites were determined from the peaks in difference Fourier maps. Phase refinement was performed with the program package PHASES (24). The results of heavy atom refinement and phasing by m.i.r. at 3.0 Å resolution are presented in Table II. The phase was improved greatly and the figure-of-merit increased to 0.854 after solvent flattening with PHASES (25). The model was built using the program TURBO-FRODO (AFMB-CNRS, Marseille, France) on a Silicon Graphics Octane computer. Simulated annealing refinement was carried out with this model using 50 -2.3 Å resolution data obtained with CNS (26). The model was heated to 2500 K and then slowly cooled to 300 K (time-step, 0.5 fs; decrease in temperature, 25 K; number of steps at each temperature, 50), and then 150 cycles of Powell minimization were carried out. F o Ϫ F c and 2 F o Ϫ F c maps were used to locate the correct model. Several rounds of positional and B-factor refinement followed by manual model building were carried out to improve the model by increasing the data to 2.3 Å resolution. Water molecules were incorporated where the difference in density was more than 3.0 above the mean and the 2 F o Ϫ F c map showed a density of more than 1.0 . The final R-factor was 17.5% for 30,582 data points in the 50.0 -2.3 Å resolution range (83.9% completeness). The R-free value calculated for the randomly separated 10% data was 24.0%.
A crystal soaked with PyrMan was isomorphous with the crystal used for the native set. A F o Ϫ F c map (contoured at 3.0 ) at 2.4 Å resolution was obtained using the reflection data for the sugar-soaked crystal, and the phase was calculated from the final model of xanthan lyase.
The stereo quality of the model was assessed using the programs PROCHECK (27) and WHAT-CHECK (28). Ribbon plots were prepared using the programs MOLSCRIPT (29), BOBSCRIPT (30), RASTER3D (31), and GRASP (32). The coordinates of lyases for hyaluronate and chondroitin AC were taken from the RCSB Protein Data Bank (33). These molecular models were superimposed by means of fitting the programs RIGID and TOP included in TURBO-FRODO and CCP4, respectively.

RESULTS AND DISCUSSION
Crystallization and Structure Determination-The mature form (about 75 kDa) of xanthan lyase of Bacillus sp. GL1 was purified from recombinant E. coli cells harboring plasmid pET17b-XL4 (21). A crystal of xanthan lyase (0.3 ϫ 0.2 ϫ 0.05 mm) was obtained by the hanging-drop vapor diffusion method. The space group was determined to be P2 1 2 1 2 1 (orthorhombic) with unit cell dimensions of a ϭ 54.3 Å, b ϭ 91.4 Å, and c ϭ 160.7 Å; the solvent content was 50.2% assuming one molecule/ asymmetric unit. The results of the x-ray data collection are summarized in Table I. The structure of the enzyme was determined by the m.i.r. method. Table II shows the refinement statistics for the heavy atoms at 3.0 Å resolution. The protein model was built after solvent flattening of the m.i.r. phase with the PHASES program (24), and the model was refined by means of simulated annealing and the restrained least-squares method using CNS (26), as shown in Table I.
Quality of the Refined Model-The refined model of xanthan lyase comprises 752 aa residues, 248 water molecules, and one calcium ion. The N-and C-terminal aa residues of the mature form produced from the preproform through posttranslational processing were confirmed to be Ser 26 and Gly 777 , respectively, by electron density mapping. All of the polypeptide chain sequences could be well traced, and the electron density of the main and side chains was generally very well defined in the 2 F o Ϫ F c map. The final overall R-factor for the refined model was 0.175, with 30,582 unique reflections within the 50.0 -2.3 Å resolution range. The final free R-factor calculated with the randomly selected 10% data was 0.240. The final root-meansquare (r.m.s.) deviations from the standard geometry were 0.0060 Å for bond lengths and 1.29 o for bond angles. Based on the theoretical curves in the plot calculated according to Luz- zati (34), the absolute positional error was estimated to be close to 0.25 Å of 5.0 -2.3 Å resolution. Judging from the results of Ramachandran plot analysis, in which the stereochemical correctness of the backbone structure is indicated by the (, ) torsion angles (35), most of the non-glycine residues (86.1%) lay within the most favored regions, and the other residues (13.6%) fell in the additional and generously allowed regions, except for the Thr 247 and Asp 695 residues. The Thr 247 and Asp 695 residues are in ␤-turns. In particular, the latter turn, containing the Asp 695 residue, is similar to a ␤-hairpin consisting of four amino acid residues with the conformation of ␤-⑀-␥-␤ in a Ramachandran plot (36), because the Ala 694 , Asp 695 , Leu 696 , and Ile 697 residues fell in or near the ␤, ⑀, ␥, and ␤ regions of the plot, respectively. Furthermore, there is one cis-peptide between the Ala 753 and Pro 754 residues.
Overall Structure of Xanthan Lyase-Figs. 2 and 3 depict a ribbon model of the overall structure and topology of the secondary structure elements of xanthan lyase, respectively. The enzyme has approximate dimensions of 100 ϫ 70 ϫ 50 Å and is composed of two globular domains (N-and C-terminal domains) that form ␣and ␤-structures, respectively. The Nterminal domain comprises the 352 aa residues from Ser 26 to Asp 377 and is composed predominantly of 13 ␣-helices, 10 of which form an ␣/␣ barrel structure. The C-terminal domain comprises the 389 aa residues from Leu 389 to Gly 777 and one calcium ion and consists of 30 ␤-strands arranged in five antiparallel ␤-sheets. A peptide linker composed of the 11 aa residues from Asp 378 to Asn 388 connects the N-and C-terminal domains. In the structure of xanthan lyase, 25.7% of all aa residues are in ␣-helices, 26.2% in ␤-strands, and the remaining 48.1% in turns and coils.
The N-terminal ␣-helical domain includes an incomplete ␣ 5 / ␣ 5 -barrel formed by five inner and five outer ␣-helices, and the 10 ␣-helices (from HA3 to HA12) constituting the ␣ 5 /␣ 5 -barrel are located within the core of the domain (Fig. 4A). These 10 helices are connected by short and long loops in a nearest neighbor, up-and-down pattern. This arrangement is described as a "twisted ␣/␣-barrel" with five inner ␣-helices (HA3, HA5, HA7, HA9, and HA11), which are oriented in roughly the same direction, and five outer ␣-helices (HA4, HA6, HA8, HA10, and HA12) running in the opposite direction. There are 29 loops (from LB1 to LB29) connecting a ␤-strand to the following ␤-strand in the C-terminal domain (Fig. 3). The loop (LB18) between SC5 and SD1 includes one ␣-helix (HB1, aa residues 650 -658) with two turns. Therefore, in the Cterminal domain, 48.8% of all aa residues are in ␤-strands, 2.3% in ␣-helices, and the remaining 48.9% in turns and coils.
The C-terminal domain contains a calcium ion (Fig. 2). The site is located within a loop (LB8) and a ␤-strand (SD2) (Figs.  3 and 4B). The six oxygen atoms, OD1 of Asp 515 , OD2 of Asp 516 , OE1 and OE2 of Glu 517 , OE1 of Glu 676 , and O of WAT 951 , are coordinated to the calcium ion, and the coordination geometry comprises a distorted octahedron (Fig. 5). The distance between the calcium ion and the oxygen atoms ranges from 1.92 to 2.73 Å (average, 2.31 Å).
In the C-terminal domain, five anti-parallel ␤-sheets (sheets A-E) are formed by the 30 ␤-strands, all of which are antiparallel in the ␤-sheets (Fig. 3). The ␤-sheets are composed of four to nine ␤-strands (sheet A, SA1-4; sheet B, SB1-5; sheet C, SC1-9; sheet D, SD1-7; and sheet E, SE1-5). Sheet A is parallel to the small ␤-sheet consisting of S1 and S2 in the N-terminal ␣-helical domain. As a result, the C-terminal ␤-sheet domain shows a five-layered ␤-sheet sandwich structure (Fig. 4B). Structural Comparison of Polysaccharide Lyases-On the basis of their sequence similarity, polysaccharide lyases are classified into 12 families. The three-dimensional structures of lyases belonging to families 1, 3, 5, 6, and 8 have been determined and divided into three groups (parallel ␤-helix, ␣/␣barrel, and ␣/␣-barrel ϩ anti-parallel ␤-sheets). 1 Xanthan lyase belongs to polysaccharide lyase family 8, together with lyases for hyaluronate and chondroitin AC, although the sequence identity of xanthan lyase with the other lyases is less than 30% (Fig. 6).
The overall structure of xanthan lyase is similar to that of the family 8 lyases for hyaluronate (10, 11) and chondroitin AC (13), which consist of N-terminal ␣-helical and C-terminal ␤-sheet domains. The crystal structures of xanthan lyase and the other enzymes were superimposed by means of a fitting program, RIGID, included in TURBO-FRODO (Fig. 7). The aa sequences of the lyases for xanthan, hyaluronate, and chondroitin AC were aligned through analyses of the aa similarity and three-dimensional structures (Fig. 6). The r.m.s. deviations of C ␣ atoms between xanthan lyase and the other enzymes were determined by means of a fitting program, TOP, included in CCP4 (37) ( Table III). The overall structure of xanthan lyase is more similar to that of hyaluronate lyase than of chondroitin AC lyase, and among the family 8 lyases, the geometries of the ␤-domains are more well conserved than those of the ␣-domains.
However, the following structural differences were observed among the lyases for xanthan, hyaluronate, and chondroitin AC. Hyaluronate lyase of Streptococcus agalactiae has a small N-terminal ␤-domain composed of seven ␤-strands preceding the ␣-helical domain (11). It should be noted that in the case of hyaluronate lyase of Streptococcus pneumoniae, the presence of an N-terminal ␤-domain has not been reported due to the crystal structure of the truncated enzyme (10). The C-terminal ␤-sheet domain of xanthan lyase is slightly larger than those of the lyases for hyaluronate and chondroitin AC; i.e. xanthan lyase has 30 ␤-strands in the C-terminal domain, whereas the C-terminal domains of the lyases for hyaluronate and chondroitin AC contain 24 and 28 ␤-strands, respectively (10, 13). The ␣-helical domain of xanthan lyase is similar to that of hyaluronate lyases but differs from that of chondroitin AC lyase, as the latter has 12 ␣-helices in its N-terminal domain (Fig. 6).
Although the C-terminal domain of chondroitin AC lyase contains a calcium ion (13), the localization and coordination of the calcium ion in xanthan lyase are different from those in the AC lyase in that the calcium ion of the AC lyase coordinates with seven oxygen atoms in two water molecules and in the side chains of four aa residues (Glu 405 , Asp 407 , Asp 416 , and Tyr 417 ) in strand ␤5 corresponding to SB1 of xanthan lyase. Furthermore, structural differences between xanthan lyase and the other enzymes were found in the loops over the deep cleft formed in the N-terminal domain. In particular, loop LB16 of xanthan lyase connecting SB4 and SB3 in the C-terminal domain protrudes from the C-terminal domain and covers the cleft in the N-terminal domain (Fig. 2). The extreme protrusion of the loop is caused by the Arg 612 -Thr 615 residues; there are several gaps in the corresponding sites of the lyases for hyaluronate and chondroitin AC (Fig. 6). The significance of the loop is described below.
The topology of the secondary structure elements of the N-terminal ␣-helical domain of xanthan lyase resembles that of FIG. 2. Overall structure of xanthan lyase (ribbon stereodiagram). The colors denote elements with a secondary structure (blue, ␣-helices; red, ␤-strands; cyan, turns and coils). A calcium ion in the C-terminal domain is shown as a yellow ball. This figure was prepared using the programs MOLSCRIPT (29) and RASTER3D (31).
Structure of Xanthan Lyase Complexed with PyrMan-Although almost all polysaccharide lyases analyzed thus far, including those for hyaluronate and chondroitin AC, attack the main chains of polysaccharides in an endolytic manner and release the oligosaccharides from the polysaccharides, xanthan lyase is characteristic in that it attacks the side chains of a polysaccharide exolytically. To identify the substrate-binding site, and to clarify the structural features causing the different substrate specificities of xanthan lyase and the other lyases, the crystal structure of the enzyme complexed with PyrMan (product) was determined at 2.4 Å resolution.
structure of the native enzyme as a primary model. The results of the x-ray data collection and refinement are summarized in Table I.
The refined model of the enzyme-product complex consists of 752 aa residues (Ser 26 -Gly 777 ), 261 water molecules, one calcium ion, and one PyrMan. A stereodiagram of a ribbon presentation of the complex is shown in Fig. 8A. The final overall R-factor for the refined model was calculated as 0.169 (free R-factor, 0.242) using the data from 50 to 2.4 Å resolution (26,294 reflections).
The difference Fourier map contoured at the 3 level calculated with the 10 -2.4 Å resolution data exhibited the highest densities in the product region (Fig. 8B). The average B-factor of PyrMan was 20.6 Å 2 . The nomenclature for the sugar-binding site in the enzyme proposed by Davies et al. (47) was used here, and the sugars are numbered starting from the cleavage site, with positive numbers increasing toward the reducing terminus. Because xanthan lyase cleaves the glycosidic bond between PyrMan and GlcUA residues (Fig. 1), the position of PyrMan is designated as "Ϫ1." Xanthan lyase and its complex with PyrMan were superim-posed by means of a fitting program included in TURBO-FRODO. The r.m.s. deviation was 0.305 Å for the 752 common C ␣ atoms. There was no significant conformational change between the protein structures with and without PyrMan. Pyr-Man in the complex structure is bound in the deep cleft formed on the N-terminal domain of the enzyme facing the interface between the N-and C-terminal domains (Fig. 8A), indicating that the active center (substrate-binding site) is located in the cleft. Some aromatic and positively charged aa residues are arranged in the active cleft, suggesting that these aa residues are responsible for the binding and depolymerization of acidic polysaccharide xanthan (Fig. 9). This feature is common to family 8 polysaccharide lyases, which depolymerize acidic polysaccharides (10,13). Structure of the Active Cleft-Several aa residues and water molecules have been shown to be crucial for the binding of PyrMan (Fig. 10A). The data listed in Table IV represent the interaction of the enzyme with the bound PyrMan molecule in the complex. There are six direct hydrogen bonds between the protein and PyrMan atoms (Table IV). In particular, the carboxyl group of the pyruvate moiety in PyrMan is directly bound to the Arg 313 , Tyr 315 , and Arg 612 residues through the formation of four hydrogen bonds. This conformation accounts for why the xanthan lyase of Bacillus sp. GL1 specifically liberates the nonreducing terminal saccharide, PyrMan, from the side chains of xanthan and is inactive on nonpyruvylated xanthan (16). The Arg 313 and Tyr 315 residues are located in the LA10 loop of the N-terminal domain, whereas the Arg 612 residue is in the LB16 loop of the C-terminal domain. As we described above, there are differences between xanthan lyase and family 8 lyases as regards the loop structure over the active cleft, and there are no direct hydrogen bonds between aa residue atoms in the C-terminal domain and sugar atoms at position Ϫ1 are present in lyases for hyaluronate (11,48) and chondroitin AC (49) complexed with the substrate or product. Therefore, the extreme protrusion of the LB16 loop of xanthan lyase from the C-terminal domain is responsible for the recognition and binding of PyrMan. Apart from their interaction with the carboxyl group of PyrMan, the Tyr 255 and Arg 313 residues directly interact with the pyranose ring of PyrMan through the formation of hydrogen bonds. The side chain (OH) of the Tyr 255 residue forms a hydrogen bond with an oxygen atom (O-1) of PyrMan, which forms a glycosidic bond with the GlcUA residue before the enzyme reaction, suggesting that the Tyr 255 residue plays an important role in the catalytic reaction. In addition to direct hydrogen bonds, there are six hydrogen bonds between water molecules and PyrMan atoms (Table IV) Carbon-carbon (C-C) contacts were also observed between the protein (Trp 148 , Trp 197 , Tyr 255 , and Arg 313 residues) and PyrMan atoms (Table IV). The Trp 148 residue is parallel to the pyranose ring of the mannose moiety of PyrMan, indicating that the residue undergoes a stacked interaction with the sugar ring (Fig. 10A). This stacking is thought to be common to family 8 lyases, because the Trp 127 and Trp 292 residues of chondroitin AC and hyaluronate lyases, respectively, corresponding to the  Trp 148 residue of xanthan lyase, show a stacked interaction with the sugar positioned at Ϫ1 (48, 49) (Fig. 10, B and C).
The arrangements of aa residues at the Ϫ1 position of the family 8 lyases were compared (Fig. 10, B and C). The activesite architecture has been reported to be well conserved in lyases for hyaluronate and chondroitin AC (48). The geometry of the Arg 313 residue of xanthan lyase is similar to that of the Arg 466 and Arg 292 residues of lyases for hyaluronate and chondroitin AC, respectively, which are essential for the direct binding of sugars and which correspond to the Arg 313 residue of xanthan lyase (Figs. 6 and 10, B and C). Recently, the Arg 292 residue of chondroitin AC lyase responsible for the binding of GlcNAc positioned at Ϫ1 was found to be involved in the subsequent, processive, stepwise, and exolytic cleavage reaction of the enzyme (50). On the other hand, the following differences among the lyases for xanthan, hyaluronate, and chondroitin AC were observed in the arrangement of aa residues. In the case of xanthan lyase, the Arg 313 and Tyr 315 residues in the N-terminal domain and the Arg 612 residue in the C-terminal domain directly bind to the carboxyl group of PyrMan. However, no direct interaction of lyases for hyaluronate and chondroitin AC with the sugar positioned at Ϫ1 involves residues in the C-terminal domain (11,48,49), although the Asn 374 , Glu 376 , Ser 552 , and His 553 residues of the C-terminal domain of chondroitin AC lyase associate with the sugar via water molecules (49). No aa residues corresponding to the Tyr 315 and Arg 612 residues of xanthan lyase are conserved in lyases for hyaluronate and chondroitin AC (Fig. 6). The Arg 462 residue of hyaluronate lyase directly binds to O-4 of GlcNAc positioned at Ϫ1, whereas the Arg 309 residue of xanthan lyase, corresponding to the Arg 462 residue of hyaluronate lyase, undergoes no interactions with PyrMan. Therefore, these differences in the arrangement of aa residues at the Ϫ1 position determine the substrate specificities of the family 8 lyases, because the substrates of lyases for xanthan, hyaluronate, and chondroitin AC have PyrMan, GlcNAc, and GalNAc residues, respectively, at-FIG. 10. A, PyrMan bound in the active site of xanthan lyase (ribbon stereodiagram). The figure shows the bound sugar and the surrounding aa residues (positive, purple; aromatic, yellow) and water molecules (w, black ball) interacting with the sugar. The sugar is represented by a gray ball-and-stick model (red ball, oxygen atom). Direct hydrogen bonds (Յ3.2 Å), shown as dotted lines, are formed between the sugar atoms (red) and the aa residues. B, superpositioning of the active-site structures of xanthan lyase and hyaluronate lyase (PDB code, 1C82; stereodiagram). The aa residues of xanthan lyase (purple) and hyaluronate lyase (green) are responsible for the direct interaction with sugars and the catalytic reaction. PyrMan at position Ϫ1 of xanthan lyase is shown as a gray ball-andstick model (red ball, oxygen atom). Glc-NAc at position Ϫ1 of hyaluronate lyase is shown as a black ball-and-stick model (red ball, oxygen atom; blue ball, nitrogen atom). C, superpositioning of the activesite structures of xanthan lyase and chondroitin AC lyase (PDB code, 1HM2; stereodiagram). The aa residues of xanthan lyase (purple) and chondroitin AC lyase (yellow) are responsible for the direct interaction with sugars and the catalytic reaction. PyrMan at position Ϫ1 of xanthan lyase is shown as a gray ball-andstick model (red ball, oxygen atom). The GalNAc and GlcUA at positions Ϫ1 and ϩ1, respectively, of chondroitin AC lyase are shown as a black ball-and-stick model (red ball, oxygen atom; blue ball, nitrogen atom; yellow ball, sulfur atom). This figure was prepared using the programs MOLSCRIPT (29) and RASTER3D (31).
The Tyr 255 residue interacts directly in the active cleft with the O-1 oxygen atom of PyrMan involved in the formation of the glycosidic bond between the Ϫ1 and ϩ1 sugars. This finding indicates that the residue is responsible for the catalytic reaction. Several structural studies on catalytic residues in the clefts of lyases for hyaluronate and chondroitin AC have been reported (11,48,49). More recently, the Asn 349 , His 399 , and Tyr 408 residues of hyaluronate lyase from S. pneumoniae were shown to participate in the catalytic reaction through x-ray crystallographic analysis of a mutant enzyme (Y408F) complexed with hyaluronate tetra-and hexasaccharides (51). Asn 349 interacts with the carboxyl group of the GlcUA residue, His 399 functions as a base and withdraws a proton from the C-5 carbon of the GlcUA residue, and Tyr 408 , acting as an acid, donates a proton to the glycosidic oxygen to be cleaved. These three residues are conserved in xanthan lyase of Bacillus sp. GL1, and the Asn 194 , His 246 , and Tyr 255 residues of the xanthan lyase correspond to the respective residues of hyaluronate lyase (Fig. 6). The His 225 and Tyr 234 residues of chondroitin AC lyase corresponding to the His 399 and Tyr 408 residues of hyaluronate lyase have also been reported as crucial for the catalytic reaction (49). The arrangement of these residues is highly conserved (Fig. 10, B and C) in the family 8 lyases; xanthan lyases (N194A, H246A, and Y255F) in which the Asn 194 , His 246 , and Tyr 255 residues were substituted with Ala, Ala, and Phe, respectively, exhibited little enzymatic activity. 3 These results suggest that the Asn 194 , His 246 , and Tyr 255 residues play a crucial role in the ␤-elimination reaction of xanthan lyase, as seen for the lyases for hyaluronate and chondroitin AC. To clarify the reaction mechanism of the exotype xanthan lyase in more detail and to establish common structural rules for polysaccharide lyases, we have attempted to determine the structures of the wild type and mutant enzymes complexed with xanthan-branched pentasaccharide as a substrate (52).
Conclusions and Implications-To the best of our knowledge, this is the first report on the determination of the crystal structure of an exotype polysaccharide lyase that can act on the side chains of a polysaccharide. The enzyme consists of Nterminal ␣-helical and C-terminal ␤-sheet domains and has a deep cleft in the N-terminal domain facing the interface between the N-and C-terminal domains. Because the basic frames of lyases for xanthan, hyaluronate, and chondroitin AC are similar to each other, all of the polysaccharide lyases belonging to family 8 are considered to share a common structure consisting of N-terminal ␣-helical and C-terminal ␤-sheet domains. Furthermore, their active sites are all located in a deep cleft.
Based on the crystal structure of the enzyme complexed with PyrMan, the deep cleft in the enzyme was revealed to be responsible for the recognition of the substrate and the catalytic reaction. The enzyme specifically binds to the nonreducing terminal PyrMan of xanthan side chains in the cleft, as the aromatic and positively charged aa residues in the active cleft directly interact with the carboxyl group of PyrMan through the formation of hydrogen bonds. The arrangement of aa residues of xanthan lyase in the recognition site for PyrMan attached to the GlcUA residue differs from that in lyases for hyaluronate and chondroitin AC in the corresponding sites for GlcNAc and GalNAc, respectively, attached to the GlcUA residue. These differences in the aa arrangement are thought to determine the substrate specificity.
As seen for lyases for hyaluronate and chondroitin AC, the Asn 194 , His 246 , and Tyr 255 residues in the active cleft of xanthan lyase are thought to be involved in the catalytic reaction. As regards family 5 alginate lyase A1-III, similar to the case of the N-terminal ␣-helical domain of family 8 lyases, we have clarified that the activated Tyr 246 residue homologous to the Tyr 255 residue of xanthan lyase is bifunctional as a base and an acid (53). Therefore, the Tyr residue is considered important for the catalytic reactions of these polysaccharide lyases including an ␣/␣-barrel structure.