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

Wataru HashimotoDagger , Hirokazu NankaiDagger , Bunzo Mikami§, and Kousaku MurataDagger ||

From the Dagger  Department of Basic and Applied Molecular Biotechnology, Division of Food and Biological Science, Graduate School of Agriculture, Kyoto University and the § Laboratory of Quality Design of Exploitation, Division of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

Received for publication, August 8, 2002, and in revised form, November 27, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 final R-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 alpha -helical and C-terminal beta -sheet domains, which constitute incomplete alpha 5/alpha 5-barrel and anti-parallel beta -sheet structures, respectively. A deep cleft is located in the N-terminal alpha -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.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 polysaccharides. The properties of glycosyl hydrolases that act on poly- and 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).

As regards the second group, the lyases, it is known that they recognize uronic acid residues in polysaccharides, catalyze the beta -elimination reaction, and produce unsaturated saccharides with C=C double bonds at the nonreducing terminal uronate residues (Fig. 1). These characteristics of lyases indicate that they share common structural features determining their uronate recognition sites and reaction modes (beta -elimination reaction). Although structural analyses of lyases for pectate (3-8), alginate (9), hyaluronate (10, 11), and chondroitin (12, 13) have been made, there is little information regarding the structural rules common to polysaccharide lyases.

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 beta -elimination reaction (Fig. 1A) (16). The enzyme is synthesized as a precursor form (99 kDa) and is then converted into the mature form (~75 kDa) through posttranslational excision of the signal peptide (2 kDa) and C-terminal polypeptide (~22 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).


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Fig. 1.   Structures of polysaccharides and modes of action of polysaccharide lyases. A, xanthan; B, hyaluronate; C, chondroitin. Thick and dotted-line arrows indicate the cleavage sites for polysaccharide lyases and the degradation reactions of polysaccharides, respectively.

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 beta -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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Materials-- Pyruvylated xanthan (average molecular mass, 2 × 106; pyruvylation of the terminal mannosyl residue in the side chain, ~50%) was obtained from Kohjin Co., Tokyo, Japan. Polyethylene glycol 4000 was purchased from Nacalai Tesque, Kyoto, Japan. DEAE-Toyopearl 650 M and Super Q-Toyopearl 650C were from Tosoh Co., Tokyo, Japan. Bio-Gel P2 was from Bio-Rad. The restriction endonucleases and DNA-modifying enzymes were from Takara Shuzo Co., Kyoto, and Toyobo Co., Tokyo, respectively.

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 = 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 × 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 × 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 µM pepstatin A, was fractionated with ammonium sulfate. The precipitate (0-30% saturation) was collected by centrifugation at 15,000 × 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 × 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 (~75 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 × 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 (~75 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 × 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 (~75 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 µl of protein solution (7.18 mg of protein/ml) with 3 µl 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 NaAuCl4, 0.2 mM AgNO3, 1 mM Ac2UO2, 2 mM SmCl3, 1 mM GdCl3, 1 mM HoCl3, and 1 mM CdCl2 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 CuKalpha 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.  Fo  -  Fc  and 2 Fo  -  Fc  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 sigma  above the mean and the 2 Fo  -  Fc  map showed a density of more than 1.0 sigma . 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  Fo  -  Fc  map (contoured at 3.0 sigma ) 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 × 0.2 × 0.05 mm) was obtained by the hanging-drop vapor diffusion method. The space group was determined to be P212121 (orthorhombic) with unit cell dimensions of a = 54.3 Å, b = 91.4 Å, and c = 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.

                              
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Table I
Data collection and refinement statistics for xanthan lyase

                              
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Table II
Statistics for heavy atom derivatives of xanthan lyase

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 Ser26 and Gly777, 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 Fo  -  Fc  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-mean-square (r.m.s.) deviations from the standard geometry were 0.0060 Å for bond lengths and 1.29o for bond angles. Based on the theoretical curves in the plot calculated according to Luzzati (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 (phi , psi ) 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 Thr247 and Asp695 residues. The Thr247 and Asp695 residues are in beta -turns. In particular, the latter turn, containing the Asp695 residue, is similar to a beta -hairpin consisting of four amino acid residues with the conformation of beta -epsilon -gamma -beta in a Ramachandran plot (36), because the Ala694, Asp695, Leu696, and Ile697 residues fell in or near the beta , epsilon , gamma , and beta  regions of the plot, respectively. Furthermore, there is one cis-peptide between the Ala753 and Pro754 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 × 70 × 50 Å and is composed of two globular domains (N- and C-terminal domains) that form alpha - and beta -structures, respectively. The N-terminal domain comprises the 352 aa residues from Ser26 to Asp377 and is composed predominantly of 13 alpha -helices, 10 of which form an alpha /alpha barrel structure. The C-terminal domain comprises the 389 aa residues from Leu389 to Gly777 and one calcium ion and consists of 30 beta -strands arranged in five anti-parallel beta -sheets. A peptide linker composed of the 11 aa residues from Asp378 to Asn388 connects the N- and C-terminal domains. In the structure of xanthan lyase, 25.7% of all aa residues are in alpha -helices, 26.2% in beta -strands, and the remaining 48.1% in turns and coils.


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Fig. 2.   Overall structure of xanthan lyase (ribbon stereodiagram). The colors denote elements with a secondary structure (blue, alpha -helices; red, beta -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).


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Fig. 3.   Topology diagram of xanthan lyase. The enzyme consists of two domains (N- and C-terminal domains). The 13 alpha -helices (HA1-HA13) and two short beta -strands (S1 and S2) in the N-terminal domain are shown as boxes and arrows, respectively. The inner helices (HA3, HA5, HA7, HA9, and HA11) and outer helices (HA4, HA6, HA8, HA10, and HA12) in the alpha /alpha -barrel structure are shown in green and purple, respectively, and the other helices (HA1, HA2, and HA13) are in gray. The 30 beta -strands arranged into sheets A, B, C, D, and E in the C-terminal domain are shown as blue, purple, cyan, green, and red arrows, respectively. A short alpha -helix (HB1) and calcium ion (Ca) are shown as a gray box and a yellow ball, respectively.

N-terminal alpha -Helical Domain-- The N-terminal domain is composed primarily of two short beta -strands and 13 alpha -helices. The latter contribute to the formation of an alpha /alpha -barrel structure with a deep cleft, which is considered to be an active site (Figs. 2 and 4A). The 13 major alpha -helices, numbered sequentially from HA1 to HA13 (HA1, aa residues 28-41; HA2, 51-69; HA3, 90-106; HA4, 118-131; HA5, 153-165; HA6, 173-184; HA7, 192-208; HA8, 212-221; HA9, 255-270; HA10, 284-291; HA11, 318-333; HA12, 338-354; and HA13, 366-376), vary in length between 8 and 19 aa residues and consist of 187 aa residues (Fig. 3). These alpha -helices are arranged as follows: four (HA5, HA6, HA8, and HA13) with three turns; seven (HA1, HA3, HA4, HA7, HA9, HA11, and HA12) with four turns; one (HA2) with five turns; and one (HA10) with two turns.


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Fig. 4.   A, N-terminal alpha -helical domain (ribbon stereodiagram). Five pairs of alpha -helices (HA3 and HA4 (blue), HA5 and HA6 (green), HA7 and HA8 (yellow), HA9 and HA10 (purple), and HA11 and HA12 (cyan)) form an incomplete alpha 5/alpha 5 barrel structure. The other helices are colored gray. Two short beta -strands (S1 and S2) are shown as red arrows. B, C-terminal beta -sheet domain (ribbon stereodiagram). The 30 beta -strands are arranged in five anti-parallel beta -sheets shown in blue (sheet A), purple (sheet B), cyan (sheet C), green (sheet D), and red (sheet E), respectively. A helix (HB1), turns, and coils are gray. This figure was prepared using the programs MOLSCRIPT (29) and RASTER3D (31).

There are 12 loops (from LA1 to LA12) connecting an alpha -helix to the following alpha -helix in the N-terminal domain (Fig. 3). The loop (LA8) between HA8 and HA9 includes two short beta -strands (S1, aa residues 235-238, and S2, 243-245). Therefore, in the N-terminal domain, 52.3% of all aa residues are in alpha -helices, 2.0% in beta -strands, and the remaining 45.7% in turns and coils.

The N-terminal alpha -helical domain includes an incomplete alpha 5/alpha 5-barrel formed by five inner and five outer alpha -helices, and the 10 alpha -helices (from HA3 to HA12) constituting the alpha 5/alpha 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 alpha /alpha -barrel" with five inner alpha -helices (HA3, HA5, HA7, HA9, and HA11), which are oriented in roughly the same direction, and five outer alpha -helices (HA4, HA6, HA8, HA10, and HA12) running in the opposite direction.

C-terminal beta -Sheet Domain-- The C-terminal domain consists predominantly of 30 beta -strands (SA1, aa residues 389-396; SA2, 398-402; SA3, 407-411; SA4, 437-441; SB1, 465-467; SC1, 482-487; SC2, 491-499; SC3, 506-513; SC4, 518-526; SB2, 533-541; SC8, 550-552; SC9, 554-557; SB5, 563-567; SC7, 571-575; SC6, 585-589; SB4, 593-604; SB3, 620-632; SC5, 638-646; SD1, 662-667; SD2, 671-676; SD3, 681-686; SE1, 692-694; SE2, 697-699; SD4, 703-710; SD5, 714-720; SE3, 729-734; SD6, 740-743; SE5, 747-751; SE4, 756-761; and SD7, 769-775) (Figs. 2, 3, and 4B). The beta -strands vary in length between 3 and 13 aa residues and consist of 190 aa residues.

There are 29 loops (from LB1 to LB29) connecting a beta -strand to the following beta -strand in the C-terminal domain (Fig. 3). The loop (LB18) between SC5 and SD1 includes one alpha -helix (HB1, aa residues 650-658) with two turns. Therefore, in the C-terminal domain, 48.8% of all aa residues are in beta -strands, 2.3% in alpha -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 beta -strand (SD2) (Figs. 3 and 4B). The six oxygen atoms, OD1 of Asp515, OD2 of Asp516, OE1 and OE2 of Glu517, OE1 of Glu676, and O of WAT951, 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).


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Fig. 5.   Coordination of the calcium ion in the C-terminal domain (stereodiagram). The calcium ion (yellow ball) coordinates to the oxygen atoms of Asp515, Asp516, Asp517, Glu676, and WAT951, which are shown in purple. These interactions are indicated by dotted lines. This figure was prepared using the programs MOLSCRIPT (29) and RASTER3D (31).

In the C-terminal domain, five anti-parallel beta -sheets (sheets A-E) are formed by the 30 beta -strands, all of which are anti-parallel in the beta -sheets (Fig. 3). The beta -sheets are composed of four to nine beta -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 beta -sheet consisting of S1 and S2 in the N-terminal alpha -helical domain. As a result, the C-terminal beta -sheet domain shows a five-layered beta -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 beta -helix, alpha /alpha -barrel, and alpha /alpha -barrel + anti-parallel beta -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).


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Fig. 6.   Sequence alignment of polysaccharide lyases (family 8). The aa sequences of the lyases were aligned through analyses of aa similarity using the ClustalW program (clustalw.genome.ad.jp) and the three-dimensional structures. XLY, xanthan lyase (GenBankTM accession No. AB037178) of Bacillus sp. GL1; HLY, hyaluronate lyase of S. pneumoniae (identity score with XLY, 27.2%; GenBankTM accession No. L20670; PDB code, 1EGU); and CLY, chondroitin AC lyase of Flavobacterium heparinum (identity score with XLY, 25.9%; GenBankTM accession No. U27583; PDB code, 1CB8). The N- and C-terminal aa residues Ser26 and Gly777, respectively, of the refined model of xanthan lyase are indicated by arrows. A green box indicates the residues for the edge of the protrusion loop LB16. The aa residues responsible for the binding of PyrMan and the catalytic reaction are shown in red and green, respectively. The regions shown in cyan and purple constitute the alpha -helices and beta -strands, respectively. Identical and similar aa among the three proteins are denoted by asterisks and dots, respectively.

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 alpha -helical and C-terminal beta -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 Calpha 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 beta -domains are more well conserved than those of the alpha -domains.


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Fig. 7.   Superpositioning of the overall structures of xanthan lyase (blue, Calpha backbone) and hyaluronate lyase (PDB code, 1EGU; purple, Calpha backbone) (stereodiagram). This figure was prepared using the programs MOLSCRIPT (29) and RASTER3D (31).

                              
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Table III
The r.m.s. deviation of Calpha atoms between xanthan lyase and other lyases

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 beta -domain composed of seven beta -strands preceding the alpha -helical domain (11). It should be noted that in the case of hyaluronate lyase of Streptococcus pneumoniae, the presence of an N-terminal beta -domain has not been reported due to the crystal structure of the truncated enzyme (10). The C-terminal beta -sheet domain of xanthan lyase is slightly larger than those of the lyases for hyaluronate and chondroitin AC; i.e. xanthan lyase has 30 beta -strands in the C-terminal domain, whereas the C-terminal domains of the lyases for hyaluronate and chondroitin AC contain 24 and 28 beta -strands, respectively (10, 13). The alpha -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 alpha -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 (Glu405, Asp407, Asp416, and Tyr417) in strand beta 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 Arg612-Thr615 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 alpha -helical domain of xanthan lyase resembles that of alginate lyase, A1-III (polysaccharide lyase family 5), with an alpha 6/alpha 5-barrel structure, which we have determined to be an example of an endotype polysaccharide lyase (9). The alpha /alpha -barrel structure is found in sugar-related enzymes such as glucoamylases (38, 39) endoglucanase (40, 41), endo/exocellulase (42), N-acyl-D-glucosamine 2-epimerase (43), alpha -1,2-mannosidase (44), and maltose phosphorylase (45), as well as polysaccharide lyases. These enzymes form the alpha /alpha -toroid family in the SCOP data base (scop.berkeley.edu/data/scop.b.b.bbc.html). Although these enzymes catalyze different reactions (hydrolysis, epimerization, and beta -elimination), their alpha /alpha -barrel structures possibly are responsible for the binding of polysaccharides in the hydrolytic and eliminative depolymerization reactions and for the production of common intermediates in the epimerization and beta -elimination reactions. In fact, the occurrence of a common step in the catalytic reactions of two types of alginate-modifying enzymes, lyases and epimerases, has been reported (46).

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.

PyrMan was soaked in a native crystal of xanthan lyase. Diffraction data for the crystal of xanthan lyase complexed with the product up to 2.40 Å resolution were collected and refined to 2.4 Å resolution with CNS (26) using the refined 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 (Ser26-Gly777), 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).


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Fig. 8.   A, overall structure of xanthan lyase complexed with PyrMan (ribbon stereodiagram). The colors denote the secondary structure elements (blue, alpha -helices; red, beta -strands; cyan, turns and coils). PyrMan is shown as a gray ball-and-stick model (red, oxygen atom). B, stereodiagram of the electron density map of PyrMan and the surrounding aa residues. The omit and 2 Fo  -  Fc  maps in PyrMan are shown as thin cyan and red lines, respectively. PyrMan is shown as a gray ball-and-stick model (red, oxygen atom). Aromatic and positively charged aa residues are shown in yellow and purple, respectively. This figure was prepared using the programs MOLSCRIPT (29), BOBSCRIPT (30), and RASTER3D (31).

The difference Fourier map contoured at the 3sigma 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 Å2. 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 "-1."

Xanthan lyase and its complex with PyrMan were superimposed by means of a fitting program included in TURBO-FRODO. The r.m.s. deviation was 0.305 Å for the 752 common Calpha atoms. There was no significant conformational change between the protein structures with and without PyrMan. PyrMan 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).


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Fig. 9.   Molecular surface of the active cleft. Aromatic, positively, and negatively charged aa residues are colored yellow, cyan, and purple, respectively. PyrMan is shown as a stick model. A, the mannose moiety of PyrMan is on the front side, and the pyruvate moiety of PyrMan on the back side. The catalytic site is thought to be located in front of the tunnel. B, this view is from the opposite direction of A. This figure was prepared using the program GRASP (32).

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 Arg313, Tyr315, and Arg612 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 Arg313 and Tyr315 residues are located in the LA10 loop of the N-terminal domain, whereas the Arg612 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 -1 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 Tyr255 and Arg313 residues directly interact with the pyranose ring of PyrMan through the formation of hydrogen bonds. The side chain (OH) of the Tyr255 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 Tyr255 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). Furthermore, there are six water-mediated hydrogen bonds between the protein and PyrMan atoms: O-1=WAT812=Tyr255OH (2.9 Å), O-2=WAT818=Ser258 OG (3.0 Å), O-2=WAT859=Glu310 OE2 (2.8 Å), O-3=WAT818=Glu310 OE2 (2.8 Å), O-6=WAT1066=Arg612 NH2 (3.1 Å), and O-8=WAT1073=Tyr315 OH (3.2 Å).


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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 (<= 3.2 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 -1 of xanthan lyase is shown as a gray ball-and-stick model (red ball, oxygen atom). GlcNAc at position -1 of hyaluronate lyase is shown as a black ball-and-stick model (red ball, oxygen atom; blue ball, nitrogen atom). C, superpositioning of the active-site 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 -1 of xanthan lyase is shown as a gray ball-and-stick model (red ball, oxygen atom). The GalNAc and GlcUA at positions -1 and +1, 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).

                              
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Table IV
Interactions between xanthan lyase and PyrMan

Carbon-carbon (C-C) contacts were also observed between the protein (Trp148, Trp197, Tyr255, and Arg313 residues) and PyrMan atoms (Table IV). The Trp148 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 Trp127 and Trp292 residues of chondroitin AC and hyaluronate lyases, respectively, corresponding to the Trp148 residue of xanthan lyase, show a stacked interaction with the sugar positioned at -1 (48, 49) (Fig. 10, B and C).

The arrangements of aa residues at the -1 position of the family 8 lyases were compared (Fig. 10, B and C). The active-site architecture has been reported to be well conserved in lyases for hyaluronate and chondroitin AC (48). The geometry of the Arg313 residue of xanthan lyase is similar to that of the Arg466 and Arg292 residues of lyases for hyaluronate and chondroitin AC, respectively, which are essential for the direct binding of sugars and which correspond to the Arg313 residue of xanthan lyase (Figs. 6 and 10, B and C). Recently, the Arg292 residue of chondroitin AC lyase responsible for the binding of GlcNAc positioned at -1 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 Arg313 and Tyr315 residues in the N-terminal domain and the Arg612 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 -1 involves residues in the C-terminal domain (11, 48, 49), although the Asn374, Glu376, Ser552, and His553 residues of the C-terminal domain of chondroitin AC lyase associate with the sugar via water molecules (49). No aa residues corresponding to the Tyr315 and Arg612 residues of xanthan lyase are conserved in lyases for hyaluronate and chondroitin AC (Fig. 6). The Arg462 residue of hyaluronate lyase directly binds to O-4 of GlcNAc positioned at -1, whereas the Arg309 residue of xanthan lyase, corresponding to the Arg462 residue of hyaluronate lyase, undergoes no interactions with PyrMan. Therefore, these differences in the arrangement of aa residues at the -1 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, attached to the common GlcUA residues (Fig. 1).

The Tyr255 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 -1 and +1 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 Asn349, His399, and Tyr408 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). Asn349 interacts with the carboxyl group of the GlcUA residue, His399 functions as a base and withdraws a proton from the C-5 carbon of the GlcUA residue, and Tyr408, 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 Asn194, His246, and Tyr255 residues of the xanthan lyase correspond to the respective residues of hyaluronate lyase (Fig. 6). The His225 and Tyr234 residues of chondroitin AC lyase corresponding to the His399 and Tyr408 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 Asn194, His246, and Tyr255 residues were substituted with Ala, Ala, and Phe, respectively, exhibited little enzymatic activity.3 These results suggest that the Asn194, His246, and Tyr255 residues play a crucial role in the beta -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 N-terminal alpha -helical and C-terminal beta -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 alpha -helical and C-terminal beta -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 Asn194, His246, and Tyr255 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 alpha -helical domain of family 8 lyases, we have clarified that the activated Tyr246 residue homologous to the Tyr255 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 alpha /alpha -barrel structure.

    ACKNOWLEDGEMENT

Computation time was provided by the Supercomputer Laboratory at the Institute for Chemical Research, Kyoto University.

    FOOTNOTES

* This work was supported in part by the Bio-oriented Technology Research Advancement Institution (BRAIN) of Japan and by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (to K. M., B. M., and W. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and structure factors of xanthan lyase and its complex with PyrMan (codes 1J0M and 1J0N, respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

These authors contributed equally to this work.

|| To whom correspondence should be addressed. Tel.: 81-774-38-3766; Fax: 81-774-38-3767; E-mail: murata@food2.food.kyoto-u.ac.jp.

Published, JBC Papers in Press, December 9, 2002, DOI 10.1074/jbc.M208100200

1 B. Henrissat, P. Coutinho, and E. Deleury, afmb.cnrs-mrs.fr/~cazy/CAZY/index.html.

3 W. Hashimoto, H. Nankai, B. Mikami, and K. Murata, unpublished results.

    ABBREVIATIONS

The abbreviations used are: PyrMan, pyruvylated mannose; Glc, D-glucose; GlcUA, D-glucuronic acid; Man, D-mannose; GlcNAc, N-acetyl-D-glucosamine; GalNAc, N-acetyl-D-galactosamine; m.i.r., multiple isomorphous replacement; aa, amino acid; r.m.s., root-mean-square; WAT, water molecule; Ac, acetate; KPB, potassium phosphate buffer; Bicine, N,N-bis(2-hydroxyethyl)glycine; CNS, crystallography NMR software; PDB, protein data bank.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. Bourne, Y., and Henrissat, B. (2001) Curr. Opin. Struct. Biol. 11, 593-600[CrossRef][Medline] [Order article via Infotrieve]
2. Davies, G. J., and Henrissat, B. (2002) Biochem. Soc. Trans. 30, 291-297[CrossRef][Medline] [Order article via Infotrieve]
3. Yoder, M. D., Keen, N. T., and Jurnak, F. (1993) Science 260, 1503-1507[Medline] [Order article via Infotrieve]
4. Yoder, M. D., Lietzke, S. E., and Jurnak, F. (1993) Structure 1, 241-251[Medline] [Order article via Infotrieve]
5. Pickersgill, R., Jenkins, J., Harris, G., Nasser, W., and Robert-Baudouy, J. (1994) Nat. Struct. Biol. 1, 717-723[Medline] [Order article via Infotrieve]
6. Mayans, O., Scott, M., Connerton, I., Gravesen, T., Benen, J., Visser, J., Pickersgill, R., and Jenkins, J. (1997) Structure 15, 677-689
7. Vitali, J, Schick, B., Kester, H. C., Visser, J., and Jurnak, F. (1998) Plant Physiol. 116, 69-80[Abstract/Free Full Text]
8. Akita, M., Suzuki, A., Kobayashi, T., Ito, S., and Yamane, T. (2001) Acta Crystallogr. Sect. D Biol. Crystallogr. 57, 1786-1792[CrossRef][Medline] [Order article via Infotrieve]
9. Yoon, H.-J., Mikami, B., Hashimoto, W., and Murata, K. (1999) J. Mol. Biol. 290, 505-514[CrossRef][Medline] [Order article via Infotrieve]
10. Li, S., Kelly, S. J., Lamani, E., Ferraroni, M., and Jedrzejas, M. J. (2000) EMBO J. 15, 1228-1240[CrossRef]
11. Li, S., and Jedrzejas, M. J. (2001) J. Biol. Chem. 276, 41407-41416[Abstract/Free Full Text]
12. Huang, W., Matte, A., Li, Y., Kim, Y. S., Linhardt, R. J., Su, H., and Cygler, M. (1999) J. Mol. Biol. 294, 1257-1269[CrossRef][Medline] [Order article via Infotrieve]
13. Féthiere, J., Eggimann, B., and Cygler, M. (1999) J. Mol. Biol. 14, 635-647[CrossRef]
14. Yoon, H.-J., Hashimoto, W., Miyake, O., Okamoto, M., Mikami, B., and Murata, K. (2000) Protein Expression Purif. 19, 84-90[CrossRef][Medline] [Order article via Infotrieve]
15. Hashimoto, W., Sato, N., Kimura, S., and Murata, K. (1998) Arch. Biochem. Biophys. 354, 31-39[CrossRef][Medline] [Order article via Infotrieve]
16. Hashimoto, W., Miki, H., Tsuchiya, N., Nankai, H., and Murata, K. (1998) Appl. Environ. Microbiol. 64, 3765-3768[Abstract/Free Full Text]
17. Rogovin, S. P., Anderson, R. F., and Cadmus, M. C. (1961) J. Biochem. Microbiol. Technol. Eng. 3, 51-63
18. Jansson, P.-E., Kenne, L., and Lindberg, B. (1975) Carbohydr. Res. 45, 275-282[CrossRef][Medline] [Order article via Infotrieve]
19. Sandford, P. A., Pittsley, J. E., Knutson, C. A., Watson, P. R., Cadmus, M. C., and Janes, A. (1977) in Extracellular Microbial Polysaccharides Symposium Series No. 45 (Sandford, P. A. , and Laskin, A., eds) , pp. 192-210, American Chemical Society, Washington, D. C.
20. Becker, A., Katzen, F., Puhler, A., and Ielpi, L. (1998) Appl. Microbiol. Biotechnol. 50, 145-152[CrossRef][Medline] [Order article via Infotrieve]
21. Hashimoto, W., Miki, H., Tsuchiya, N., Nankai, H., and Murata, K. (2001) Appl. Environ. Microbiol. 67, 713-720[Abstract/Free Full Text]
22. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
23. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
24. Furey, W., and Swaminathan, S. (1997) Methods Enzymol. 277, 590-620
25. Wang, B. C. (1985) Methods Enzymol. 115, 90-112[Medline] [Order article via Infotrieve]
26. Brünger, A. T., Adams, P. D., Clore, G. M., Delano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, N., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
27. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
28. Hooft, R. W., Vriend, G., Sander, C., and Abola, E. E. (1996) Nature 381, 272[Medline] [Order article via Infotrieve]
29. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
30. Esnouf, R. M. (1997) J. Mol. Graph. Model. 15, 132-134[CrossRef][Medline] [Order article via Infotrieve], 112-113
31. Merrit, E. A., and Murphy, M. E. P. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 869-873[CrossRef][Medline] [Order article via Infotrieve]
32. Nicholls, A., Sharp, K., and Honig, B. (1991) Proteins Struct. Funct. Genet. 11, 281-296[Medline] [Order article via Infotrieve]
33. Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E. (2000) Nucleic Acids Res. 28, 235-242[Abstract/Free Full Text]
34. Luzzati, V. (1952) Acta Crystallogr. 5, 802-810[CrossRef]
35. Ramachandran, G. N., and Sasisekharan, V. (1968) Adv. Protein Chem. 23, 283-437[Medline] [Order article via Infotrieve]
36. Sibanda, B. L., and Thornton, J. M. (1985) Nature 316, 170-174[Medline] [Order article via Infotrieve]
37. Collaborative Computational Project 4. (1994) Acta Crystallogr. Sect. D Biol. Crystallogr. 50, 760-763[CrossRef][Medline] [Order article via Infotrieve]
38. Aleshin, E. A., Golubev, A., Firsov, L. M., and Honzatko, R. B. (1992) J. Biol. Chem. 267, 19291-19298[Abstract/Free Full Text]
39. Sevcik, J., Solovicová, A., Hostinová, E., Gasperik, J., Wilson, K. S., and Dauter, Z. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 854-866[CrossRef][Medline] [Order article via Infotrieve]
40. Juy, M., Amit, A. G., Alzari, P. M., Poljak, R. J., Claeyssens, M., Bguin, P., and Aubert, J. (1992) Nature 357, 89-91[CrossRef]
41. Alzari, P. M., Souchon, H., and Dominguez, R. (1996) Structure 15, 265-275
42. Sakon, J., Irwin, D., Wilson, D. B., and Karplus, P. A. (1997) Nat. Struct. Biol. 4, 810-818[Medline] [Order article via Infotrieve]
43. Itoh, T., Mikami, B., Maru, I., Ohta, Y., Hashimoto, W., and Murata, K. (2000) J. Mol. Biol. 303, 733-744[CrossRef][Medline] [Order article via Infotrieve]
44. Vallee, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581-588[Abstract/Free Full Text]
45. Egloff, M.-P., Uppenberg, J., Haalck, L., and Van Tilbeurgh, H. (2001) Structure 9, 689-697[CrossRef][Medline] [Order article via Infotrieve]
46. Gacesa, P. (1987) FEBS Lett. 212, 199-202[CrossRef]
47. Davies, G. J., Wilson, K. S., and Henrissat, B. (1997) Biochem. J. 321, 557-559[Medline] [Order article via Infotrieve]
48. Ponnuraj, K., and Jedrzejas, M. J. (2000) J. Mol. Biol. 299, 885-895[CrossRef][Medline] [Order article via Infotrieve]
49. Huang, W., Boju, L., Tkalec, L., Su, H., Yang, H. O., Gunay, N. S., Linhardt, R. J., Kim, Y. S., Matte, A., and Cygler, M. (2001) Biochemistry 40, 2359-2372[CrossRef][Medline] [Order article via Infotrieve]
50. Capila, I., Wu, Y., Rethwisch, D. W., Matte, A., Cygler, M., and Linhardt, R. J. (2002) Biochim. Biophys. Acta 1597, 260-270[Medline] [Order article via Infotrieve]
51. Jedrzejas, M. J., Mello, L. V., De, Groot, B. L., and Li, S. (2002) J. Biol. Chem. 277, 28287-28297[Abstract/Free Full Text]
52. Nankai, H., Hashimoto, W., Miki, H., Kawai, S., and Murata, K. (1999) Appl. Environ. Microbiol. 65, 2520-2526[Abstract/Free Full Text]
53. Yoon, H.-J., Hashimoto, W., Miyake, O., Murata, K., and Mikami, B. (2001) J. Mol. Biol. 307, 9-16[CrossRef][Medline] [Order article via Infotrieve]


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