Crystal Structure of Exotype Alginate Lyase Atu3025 from Agrobacterium tumefaciens*

Alginate, a major component of the cell wall matrix in brown seaweeds, is degraded by alginate lyases through a β-elimination reaction. Almost all alginate lyases act endolytically on substrate, thereby yielding unsaturated oligouronic acids having 4-deoxy-l-erythro-hex-4-enepyranosyluronic acid at the nonreducing end. In contrast, Agrobacterium tumefaciens alginate lyase Atu3025, a member of polysaccharide lyase family 15, acts on alginate polysaccharides and oligosaccharides exolytically and releases unsaturated monosaccharides from the substrate terminal. The crystal structures of Atu3025 and its inactive mutant in complex with alginate trisaccharide (H531A/ΔGGG) were determined at 2.10- and 2.99-Å resolutions with final R-factors of 18.3 and 19.9%, respectively, by x-ray crystallography. The enzyme is comprised of an α/α-barrel + anti-parallel β-sheet as a basic scaffold, and its structural fold has not been seen in alginate lyases analyzed thus far. The structural analysis of H531A/ΔGGG and subsequent site-directed mutagenesis studies proposed the enzyme reaction mechanism, with His311 and Tyr365 as the catalytic base and acid, respectively. Two structural determinants, i.e. a short α-helix in the central α/α-barrel domain and a conformational change at the interface between the central and C-terminal domains, are essential for the exolytic mode of action. This is, to our knowledge, the first report on the structure of the family 15 enzyme.

Carbohydrate-active enzymes such as glycoside hydrolases, polysaccharide lyases, glycosyl transferases, and carbohydrate esterases are categorized into more than 200 families based on amino acid sequences in the Carbohydrate-Active enZYmes (CAZy) data base (1). Polysaccharide lyases (PLs) 2 are classified into 21 PL families and they play an important role in degrading acidic polysaccharides such as polygalacturonan, rhamnogalacturonan, alginate, chondroitin, hyaluronan, heparin, heparan, and xanthan. These lyases commonly recognize uronic acid residues in polysaccharides, catalyze ␤-elimination, and produce unsaturated saccharides with CϭC double bonds at nonreducing terminal uronate residues.
The crystal structures of PLs in 18 families have been determined, and the structure and functional relationships of enzymes such as pectin lyase in family PL-1 (14); pectate lyases in families PL-2, PL-3, and PL-10 (15)(16)(17); alginate lyases in families PL-5, PL-7, and PL-14 (13,18,19); rhamnogalacturonan lyases in families PL-4 and PL-11 (20,21); chondroitin lyases in families PL-6 and PL-8 (22); hyaluronan lyases in families PL-8 and PL-16 (23,24); heparin lyases in families PL-13 and PL-21 (25,26); and xanthan lyase in family PL-8 (12) have been demonstrated. On the other hand, no structural information on family PL-15 alginate lyases has been accumulated, and the enzyme mechanism for releasing unsaturated monosaccharides from the polysaccharide main chain remains to be clarified. This article deals with the determination of crystal structures of family PL-15 Atu3025 and its complex with unsaturated alginate trisaccharide through x-ray crystallography, and identification of the active site structure responsible for substrate recognition and exolytic reaction.
Enzyme and Protein Assays-The assay for alginate lyase was conducted as follows. The enzyme was incubated at 30°C for 5 min in a reaction mixture (1 ml) consisting of 0.05% sodium alginate and 50 mM Tris-HCl (pH 7.5). The reaction mixture was heated at 100°C for 5 min to terminate the reaction, and the amount of unsaturated uronic acids produced in the reaction mixture was determined by the thiobarbituric acid method (27). In each assay, the enzyme was stable and the reaction products were confirmed to increase proportionally with time and enzyme concentration in the reaction mixture. Enzyme activity was determined by measuring the increase in absorbance at 548 nm, resulting from the condensation of ␤-formylpyruvic acid with thiobarbituric acid (⑀ ϭ 2.9 ϫ 10 4 /M cm). One unit of enzyme activity was defined as the amount of enzyme required to liberate 1 mol of ␤-formylpyruvic acid per minute at 30°C. For site-directed mutants, the enzyme assay was done as follows. To estimate the specific activity of the mutants, a large amount of the enzyme was incubated at 30°C for 60 min in a reaction mixture (1 ml) consisting of 0.05% unsaturated ␤-Dmannuronate disaccharide (⌬MM) and 50 mM Tris-HCl (pH 7.5). One unit of enzyme activity was defined as the amount of enzyme required to degrade 1 mol of ⌬MM/min at 30°C. Quantification of the residual ⌬MM was performed using high performance liquid chromatography. An Ä KTA purifier (GE Healthcare) equipped with a Superdex Peptide 10/300 GL was used for high performance liquid chromatography analysis. Reaction products were eluted at a flow rate of 0.5 ml/min with 10 mM potassium phosphate (pH 7.0) and detected by measuring the absorbance at 235 nm on the basis of CϭC double bonds in the ⌬MM. Protein content was determined by the Bradford method (28), with bovine serum albumin as the standard.
Expression and Purification of Atu3025 and Its Derivative with Selenomethionine-Protein expression and purification of Atu3025 were conducted as described previously (5). The DNA fragment encoding the complete sequence of the Atu3025 gene (GenBank number AE007870) with the original stop codon was ligated with pET21b. Thus, the polypeptide expressed from the resultant plasmid (pET21b/Atu3025) was not fused to the histidine-tagged sequence. An overexpression system for the Atu3025 derivative with selenomethionine (SeMet Atu3025) was also constructed in Escherichia coli cells. Unless specified otherwise, all purification procedures were performed at 0 -4°C. E. coli B834(DE3) cells harboring pET21b/Atu3025 were grown in 6 liters of minimal medium (29) (1.0 liter/flask) implemented with 25 mg of selenomethionine/liter. The cells were collected by centrifugation at 6,000 ϫ g for 5 min, washed with 20 mM Tris-HCl (pH 7.5) supplemented with 0.5 mM dithiothreitol (DTT), and resuspended in the same buffer. After ultrasonic disruption of cells at 9 kHz for 20 min, the clear solution obtained on centrifugation at 20,000 ϫ g for 20 min was used as the cell extract. After an overnight dialysis against 20 mM Tris-HCl (pH 7.5) supplemented with 0.5 mM DTT, the cell extract was applied to a DEAE-Toyopearl 650M column (2.5 ϫ 10 cm) previously equilibrated with 20 mM Tris-HCl (pH 7.5) supplemented with 0.5 mM DTT. The absorbed proteins were eluted with a linear gradient of NaCl (0 -700 mM) in 20 mM Tris-HCl (pH 7.5) supplemented with 0.5 mM DTT (200 ml), and 4-ml fractions were collected every 4 min. Fractions containing SeMet Atu3025, which were eluted between 300 and 500 mM NaCl, were combined and dialyzed overnight against 20 mM Tris-HCl (pH 7.5) supplemented with 0.5 mM DTT. The dialysate was applied to a HiLoad 26/10 Q-Sepharose High Performance column (2.6 ϫ 10 cm) previously equilibrated with 20 mM Tris-HCl (pH 7.5) containing 300 mM NaCl and 0.5 mM DTT. The absorbed proteins were eluted with a linear gradient of NaCl (300 -500 mM) in 20 mM Tris-HCl (pH 7.5) supplemented with 0.5 mM DTT (150 ml), and 4-ml fractions were collected every 1 min. Fractions containing SeMet Atu3025, which were eluted between 380 and 420 mM NaCl, were combined and applied to a HiLoad 16/60 Superdex 200 pg column (1.6 ϫ 60 cm) previously equilibrated with 20 mM Tris-HCl (pH 7.5) containing 0.15 M NaCl and 0.5 mM DTT. The proteins were eluted with the same buffer (120 ml), and 2-ml fractions were collected every 2 min. These two purification steps, such as Q-Sepharose High Performance and Superdex 200 pg, were done by the using Ä KTA purifier at room temperature. Fractions containing SeMet Atu3025 were combined and dialyzed overnight against 20 mM Tris-HCl (pH 7.5), and used as the purified SeMet Atu3025. The purified SeMet Atu3025 had the same N-terminal amino acid sequence as Atu3025, with a calculated molecular mass of 87,871 Da from the predicted amino acid sequence (776 residues).
Crystallization and X-ray Diffraction-Atu3025 (20 mg/ml) was crystallized by sitting-drop vapor diffusion on Linbro tissue culture plates as described previously (30). The condition most suitable for Atu3025 crystallization was determined to be a mixture of 80 mM Tris-HCl (pH 8.5), 24% (w/v) polyethylene glycol 4,000, 0.16 M magnesium chloride, and 20% (v/v) glycerol. The SeMet Atu3025 was crystallized under the same condition as the native Atu3025 crystal. A crystal for the site-directed mutant (H531A) in complex with alginate trisaccharide (⌬GGG) was prepared by sitting drop vapor diffusion on Intelli plates (Veritas). The droplet (2 l) was prepared by mixing 1 l of the protein solution with 1 l of the reservoir solution and equilibrated at 20°C against 0.05 ml of reservoir solution. The H531A/⌬GGG complex crystal appeared through sparse matrix screening using a Crystal Screen Cryo kit from Hampton Research in about six months. The condition most suitable for H531A/⌬GGG crystallization was determined to be a mixture of 85 mM HEPES-Na (pH 7.5), 17% (w/v) polyethylene glycol 4,000, 8.5% (v/v) isopropyl alcohol, 15% (v/v) glycerol, and 10 mM ⌬GGG. The crystal on a nylon loop (Hampton Research) was placed directly in a cold nitrogen gas stream at Ϫ173°C, and x-ray diffraction images of the crystal were collected at Ϫ173°C under a nitrogen gas stream with a Jupiter 210 CCD detector and synchrotron radiation of wavelength 1.000 Å at the BL-38B1 station of SPring-8 (Hyogo, Japan). During data collection for single-wavelength anomalous diffraction analysis using the SeMet Atu3025 crystal, the synchrotron radiation wavelength was adjusted to 0.9792 Å. The wavelength was defined from XAFS measurement of the SeMet Atu3025 crystal. The 240 diffraction images (total 240°) with 1.0°oscillation were collected as a consecutive series of datasets. Diffraction data were processed using the HKL2000 program package (31). Data collection statistics are summarized in Table 1.
Structure Determination and Refinement-The crystal structure of Atu3025 was solved by the single-wavelength anomalous diffraction method using the SeMet Atu3025 crystal. Selenium sites and initial phasing were determined by the SOLVE program (32). Density modifications (solvent flattening and histogram matching) were done using the program RESOLVE (33). The initial model was built to consist of 2,812 amino acid residues without side chains through manual modeling and refinement. The Coot program (34) was used for the modification of the initial model. The 50.0 -2.10-Å resolution dataset was truncated with the CCP4 program package (35) and used for subsequent refinement. The H531A/⌬GGG crystal was solved by molecular replacement using the Molrep program in the CCP4 program package with the native Atu3025 structure as a reference model. Initial rigid body refinement and several rounds of restrained refinement against the dataset were done using the Refmac5 program (36). Water molecules were incorporated where the difference in density exceeded 3.0 above the mean and the 2F o Ϫ F c map showed a density of more than 1.0 . The structure of the enzyme-sugar complex was refined using the Coot program with the parameter file for gulurononic acid at the PRODRG site. Protein models were superimposed, and their root mean square (r.m.s.) deviations were determined with the LSQKAB program (37), a part of the CCP4 program package. Final model quality was checked with the PROCHECK program (38). Ribbon plots were prepared using the PyMOL program (39). Coordinates used in this work were taken from the RCSB Protein Data Bank (PDB) (40).

RESULTS AND DISCUSSION
Structure Determination and Refined Model Quality-Data collection and refinement statistics are summarized in Table 1. The refined model from the Atu3025 crystal includes 1,527 amino acid residues, 1,067 water molecules, two chloride ions for two protein molecules, designated as Atu3025 molecules A and B, in an asymmetric unit. 10 amino acid residues ( 1 MRP-SAPAISR 10 ) in molecule A and 11 amino acid residues ( 1 MRP-SAPAISRQ 11 ) in molecule B N-terminal, and 2 amino acid residues ( 775 QF 776 ) C-terminal in each molecule could not be assigned in the 2F o Ϫ F c map possibly due to disorder. The final overall R-factor for the refined model was 18.3% with 95,821 unique reflections within a 40.9 -2.11-Å resolution range. The final overall free R-factor calculated with randomly selected 5% reflection was 22.4%. Based on theoretical curves in the plot calculated according to Luzzati (41), the absolute positional error was estimated to be 0.23 Å at a resolution of 2.11 Å. Ramachandran plot analysis (42), in which the stereochemical correctness of the backbone structure is indicated by (, ) torsion angles (43), shows that 97.1% of nonglycine residues are in the favored region and 2.9% are in the allowed regions. Four cispeptides were observed between Glu 27 and Pro 28 , Asn 37 and Pro 38 , His 139 and Pro 104 , and Tyr 349 and Ser 350 residues in each molecule.
Overall Structure-The overall structure (Fig. 2, A-C) and topology of the secondary structure elements (Fig. 2D) indicate that alginate lyase Atu3025 consists of three globular domains: N-terminal small ␤-sheet domain, central ␣-domain, and C-terminal ␤-sheet domain. The N-terminal small ␤-sheet domain consists of 106 amino acid residues from Gln 11 to Ile 116 and is composed of two antiparallel ␤-sheets consisting of seven ␤-strands (S1 to S7). The central ␣-domain consists of 364 amino acid residues from Arg 128 to Asp 491 and is constituted by 15 ␣-helices (HA1 to HA12 and H1 to H3). The three helices,

JOURNAL OF BIOLOGICAL CHEMISTRY 24523
Conformational Change by Substrate Binding-To identify the catalytic residues and structural determinants for the exolytic mode of action in Atu3025, a crystal of H531A/⌬GGG was prepared. His 531 comprises a component of the active pocket, and the mutant H531A shows a significantly lower activity than the wild-type enzyme. Data collection and refinement statistics are summarized in Table 1. The crystal structure of H531A/ ⌬GGG determined at 2.99-Å resolution consists of 766 amino acid residues, which are derived from an Atu3025 monomer, and one ⌬GGG molecule in an asymmetric unit. The ⌬GGG molecule was bound to the deep pocket between central ␣/␣barrel and C-terminal ␤-sheet domains (Fig. 3A). The nonreducing terminal and central residues of ⌬GGG are well fitted in the electron density map with average B-factors of 42.0 and 47.4 Å 2 , whereas the reducing terminal residue are poorly fitted (average B-factor of 55.5 Å 2 ) (Fig. 3B).
The r.m.s. deviation between ligand-free Atu3025 and H531A/⌬GGG complex structures was calculated as 1.59 Å for all residues (764 C␣ atoms), indicating that significant conformational changes occur between protein structures with and without ⌬GGG. Fig. 3C shows the superimposition of ligandfree Atu3025 and H531A/⌬GGG. The protein structures have been aligned by use of only the central ␣/␣ barrel domain (amino acid residues from Arg 128 to Asp 491 ). The C-terminal ␤-sheet domain of Atu3025 can adopt two different conformations through a rigid-body rotation of ϳ10°. This hinge bending motion was observed in the plane formed at the interface of the ␣/␣-barrel and C-terminal ␤-sheet domains. ⌬GGG is included in the pocket-like active center, causing the conformational rearrangement (Fig. 3E). Several amino acid residues such as Arg 193 , Trp 198 , Arg 199 , Tyr 202 , Glu 254 , His 311 , Arg 314 , His 364 , Tyr 365 , Ser 418 , Phe 462 , Tyr 463 , Trp 467 , His 531 , Tyr 555 , and Phe 558 are arranged around the active pocket. Structure comparison of ligand-free Atu3025 and H531A/⌬GGG demonstrates that the side chain of Phe 462 is turned away and interacts with Ser 418 , which is positioned at the opposite side of the active pocket (Fig.  3, D and E), suggesting that Phe 462 plays an important role in forming the pocket. Enzyme flexibility as seen in Atu3025 has been reported in family PL-8 hyaluronate lyase from Streptococcus pneumoniae (47); this flexibility is essential to construct the active center for enzymes with an ␣/␣-barrel ϩ anti-parallel ␤-sheet topology.
Active Site Structure-The crystal structure of H531A/ ⌬GGG revealed the binding mode of the substrate ⌬GGG molecule to the active cleft (Fig. 3F). Subsites are labeled so that Ϫn represents the nonreducing terminus and n the reducing terminus, and cleavage occurs between the Ϫ1 and ϩ1 sites (48). Atu3025 depolymerizes alginate exolytically, releasing monosaccharides from the nonreducing end of the polymer through a ␤-elimination reaction. Based on the location of the nonreducing terminus residue of ⌬GGG in the deepest spot of the active pocket, the saccharides are considered to be positioned at subsites Ϫ1, ϩ1, and ϩ2. Therefore, the constituent guluronic acid residues of ⌬GGG are designated as ⌬GϪ1, Gϩ1, and Gϩ2 from the nonreducing end. Several amino acid residues are responsible for binding to ⌬GGG (Table 3, Fig. 3F). ⌬GϪ1 is accommodated at subsite Ϫ1 through seven hydrogen bonds by four residues, Arg 199 , Tyr 202 , His 364 , and Tyr 555 , and 26 carboncarbon (C-C) contacts by six residues, Trp 198 , Arg 199 , Ser 418 , Phe 462 , Trp 467 , and Tyr 555 . Residue Trp 467 interacts with the pyranose ring of ⌬GϪ1 through a stacking interaction by 12 C-C contacts. At subsite ϩ1, seven hydrogen bonds by five residues, Glu 254 , Ser 310 , His 311 , Arg 314 , and Tyr 365 , and 23 C-C contacts by four residues, Trp 198 , His 311 , Tyr 365 , and Phe 558 , are formed between Gϩ1 and protein. At subsite ϩ2, Gϩ2, the reducing terminal residue of ⌬GGG, is hydrogen bonded to three residues, Ser 310 , His 311 , and Ser 530 , and there are 14 C-C contacts by three residues, Ser 310 , His 311 , and Phe 558 . Phe 558 is partially parallel to the pyranose ring of both Gϩ1 and Gϩ2, indicating that the residue undergoes a partially stacking interaction with the sugar ring of these two residues. Positively charged residues such as Arg 199 and Arg 314 are supposed to be crucial for binding acidic polysaccharides and/or neutralizing the negative charge of the carboxyl groups in ⌬GϪ1 and Gϩ1. A multiple sequence alignment of family PL-15 exotype alginate lyases using the ClustalW program is shown in Fig. 4. Atu3025 shows a significant sequence identity of 55.0% with  A1-IV from Sphingomonas sp. A1. The residues outlined above are completely conserved between the two.
Structural Insights into the Catalytic Reaction and Exolytic Mode of Action-The catalytic reaction of polysaccharide lyases is divided into three steps as follows (49): (i) positively charged residues stabilize or neutralize the negative charge on the C-6 carboxylate anion, (ii) a general base catalyst abstracts the proton from C-5 of the uronic acid residue, and (iii) a general acid catalyst donates the proton to the glycoside bond to be cleaved. To identify the role of Atu3025 residues in the active site, we constructed the mutants, R199A, H311A, Y365F, W467A, and H531A, in which Arg 199 , His 311 , Tyr 365 , Trp 467 , and His 531 were replaced by Ala, Ala, Phe, Ala, and Ala, respectively. These residues for mutation were supposed to play an important role in recognizing the acidic polysaccharide and/or catalyzing the enzyme reaction based on the structure of H531A/⌬GGG. The mutants were overexpressed in E. coli cells and purified to homogeneity. Their specific activities were determined as follows (Table 4). In all mutants, especially H311A and W467A, the enzyme activity significantly decreased. Various amino acid residues are reported to act as a catalytic base or acid in PL families. Although Arg in families PL-1 and PL-10 (17), Lys in family PL-9 (50), Tyr in families PL-5 (19), and PL-8 (22), and His in family PL-7 (18) function as a catalytic base, Tyr in families PL-5 (19), PL-7 (18), and PL-8 (22) function as a catalytic acid. In the family PL-5 alginate lyase A1-III from Sphingmonas sp. strain A1, Tyr 246 is considered to act as both a catalytic base and acid (19). On the other hand, the family PL-7 alginate lyase A1-IIЈ from Sphingmonas sp. strain A1 adopts His 191 and Tyr 284 as the catalytic base and acid (18), respectively. These catalytic His and Tyr residues well conserved in families PL-5 and PL-7 correspond to His 311 and Tyr 365 in Atu3025, respectively. Based on the active site structure of H531A/ ⌬GGG (Fig. 3F), the distance between His 311 and C5 of Gϩ1 was calculated as 3.4 Å, and between Tyr 365 and O4 of Gϩ1 was less than 4.0 Å. Judging from the significant decrease in the specific activity of H311A and Y365F (Table 4), it is plausible that His 311 and Tyr 365 function as a catalytic base and acid, respectively (Fig. 5), as seen in A1-IIЈ (18). Tyr 555 is also a candidate for a catalytic acid because the distance between Tyr 555 and O4 of Gϩ1 was determined as 4.0 Å (Fig. 5), although the Tyr residue is not conserved in family PL-15 alginate lyases (Atu3025, A1-IV, and A1-IVЈ), so far characterized. His 531 is located close enough to interact with the carbohydrate group of Gϩ1, when the residue retains the similar side chain conformation as in the ligand-free Atu3025 (Fig. 3D). Due to this struc- strain A1 (GenPept accession number BAB03319). Amino acid sequences were aligned using the ClustalW program. Identical and similar amino acid residues in these alginate lyases are denoted by asterisks and dots, respectively. Secondary structure elements of Atu3025 are as shown above. Amino acid residues interacting with ⌬GGG are highlighted in black. tural feature and low activity of H531A (Table 4), His 531 is supposed to play a major role in stabilizing or neutralizing the negative charge on the carboxylate anion of Gϩ1 (Fig. 5). The reduced activity of R199A indicates that Arg 199 also functions as a stabilizer on the carboxyl group of ⌬G-1 as described above. The intrinsic function of each possible catalytic residue should be analyzed through structure determination of the enzyme and substrate complex at higher resolution. Unlike other polysaccharide lyases, family PL-15 enzymes such as Atu3025 and A1-IV can release unsaturated monosaccharides from polysaccharide. The crystal structure of H531A/ ⌬GGG shows a part of the structural determinants for the exolytic mechanism. The distinguishing characteristic of Atu3025, namely a pocket-like structure, is observed in the environment surrounding subsite Ϫ1. The pocket is formed by two structural determinants, i.e. a conformational change at the interface between the central and C-terminal domains and the presence of a short ␣-helix H3 (Fig. 3, D and E). Tyr 555 and Phe 558 in the C-terminal ␤-sheet domain and Phe 462 and Trp 467 around H3 are actually involved in accommodating the substrate (Table 3). At subsite Ϫ1, seven hydrogen bonds and 26 C-C contacts are formed between the enzyme and sugar. In particular, Trp 467 forms 12 C-C contacts with ⌬G-1 and the mutant W467A showed the significant reduction in the specific activity compared with the wild-type (Table 4), indicating that the residue is essential for recognition of a nonreducing terminal residue of the substrate. This pocket-like structure is not observed in other polysaccharide lyases, even in the family PL-8 and PL-20 enzymes with the ␣/␣-barrel ϩ anti-parallel ␤-sheet as a basic scaffold. In family PL-8 hyaluronate lyase from S. pneumoniae (47), a pocket-like structure is absent in its active site although the conformational change caused by substrate binding has been reported. Some polysaccharide lyases such as rhamnogalacturonan lyase YesX from B. subtilis and alginate lyase vAL-1 from Chorella virus have been reported to release the unsaturated disaccharide exolytically. Their previous crystallographic studies demonstrated that the steric hindrance at one end of the active cleft and the sugar-binding affinity level in subsites are crucial for the exolytic mode of action of YesX (51) and vAL-1 (13), respectively. Therefore, it is reasonable to presume that the pocket-like structure is a characteristic of the family PL-15 exotype alginate lyases, which release unsaturated monosaccharides as a sole product, and is essential for recognizing the nonreducing terminal residues of substrate. Based on the above described results, Atu3025 probably adopts the five-step cycle to exhibit the exolytic mode of action as follows: (i) alginate approaches the active site of the "open form" enzyme, (ii) the terminal saccharide of the polymer is accommodated at subsite Ϫ1 through the conformational modulation (open/close), (iii) the glycosidic linkage is cleaved in the "closed form," (iv) the enzyme takes the open form to release the product, and (v) the remaining substrate slides along the active site through the conformational modulation (open/close).
Conclusions-This is, to our knowledge, the first report on the structural determination of the family PL-15 enzyme and establishment of a new structural category in alginate lyases involving an ␣/␣-barrel ϩ anti-parallel ␤-sheet-fold. The active site structure of H531A/⌬GGG and the subsequent site-directed mutational analysis suggest that His 311 and Tyr 365 function as a catalytic base and acid, respectively. Additionally, a pocket-like structure of Atu3025, which is formed by the conformational change at the interface between the central and C-terminal domains, is essential for the exolytic mode of action involved in releasing unsaturated monosaccharides from the polysaccharide main chain.