Journal of Bacteriology, May 2002, p . 2399-2403, Vol . 184, No . 9

Novel Carbohydrate-Binding Module of ß-1,3-Xylanase from a Marine Bacterium, Alcaligenes sp . Strain XY-234

Fumiyoshi Okazaki,¹ Yutaka Tamaru,¹ Shinnosuke Hashikawa,^1, Yu-Teh Li,² and Toshiyoshi Araki^1*

Department of Life Science, Faculty of Bioresources, Mie University, 1515 Kamihama, Tsu, Mie 514-8507, Japan,¹ Department of Biochemistry, Tulane University School of Medicine, New Orleans, Louisiana 70112²

Received 21 August 2001/ Accepted 7 February 2002

Many glycoside hydrolases (GHs), particularly those involved in the hydrolysis of polysaccharides, frequently display a modular structure featuring a catalytic module (or unit) attached to one or several ancillary noncatalytic modules (NCMs), whose precise functions are often unknown (7) . The NCMs of GHs can be identified in many cases by sequence comparisons (9) . As their catalytic modules can be classified into several families based on amino acid sequence similarities, the NCMs also form a number of different families . A classification of such modules, independent and complementary to that of catalytic modules, is needed for a more meaningful description of GHs . For the enzymes acting on polysaccharides, such as cellulose, chitin, starch, ß-1,3-glucan, and ß-1,4-xylan, several NCMs have been shown to promote the attachment of the enzyme to the polysaccharide matrix, thereby facilitating the degradation of crystalline polysaccharides .

Alcaligenes sp . strain XY-234 is a marine bacterium that secretes an extracellular ß-1,3-xylanase into the growth medium in the presence of ß-1,3-xylan . We have previously reported the purification, properties, and N-terminal amino acid sequence of the enzyme from this organism (2) . We have also cloned and sequenced a ß-1,3-xylanase gene (txyA) from another bacterium, Vibrio sp . strain XY-214 (3), but have not yet characterized the unknown domain of TxyA at its C-terminal region .

To further elucidate the structure and the molecular architecture of ß-1,3-xylanases, we have cloned and sequenced a new ß-1,3-xylanase gene from Alcaligenes sp . strain XY-234 . The present paper provides data that the txyA gene belongs to family 26 of the GHs and that the enzyme encoded by this gene can specifically bind to and degrade ß-1,3-xylan . We have also identified and characterized a carbohydrate-binding module (CBM) in TxyA, which should be classified as a novel CBM .

Bacterial strains and plasmids. Alcaligenes sp . strain XY-234 isolated from sea mud was grown as described previously (2) and used as the source of chromosomal DNA . Escherichia coli DH5 (Stratagene) and E . coli BL21(DE3) (Novagen) were the host strains for cloning experiments and for the production of recombinant proteins, respectively . pBluescriptII (Stratagene) was used as a cloning vector, and pET21a and pET22b (Novagen) were used as the expression vectors .

Recombinant DNA techniques. Chromosomal DNA from Alcaligenes sp . strain XY-234 was isolated by the method of Saito et al . (18) . Plasmid DNA was purified with the Wizard Plus miniprep DNA purification system (Promega) . Restriction endonucleases were purchased from Takara (Kyoto, Japan) and Toyobo (Tokyo, Japan) and used in accordance with the manufacturer's specifications . DNA ligation was carried out by the Ligation Kit Ver.2 (Takara) . DNA fragments were recovered after electrophoresis with a GeneClean II kit (Bio 101 Inc.) .

To obtain the txyA gene from Alcaligenes sp . strain XY-234, Southern hybridization analysis was carried out for restriction enzyme digestion fragments of chromosomal DNA from strain XY-234 using the ß-1,3-xylanase gene of Vibrio sp . strain XY-214 (3) labeled with AlkPhos Direct (Pharmacia, Uppsala, Sweden) as a probe . The 5-kb ClaI and 2-kb NheI digestion fragments that hybridized with the probe were ligated into the dephosphorylated ClaI and XbaI sites of pBluescriptII SK (-), respectively . These ligation mixtures were incubated at 4°C overnight and then transformed into competent E . coli DH5 by the method of Sambrook et al . (20) .

TxyA activity and protein assays. The enzyme solution (0.1 ml) was added to a mixture of 0.4 ml of 200 mM 2-morpholinoethanesulfonic acid (MES)-NaOH buffer (pH 7.0) and 0.5 ml of 1% ß-1,3-xylan solution . After incubating at 37°C for 10 min, the reducing sugar generated was measured by the Somogyi-Nelson method (22) and expressed as xylose . One unit of enzyme activity was defined as the amount of enzyme that liberated 1 µmol of D-xylose per min under the above conditions .

Protein concentrations were measured by the method of Bradford (6) using bovine serum albumin as a standard .

Protein purification. Native TxyA from Alcaligenes sp . strain XY-234 was purified as described previously (2) . To express the recombinant enzymes, E . coli BL21(DE3) was grown at 37°C in 3 liters of Luria-Bertani (LB) medium (20) supplemented with 100 µg of ampicillin per ml . When the A₆₀₀ reached 0.6, the culture was cooled at 25°C, and isopropyl-ß-D-thiogalactopyranoside was added to a final concentration of 1 mM . Incubation was continued for another 3 h at 25°C, and the periplasmic fraction of the harvested cells was used as the crude enzyme . The crude enzyme solution derived from the recombinant pET21a was purified by a combination of Q Sepharose FF and Ether-Toyopearl 650S column chromatographies . His-tagged proteins from the recombinant pET22b were purified by HiTrap chelating (Pharmacia) .

SDS-PAGE, zymogram, and Western blot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 12.5% polyacrylamide gel by the method of Laemmli (13) . After electrophoresis, the gel was stained with Coomassie brilliant blue R . A low-molecular-weight SDS calibration kit (Pharmacia) was used as the standard . Zymogram was carried out by the method of Beguin (5) . Western blot (immunoblot) analysis was performed by using a polyclonal anti-TxyA antiserum raised against ß-1,3-xylanase (TxyA) of Vibrio sp . strain XY-214 .

TLC analysis. Purified enzyme was incubated with 1% polysaccharide solutions containing ß-1,3-xylan, ß-1,4-xylan, curdlan, CMC, ß-1,4-mannan, or glucomannan at 37°C . The reaction products were separated on a thin-layer chromatography (TLC) plate (Merck, Darmstadt, Germany) with a solvent system consisting of n-butanol-acetic acid-water (10:5:1) . For detection of the products, the plates were sprayed with the diphenylamine-aniline-phosphate reagent (4) and baked for 10 min at 100°C .

Determination of the CBM-ß-1,3-xylan dissociation constant and the ß-1,3-xylan-binding capacity. For quantitative analysis, the reaction mixture for the ß-1,3-xylan-binding assay contained 1 mg of ß-1,3-xylan and appropriate amounts of protein (100 to 500 µg/ml [total protein]) in a final volume of 1 ml of 50 mM Tris-HCl buffer (pH 7.5) . After the mixture was incubated at 4°C for 1 h with slow vertical rotation (10 rpm), ß-1,3-xylan was removed by centrifugation, and the concentration of unbound protein ([P], micromolar) in the supernatant was measured by the Bradford method (6) . The bound protein concentration ([PC], in micromoles per gram of ß-1,3-xylan) was determined by subtracting [P] from the total protein concentration . All assays were carried out in triplicate . Adsorption parameters were obtained by using the equation of Sakoda and Hiromi (19), [PC] = [P] [PC]_max/(K_d + [P]), where K_d (micromolar) and [PC]_max (micromoles per gram of ß-1,3-xylan) are the equilibrium dissociation constant and maximum amount of protein bound, respectively .

For qualitative analysis, the assay tubes contained 0.5 mg of ß-1,3-xylan and 2 µg of protein in 0.5 ml of 50 mM Tris-HCl buffer (pH 7.5) . After incubation at 4°C for 1 h with vertical rotation, the assay tubes were centrifuged to sediment the ß-1,3-xylan . After removing the buffer, the pellets were washed three times with 1 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 1 M NaCl . The pellets were then resuspended in 20 µl of SDS sample buffer and analyzed by SDS-PAGE .

Determination of binding to other polysaccharides. Avicel (ß-1,4-glucan), curdlan (ß-1,3-glucan), ß-1,4-mannan, and ß-1,4-xylan were tested to determine whether they bind to CBM protein . The method used was the same as that used in determining binding to ß-1,3-xylan .

Nucleotide sequence accession number. The nucleotide sequence data reported in this paper have been submitted to GenBank under accession no . AB039953 .

The deduced amino acid sequence encoded by the txyA gene was compared with protein sequences in the GenBank and EMBL databases as well as those in the literature . Based on the amino acid sequence similarity, TxyA was predicted to consist of a signal sequence followed by a catalytic domain, a linker sequence, and an unknown domain at the C terminus . The whole amino acid sequence of TxyA revealed 84.6% identity and 92.3% similarity with TxyA of Vibrio sp . strain XY-214 (3) .

The N-terminal region (residues 23 to 348) of strain XY-234 TxyA contained a putative catalytic domain which was homologous to a member of the family 26 ß-mannanases (11), i.e., Thermotoga neapolitana ManA (39.0% identity and 57.7% similarity among 322 amino acids; accession no . U58632) (Fig . 1A), Caldicellulosiruptor saccharolyticus ManA (24.7% identity and 45.2% similarity among 93 amino acids; accession no . U39812) (8), Clostridium thermocellum ManA (26.7% identity and 46.5% similarity among 101 amino acids; accession no . AB044406) (10) and Man26B (26.5% identity and 44.6% similarity among 83 amino acids; accession no . AJ242666), Dictyoglomus thermophilum ManA (25.8% identity and 44.9% similarity among 89 amino acids; accession no . AF013989), and Rhodothermus marinus ManA (31.1% identity and 48.9% similarity among 90 amino acids; accession no . X90947) (16) . In particular, the putative catalytic domain of TxyA from strain XY-234 showed significant homology with ß-1,3-xylanases such as Vibrio sp . strain XY-214 TxyA (88.8% identity and 96.3% similarity among 322 amino acids; accession no . AB029043) (3) and Pseudomonas sp . strain ND137 AxnB (57.2% identity and 71.4% similarity among 332 amino acids; accession no . AB063257) (Fig . 1A) .

 
As shown in Fig 1B, the C-terminal region (residues 376 to 469) of TxyA from strain XY-234 shows 77.4% identity and 87.1% similarity among 93 amino acids with Vibrio sp . strain XY-214 TxyA (3) . It also shows 35.4% identity and 57.3% similarity among 82 amino acids with Pseudomonas sp . strain ND137 AxnB . The function of the C-terminal domain is still unknown . The glycine-rich region (residues 349 to 375), containing one array of DGG and six repeats of DNGG, is similar to some linker regions of ß-1,4-xylanase XylD from Cellulomonas fimi (15) and ß-1,4-xylanases STX-I and STX-II from Streptomyces thermoviolaceus OPC-520 (25) . Interestingly, all these enzymes possess a CBM downstream of the linker region .

CBMs are discrete protein modules found in a large number of carbohydrolases and a few nonhydrolytic proteins such as cellulosomal scaffoldin proteins (21) . In rare instances, independent putative CBMs have also been described (24) . To date, about 200 different CBMs have been identified and classified into 29 families according to their similarities in aminoacid sequence (http://afmb.cnrs-mrs.fr/pedro/CAZY/index.html) .

Biochemical properties of rTxyA. The native TxyA and recombinant TxyA (rTxyA) were purified by successive column choromatographies as described in Materials and Methods . The yields of the purified TxyA and rTxyA were 0.11 mg/liter and 15.7 mg/liter of culture medium, respectively . As shown in Fig . 2A and B, both the native and recombinant enzymes moved as a single protein band in SDS-PAGE (59,000 Da) and exhibited ß-1,3-xylanase activities in a zymogram . The molecular mass of the purified rTxyA was found to be very close to that of the native enzyme . By Western blot analysis, the native and recombinant enzymes showed cross-reactivity with anti-TxyA antibodies prepared against ß-1,3-xylanase (TxyA) of Vibrio sp . strain XY-214 (Fig . 2C) . Thus, both enzymes were proved to be immunologically similar to TxyA of strain XY-214 . The rTxyA of strain XY-234 exhibited an optimum pH of 7.0 and was stable in the pH range of 5.0 to 11 at 4°C for 24 h . The optimum temperature for rTxyA activity at pH 7.0 was found to be 40°C, and the activity was stable at 40°C for 10 min .

 
The activity pattern of rTxyA was determined with several polysaccharides . The reaction solution containing 9.4 µg of purified rTxyA and 1% polysaccharide was incubated for 24 h at 37°C . TLC analysis revealed that rTxyA hydrolyzed ß-1,3-xylan to produce a series of xylooligosaccharides, with xylobiose and xylotriose as the main products . The enzyme did not cleave CMC, curdlan, glucomannan, ß-1,4-mannan, or ß-1,4-xylan . It also did not hydrolyze p-nitrophenyl-ß-D-xyloside or p-nitrophenyl-- and ß-D-mannoside (data not shown) .

Identification of CBM. To clarify whether the unknown domain at the C-terminal region of TxyA is a CBM, we constructed two deletion mutants: pCD, containing the N-terminal region from Leu23 to Ala348, and pCBM, containing the C-terminal region from Thr376 to Gln469, based on the amino acid sequence . By using these truncated proteins and the original rTxyA, we assayed ß-1,3-xylanase activity and ß-1,3-xylan-binding ability according to the procedures described in Materials and Methods . As shown in Fig . 3A, rTxyA exhibited both catalytic activity with and ability to bind ß-1,3-xylan, while the protein expressed by pCD (CD) had catalytic activity but no binding ability and the protein expressed by pCBM (CBM) had binding ability without catalytic activity . Moreover, ß-1,3-xylan-bound protein could not be set free by washing with 1 M NaCl . SDS-PAGE analysis indicating that both rTxyA and CBM could bind to ß-1,3-xylan, but this was not the case for CD (Fig . 3B) . Therefore, it was evident that the unknown domain in TxyA is a CBM and that TxyA consists of two domains, the catalytic domain at the N-terminal region (residues 23 to 348) and the CBM at the C-terminal region (residues 376 to 469) .

 
To compare the function of the proteins with and without the CBM, we determined the enzymatic properties of rTxyA and CD . The kinetic parameters obtained from Lineweaver-Burke plots showed a straight line within experimental error (Table 1) . The K_m value of CD for ß-1,3-xylan was 2.41-fold higher than that of rTxyA, while the difference in K_m value between CD and rTxyA for glycol ß-1,3-xylan was small . The overall catalytic efficiency (k_cat/K_m) of rTxyA for ß-1,3-xylan was 2.14-fold higher than that of CD . These results indicate that the CBM of TxyA contributes to the rate of depolymerization through increased binding to the polymeric substrate .

 
TxyA from Vibrio sp . strain XY-214 was also found to have ß-1,3-xylan-binding ability (unpublished data) . The CBM that has been defined here for TxyA from the Alcaligenes sp . likely has an analog in the TxyA from the Vibrio sp . Therefore, the CBMs of both the Alcaligenes and Vibrio enzymes should be classified into a new CBM family .

Characterization of CBM. Figure 4 shows a typical double reciprocal plot for the binding of pure CBM to ß-1,3-xylan . Within experimental error, the plots were linear, yielding a K_d of about 4.2 µM and [PC] _max of 18.2 µmol of CBM bound per g of ß-1,3-xylan . The latter value corresponds to approximately 221 mg of CBM protein per g of ß-1,3-xylan . The linearity of the plot suggests that there is only one type of CBM-ß-1,3-xylan interaction and that the CBM of TxyA had a high affinity for ß-1,3-xylan .

 
The ability of CBM to bind several polysaccharides, such as Avicel (ß-1,4-glucan), curdlan (ß-1,3-glucan), ß-1,4-mannan, ß-1,3-xylan, and ß-1,4-xylan, was investigated . The CBM did not show measurable binding to these polysaccharides except for ß-1,3-xylan . To determine whether soluble carbohydrates competed with ß-1,3-xylan for the CBM protein, D-xylose, ß-1,3-xylooligosaccharides, and glycol ß-1,3-xylan were included in some assays at four times the weight per volume of ß-1,3-xylan (1 mg of ß-1,3-xylan and 4 mg of soluble carbohydrate per ml of assay mix) . No appreciable differences in K_d or [PC]_max were observed, indicating that these soluble carbohydrates had little or no effect on the binding of CBM to ß-1,3-xylan .

By expression of the individual domains and activity measurements of the protein products, we have demonstrated that the unknown domain at the C terminus represents a novel CBM capable of specifically recognizing ß-1,3-xylosyl linkages . The validity of the assay is supported by the observation that [PC]_max increases linearly with the amount of ß-1,3-xylan used, while K_d is independent of the amount of ß-1,3-xylan . Since xylose and ß-1,3-xylooligosaccharides had no effect on the binding between CBM and ß-1,3-xylan, the CBM recognition site is not governed simply by the ß-1,3-xylooligosaccharide chain . A specific three-dimensional arrangement of ß-1,3-linked xylose chains must be needed for this interaction . Also, as a result of the comparison of the enzymatic properties between rTxyA and the protein expressed by pCD, removal of the CBM from TxyA caused a significant increase in the K_m of TxyA toward ß-1,3-xylan . These results indicate that this CBM plays an important role in the hydrolysis of insoluble substrates such as ß-1,3-xylan .

Y.-T . Li was supported by National Institutes of Health grant NS 09626 .

Present address: Department of Microbiology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa, Nagoya, Aichi 466-8550, Japan .

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