Báo cáo khoa học: Substrate recognition by glycoside hydrolase family 74 xyloglucanase from the basidiomycete Phanerochaete chrysosporium

Substrate Recognition by Glycoside Hydrolase Family 74 Xyloglucanase from the Basidiomycete Phanerochaete Chrysosporium

Tác giả: Takuya Ishida, Katsuro Yaoi, Ayako Hiyoshi, Kiyohiko Igarashi, và Masahiro Samejima

Lĩnh vực: Khoa học Nông nghiệp và Sinh học, Khoa Khoa học Vật liệu Sinh học, Đại học Tokyo, Nhật Bản; Viện Tài nguyên và Chức năng Sinh học, Viện Khoa học Công nghiệp và Công nghệ Tiên tiến Quốc gia (AIST), Tsukuba, Nhật Bản.

Nội dung tài liệu: Nghiên cứu này tập trung vào enzyme xyloglucanase Xgh74B từ nấm Phanerochaete chrysosporium, thuộc họ enzyme glycoside hydrolase (GH) gia đình 74. Enzyme này có một tên miền xúc tác GH gia đình 74 và một mô-đun liên kết carbohydrate (CBM) gia đình 1. Nghiên cứu đã biểu hiện enzyme tái tổ hợp và đánh giá hoạt tính thủy phân của nó đối với xyloglucan từ hạt me (TXG). Kết quả cho thấy Xgh74B có hoạt tính thủy phân cao đối với TXG, trong khi các β-1,4-glucans khác lại là chất nền kém. Mô-đun CBM của enzyme ảnh hưởng đến sự hấp phụ trên cellulose tinh thể nhưng không ảnh hưởng đến quá trình thủy phân xyloglucan, cho thấy vai trò của nó trong việc định vị enzyme. Phân tích sản phẩm thủy phân cho thấy Xgh74B phân cắt các liên kết glycosidic của các gốc glucose không phân nhánh. Tuy nhiên, phân tích động học cho thấy Xgh74B thủy phân TXG theo một cách khác biệt so với các enzyme GH gia đình 74 đã biết khác, tạo ra các oligosaccharide có mức độ trùng hợp (DP) ban đầu từ 16-18, sau đó được thủy phân chậm hơn thành các sản phẩm cuối cùng có DP 7-9. Tỷ lệ các oligosaccharide này phụ thuộc vào pH, cho thấy ái lực của enzyme đối với các oligosaccharide DP 16-18 bị ảnh hưởng bởi môi trường ion tại vị trí hoạt động.

Mục lục chi tiết:

• Substrate recognition by glycoside hydrolase family 74 xyloglucanase from the basidiomycete Phanerochaete chrysosporium
• Keywords
• Correspondence
• Database
• (Received 18 June 2007, revised 19 July 2007, accepted 5 September 2007)
• doi:10.1111/j.1742-4658.2007.06092.x
• The basidiomycete Phanerochaete chrysosporium produces xyloglucanase Xgh74B, which has the glycoside hydrolase (GH) family 74 catalytic domain and family 1 carbohydrate-binding module, in cellulose-grown culture.
• The recombinant enzyme, which was heterologously expressed in the yeast Pichia pastoris, had high hydrolytic activity toward xyloglucan from tamarind seed (TXG), whereas other β-1,4-glucans examined were poor substrates for the enzyme.
• The existence of the carbohydrate-binding module significantly affects adsorption of the enzyme on crystalline cellulose, but has no effect on the hydrolysis of xyloglucan, indicating that the domain may contribute to the localization of the enzyme.
• HPLC and MALDI-TOF MS analyses of the hydrolytic products of TXG clearly indicated that Xgh74B hydrolyzes the glycosidic bonds of unbranched glucose residues, like other GH family 74 xyloglucanases.
• However, viscometric analysis suggested that Xgh74B hydrolyzes TXG in a different manner from other known GH family 74 xyloglucanases.
• Gel permeation chromatography showed that Xgh74B initially produced oligosaccharides of degree of polymerization (DP) 16–18, and these oligosaccharides were then slowly hydrolyzed to final products of DP 7–9.
• In addition, the ratio of oligosaccharides of DP 7–9 versus those of DP 16–18 was dependent upon the pH of the reaction mixture, indicating that the affinity of Xgh74B for the oligosaccharides of DP 16-18 is affected by the ionic environment at the active site.
• Xyloglucan is a widely distributed hemicellulosic polysaccharide that is found in plant cell walls and seeds.
• In the cell wall, xyloglucan associates with cellulose microfibrils via hydrogen bonds, forming a cellulose-xyloglucan network [1-3].
• During cell expansion and development, partial disassembly of the network is required, and consequently it was proposed that xyloglucan metabolism controls plant cell elongation [4].
• In the seeds of some Leguminosae, moreover, xyloglucan acts as a deposited polysaccharide and is available for nutrition when germination occurs.
• As aqueous solutions of xyloglucan have high viscosity, they are often used as food additives to enhance viscosity and/or as stabilizers.
• The xyloglucan from tamarind seed (TXG) is one of the best-studied xyloglucans.
• It consists of a cellulose-like backbone of β-1,4-linked D-glucopyranose residues with side chains of α-D-xylopyranosyl residues attached at the C6 position.
• Galactose residues are found at the end of the side chain, and single-letter nomenclatures are used to simplify the naming of xyloglucan side chain structures; that is, G, X and L stand for β-D-Glcp, α-D-Xylp-(1 → 6)-β-D-Glcp, and β-D-Galp-(1 → 2)-α-D-Xylp-(1 → 6)-β-D-Glcp, respectively [5].
• Compositional analysis of oligosaccharide units in the polymers has shown that TXG has a repeating tetrasaccharide backbone of XXXG, XLXG, XXLG, or XLLG (Fig. 1) [6].
• Although many cellulases (EC 3.2.1.4) have been reported to hydrolyze xyloglucan as a substrate analog [7], some endo-β-1,4-glucanases have high activity toward xyloglucan, with little or no activity towards cellulose or cellulose derivatives [8,9].
• They have been assigned a new EC number (EC 3.2.1.151) and designated as xyloglucanase, xyloglucan hydrolase (XGH), or xyloglucan-specific endo-β-1,4-glucanases (XEGs) belonging to families 5, 12, 44, and 74, according to a recent classification of glycoside hydrolases (GHs) available at http://afmb.cnrs-mrs.fr/CAZY/[10-12].
• Among these enzyme families, xyloglucanases placed in GH family 74 are known to have high specific activity towards xyloglucan, with inversion of the anomeric configuration, and both endo-type and exo-type hydrolases have been found in several microorganisms [13-20].
• The exo-type enzymes recognize the reducing end of xyloglucan oligosaccharide (oligoxyloglucan reducing-end-specific cellobiohydrolase, EC 3.2.1.150, from Geotrichum sp. M128 [15] and oligoxyloglucan reducing-end-specific xyloglucanobiohydrolase from Aspergillus nidulans [20]), whereas the endo-type enzymes hydrolyze xyloglucan polymer randomly.
• In addition, XEG74 from Paenibacillus sp. KM21 and Cel74A from Trichoderma reesei have been reported to have endo-processive or dual-mode endo-like and exo-like activities [13,18].
• During the course of wood degradation, the basidiomycete Phanerochaete chrysosporium produces two GH family 74 xyloglucanases extracellularly [21].
• According to the total genome sequence of the fungus, one of these enzymes, Xgh74B, has the two-domain structure of the N-terminal GH family 74 catalytic domain and the C-terminal domain belonging to the carbohydrate-binding module (CBM) family 1 [21,22].
• In the present study, we have heterologously expressed the cDNA encoding Xgh74B in the methylotrophic yeast Pichia pastoris, and demonstrated a unique hydrolytic character of the recombinant enzyme.
• Results
• Function of CBM1 in recombinant Ph. chrysosporium Xgh74B
• The cDNA encoding Xgh74B was heterologously expressed in the yeast Pi. pastoris, and the recombinant enzymes with and without CBM1 (Xgh74B and Xgh74Bcat, respectively) were purified by column chromatography.
• As shown in Fig. 2, the molecular masses of purified Xgh74B and Xgh74Bcat were 130 kDa and 80 kDa, respectively, being apparently higher than the masses calculated from the amino acid sequences (88.4 and 75.5 kDa, respectively).
• As there are three N-glycosylation sites in the sequence of the catalytic domain according to the NetNGlyc 1.0 server (http://www.cbs.dtu.dk/services/NetNGlyc/), the N-glycan was eliminated by endo-β-N-acetylglucosaminidase H (endo-H) treatment.
• After the treatment, the molecular mass of Xgh74Bcat became close to the calculated value, whereas that of Xgh74B was still approximately 20 kDa larger than the calculated value.
• There are numerous O-glycosylation sites between the catalytic domain and the CBM, as predicted by the NetOGlyc 3.1 server (http://www.cbs.dtu.dk/services/NetOGlyc/), so the larger than calculated molecular mass of intact Xgh74B presumably reflects both N-glycosylation and O-glycosylation [23].
• The binding properties of Xgh74B and Xgh74Bcat were investigated using solid cellulosic substrates, phosphoric acid-swollen cellulose (PASC), Avicel, and bacterial microcrystalline cellulose (BMCC), as shown in Fig. 3.
• Xgh74B was adsorbed well on all three cellulose samples, whereas the amount of bound Xgh74Bcat, without CBM1, was lower than that of the intact enzyme.
• The CBM1 in Xgh74B may contribute to the binding on a crystalline, rather than an amorphous, surface, because increase of crystallinity (PASC < Avicel < BMCC) led to significant differences of adsorption between intact Xgh74B and Xgh74Bcat.
• The kinetic features of the intact enzyme and catalytic domain were compared as shown in Table 1.
• The kinetic constants for TXG of the intact enzyme and catalytic domain were all similar, and no significant difference was observed between the two proteins, suggesting that CBM1 in Xgh74B may contribute to the localization of this enzyme, but not to its function for hydrolysis of the soluble substrate.
• Substrate specificity of Xgh74B
• When TXG was used as a substrate, Xgh74B showed optimum hydrolysis at pH 6.0 and 55 °C, and was stable between pH 5.0 and 8.0 at 30 °C (data not shown).
• The Km of TXG hydrolysis by Xgh74B was estimated to be 0.25 mg mL¯¹, and the kcat was 28.1 s¯¹ when the activity was measured for the reducing sugar.
• However, Xgh74B showed very low activity (less than 5% relative activity with respect to TXG) towards other β-1,4-glycans, carboxymethyl cellulose (CMC), PASC, Avicel, BMCC, glucomannan, galactomannan, and xylan (data not shown), indicating that Xgh74B has typical characteristics of a GH family 74 xyloglucanase.
• The hydrolytic products formed from TXG by Xgh74B were analyzed by normal-phase HPLC and MALDI-TOF MS, as shown in Fig. 4.
• The results of HPLC suggested that the reaction mixture contained oligosaccharides with three different degrees of polymerization (DP) (Fig. 4B), which showed the same retention times as oligosaccharides with DPs of 7–9, XXXG, XLXG, XXLG, and XLLG.
• The molecular masses of these fragments estimated by MALDI-TOF MS (Fig. 4D) coincided with those of authentic xyloglucan oligosaccharides.
• We also analyzed the hydrolytic products of xyloglucan oligosaccharide, XXXGXXXG, and obtained a single peak at the retention time of XXXG (Fig. 4C), suggesting that Xgh74B hydrolyzes the unbranched glucose residues in TXG.
• Viscometric assay and gel permeation chromatography (GPC) analysis of TXG hydrolysis
• The viscosity of TXG was monitored during the hydrolysis with Xgh74B and XEG from Geotrichum sp. M128 (Geotrichum XEG), and the viscosity is plotted versus amount of reducing sugar in Fig. 5.
• A similar plot for XEG74 from Paenibacillus sp. strain KM21 (Paenibacillus XEG74) is also shown for reference [13].
• As described above, Xgh74B effectively hydrolyzed TXG, and a decrease in viscosity was observed, with the production of reducing sugar, indicating that Xgh74B is an endo-type enzyme that cleaves polymeric substrates in the middle of the molecule.
• However, there were differences among the plots for the three enzymes; the degree of hydrolysis-specific viscosity plot for Xgh74B is intermediate between those of Geotrichum XEG and Paenibacillus XEG74.
• Therefore, the change of the molecular mass distribution of TXG during the hydrolysis was analyzed by GPC with a refractive index detector, as shown in Fig. 6.
• In the case of Geotrichum XEG, the molecular mass decreased rapidly even at the initial stage of the reaction, suggesting that the degradation process involved random hydrolysis of β-1,4-linkages in the xyloglucan polymer chain.
• On the other hand, the degradation pattern of Xgh74B was rather similar to that of Paenibacillus XEG74, as the oligosaccharides with DP 7–9 (XXXG, XLXG, XXLG, and XLLG) were observed from the initial stage of the reaction (peak B in Figs 6B and 7D).
• However, in the case of Xgh74B, there is an additional peak at an earlier retention time (12 min, peak A).
• Peak A was fractionated and analyzed by MALDI-TOF MS analysis, and was found to consist of oligosaccharides of DP 16-18, as shown in Fig. 7A.
• In addition, the relative amount of peak A (DP 16-18) was greater when the reaction was carried out at higher pH, suggesting that the charge at the active site influences the affinity for oligosaccharides of DP 16-18.
• Discussion
• Filamentous fungi produce several extracellular xyloglucanases when they grow on plant cell walls as a carbon source.
• Before their characterization, fungal GH family 74 xyloglucanases had been thought to be cellulases, as they have apparent activity against amorphous cellulose or soluble cellulose derivatives.
• Moreover, some fungi produce GH family 74 xyloglucanases with the family 1 CBM, and this also results in enzymes that were characterized as cellulases.
• According to the total genome sequence of Ph. chrysosporium, there are two enzymes belonging to GH family 74 (Xgh74A and Xgh74B), and Xgh74B is known to have the family 1 CBM at the C-terminal region [22].
• Therefore, in the present study, we heterologously expressed recombinant Xgh74B in yeast, and the function of CBM1 in Xgh74B was characterized from adsorption and kinetic points of view.
• Apparent adsorption of intact Xgh74B was observed when solid cellulosic substrates were used, but a comparison of the kinetic parameters of intact Xgh74B and Xgh74Bcat clearly indicates that the CBM in Xgh74B does not contribute to the hydrolytic reaction of soluble xyloglucan substrates.
• The results suggest that the CBM might determine the localization of this enzyme or help in the hydrolysis of insoluble substrates.
• Recently, some diversity of substrate specificity and mode of action has been reported for GH family 74 enzymes; for example, oligoxyloglucan reducing-end specific cellobiohydrolase from Geotrichum sp. M128 and OREX from A. nidulans have oligoxyloglucan reducing-end-specific exo-activity and cannot hydrolyze xyloglucan polymer [15,20], whereas most enzymes belonging to GH family 74 are β-1,4-glucanases with the highest activity towards xyloglucan [13,14,16-18].
• Xgh74B rapidly decreases the viscosity of TXG solutions, consistent with an endohydrolase mechanism.
• On GPC, however, the final degradation products (XXXG, XLXG, XXLG, and XLLG) were observed to be formed even at the