1,2-β-Oligoglucan phosphorylase from Listeria innocua.

We characterized recombinant Lin1839 protein (Lin1839r) belonging to glycoside hydrolase family 94 from Listeria innocua. Lin1839r catalyzed the synthesis of a series of 1,2-β-oligoglucans (Sopn: n denotes degree of polymerization) using sophorose (Sop2) as the acceptor and α-D-glucose 1-phosphate (Glc1P) as the donor. Lin1839r recognized glucose as a very weak acceptor substrate to form polymeric 1,2-β-glucan. The degree of polymerization of the 1,2-β-glucan gradually decreased with long-term incubation to generate a series of Sopns. Kinetic analysis of the phosphorolytic reaction towards sophorotriose revealed that Lin1839r followed a sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of sophorotetraose and sophoropentaose were similar to those of sophorotriose, although the enzyme did not exhibit significant phosphorolytic activity on Sop2. These results indicate that the Lin1839 protein is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of 1,2-β-glucan with a degree of polymerization of ≥3. We propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein.

Introduction

‘Phosphorylase’ is the general term for enzymes that reversibly phosphorolyze glycosyl linkages to generate sugar 1-phosphates [1], [2]. Phosphorylases are generally thought to be involved in metabolism of specific oligosaccharides or storage polysaccharides in cytosol through their phosphorolytic activity. Reverse phosphorolysis is useful for preparation of specific oligosaccharides because of their strict regioselectivity [1]–[3].

This reversibility makes it possible to practically produce several oligosaccharides from abundantly available natural resources, such as sucrose and starch, by combining the reactions of two phosphorylases. Based on these features, practical preparative methods of several oligosaccharides and polysaccharides have been developed [2], [4]–[8]. These combinations enable practical production of such compounds without using expensive sugar 1-phosphates. However, the number of phosphorylases based on EC numbers is only 19, which is much smaller than those of glycoside hydrolases, and limits the usage of the enzyme class. Therefore, it would be beneficial to find new phosphorylases showing unreported substrate specificities and regioselectivities.

Phosphorylases belong to one of the glycoside hydrolase families (GH) 13, 65, 94, 112, and 130 or the glycosyl transferase families (GT) 4 and 35 on the Carbohydrate-Active EnZymes database (http://www.cazy.org) on the basis of the sequence similarity [9]. Among them, activities of GH94 members reported are cellobiose phosphorylase (EC 2.4.1.20) [10], N,N′-diacetylchitobiose phosphorylase (EC 2.4.1.280) [11], laminaribiose phosphorylase (EC 2.4.1.31) [12], [13], cellodextrin phosphorylase (EC 2.4.1.49) [14]–[16], cellobionic acid phosphorylase (EC 2.4.1.x) [17] and C-terminal domain of cyclic 1,2-β-glucan synthase (EC 2.4.1.x, CGS) possessing phosphorolytic activity on protein-bound 1,2-β-oligoglucan [18].

In the phylogenetic tree analysis of GH94, enzymes catalyzing the same reactions appeared in the same cluster, except for cellodextrin phosphorylase. We noticed that strains of genus Listeria generally possess a gene encoding a GH94 protein at a cluster in which no enzyme has been characterized. In this study, we describe the GH94 protein from Listeria innocua with phosphorylase activity specific to 1,2-β-oligoglucans that requires a new EC number.

Materials and Methods

Sequence Analysis

ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/) was used to perform multiple alignments, and MEGA5.1 was used to construct a phylogenetic tree [19].

Cloning, Expression, and Purification

Genomic DNA of L. innocua Clip 11262 (ATCC BAA-680D) was purchased from the American Type Culture Collection (Manassas, VA, USA). The gene encoding the Lin1839 protein (GenBank accession number: CAC97070.1) was amplified by PCR using KOD plus DNA polymerase (Toyobo, Osaka, Japan) and the genomic DNA as a template. Primer pair was forward primer 5′-GTGGATATccaTGgCAATGTTAAAAG-3′ and reverse primer 5′-ATACACAAAACAACCctcGAGACGG-3′ (lower case represents sequences different from original sequence of the genome sequence of L. innocua Clip 11262) containing additional NcoI and XhoI sites (underlined), respectively. The amplified lin1839 gene was inserted into the NcoI and XhoI sites of pET28a(+) (Novagen, Madison, WI, USA) to encode a His6–tag fusion at the C terminus. The constructed plasmid was used to transform Escherichia coli BL21 (DE3). The transformant was grown in 1 l of Luria–Bertani medium containing 30 μg/ml kanamycin at 37°C until the absorbance reached 0.8 at 660 nm, followed by induction using 0.1 mM IPTG at 20°C overnight. The cells were collected by centrifugation at 3,900×g for 5 min and then suspended in 20 mM MOPS–NaOH buffer (pH 7.5) containing 250 mM NaCl (buffer A). The suspended cells were disrupted by sonication and centrifuged at 27,000×g for 20 min. The obtained supernatant was loaded onto a HisTrap FF crude column (5 ml; GE Healthcare, Buckinghamshire, UK) equilibrated with buffer A containing 10 mM imidazole using a AKTA Prime Plus chromatography system (GE Healthcare). After unbound components were washed with buffer A containing 10 mM imidazole, a linear gradient of 10–250 mM imidazole in buffer A was used to elute Lin1839r (actually eluted between 25 and 100 mM of imidazole). An Amicon Ultra 30,000 molecular weight cut-off (Millipore, Billerica, MA, USA) was used to change the buffer in the pooled protein solution to 20 mM MOPS–NaOH buffer (pH 7.5). A theoretical extinction coefficient of 175,450 M−1cm−1, based on the amino acid sequence, was used to determine the protein concentration spectrophotometrically at 280 nm [20]. Finally, 88 mg of recombinant Lin1839 protein (Lin1839r) was obtained from 1 l of culture medium.

Size-exclusion Chromatography

Lin1839r (1 ml of 2.0 mg/ml solution) was loaded onto Superdex 200 (Hiload 16/60; GE Healthcare) equilibrated with 50 mM MOPS–NaOH (pH 7.0) containing 150 mM NaCl. Ovalbumin (44 kDa), conalbumin (75 kDa), aldolase (158 kDa), ferritin (440 kDa) and thyroglobulin (669 kDa; GE Healthcare) were used as standard proteins. Blue dextran 2000 (2000 kDa; GE Healthcare) was used to determine the void volume of the column.

Measurement of Phosphorolytic and Synthetic Activity

The phosphorolytic activity was determined by measuring α-glucose 1-phosphate (Glc1P) generated in a reaction mixture on the basis of the phosphoglucomutase-glucose 6-phosphate dehydrogenase method [21]. The reaction was performed in a reaction mixture (200 μl) placed in a well of a 96-well microplate (EIA/RIA plate, 96-well half area, Corning, NY, USA) containing appropriate concentrations of each sugar substrate and inorganic phosphate with 5.0 μM α-glucose 1,6-bisphosphate (Sigma–Aldrich, St. Louis, MO, USA), 5.0 IU/ml glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides (Oriental Yeast, Tokyo, Japan), 6.25 IU/ml phosphoglucomutase from rabbit muscle (Sigma–Aldrich), 0.5 mM thio-NAD+ (Oriental Yeast), and 25 mM MgCl2 in 100 mM MOPS–NaOH buffer (pH 7.5) at 30°C. The concentration of Glc1P was calculated by determining absorbance at 400 nm continuously on a Spectramax 190 (Molecular Devices, CA, USA).

The method of Lowry and Lopez [22], as described below, was used to determine the reverse phosphorolytic activity by measuring the amount of inorganic phosphate released in a reaction mixture. In brief, the enzymatic reaction was performed in a reaction mixture (160 μl) containing 10 mM Glc1P and an appropriate concentration of each acceptor in 100 mM MOPS–NaOH buffer (pH 7.5) at 30°C. An aliquot (20 μl) was mixed with 160 μl of 0.2 M sodium acetate buffer (pH 4.0) and 20 μl of 1% ammonium molybdate containing 25 mM sulfuric acid to stop the reaction every 3 minutes. Then, 20 μl of 1% ascorbic acid and 0.05% potassium bisulfate were mixed with the samples. The mixtures were incubated at 37°C for 1 h, and the concentrations of phosphate released were quantified by measuring absorbance at 700 nm. One unit of the phosphorolytic and reverse phosphorolytic activities were defined as the amount of the enzyme producing 1 μmol of Glc1P and phosphate, respectively, per minute under the above conditions with the concentrations of all substrates at 10 mM.

Temperature and pH Profile

The effect of pH on the enzymatic activity using 4.0 μg/ml Lin1839r was determined by measuring synthetic activities under the standard conditions described above and by substituting 100 mM MOPS–NaOH buffer (pH 7.5) with various 100 mM buffers. Similarly, the effect of temperature on the enzymatic activity of 4.0 μg/ml Lin1839r was determined by measuring the reverse phosphorolytic activities at various temperatures for 20 min. The thermal and pH stabilities were evaluated by measuring the residual synthetic activity of 10 mM sophorose (Sop2) and 10 mM Glc1P after incubation of Lin1839r (0.5 mg/ml) at different temperatures in 20 mM MOPS–NaOH buffer (pH 7.5) for 1 h and at different pH at 30°C for 1 h, respectively.

Thin Layer Chromatography (TLC)

An aliquot (1 μl) of the reaction mixture for the synthetic reaction was spotted onto a TLC plate (Kieselgel 60 F254; Merck, Darmstadt, Germany), and a solution of acetonitrile:water (3∶1, v/v) was used to elute the samples. The TLC plate was soaked in 5% sulfuric acid:methanol solution and heated in an oven until bands were sufficiently visible.

Structural Analysis of the Reaction Products

Reaction products (oligosaccharides) for structural determination were generated by incubation of a reaction mixture (4 ml) containing 20 mM Glc1P, 10 mM Sop2, and 40 μg/ml Lin1839r in 100 mM MOPS–NaOH (pH 7.0) at 30°C for 2 h. After using Amberlite MB4 (Organo, Tokyo, Japan) to desalt the sample, it was concentrated and loaded onto a Toyopearl HW-40F column (5.0 cm φ×80 cm; Tosoh, Tokyo, Japan), equilibrated with distilled water and eluted at a flow rate of 5.0 ml/min. Fractions containing trisaccharide and tetrasaccharide were collected, followed by lyophilization. Polysaccharide was synthesised by incubation of a reaction mixture (1 ml) containing 200 mM Glc1P, 50 mM glucose, and 125 μg/ml Lin1839r in 100 mM MOPS–NaOH (pH 7.0) at 30°C for 4 days. The products were separated on the same column as described above. Fractions containing polysaccharide were collected and desalted using Amberlite MB4, followed by lyophilization. The amounts of trisaccharide, tetrasacchride, and polysaccharide obtained were 2.3, 1.6 and 7.5 mg, respectively. One-dimensional (1H and 13C) and two-dimensional [double-quantum-filtered correlation spectroscopy (DQF-COSY), totally correlated spectroscopy (TOCSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC)] NMR spectra of the product were acquired in D2O with 2-methyl-2-propanol as an internal standard on a Bruker Avance 500 or Bruker Avance 800 spectrometer (Bruker Biospin, Rheinstetten, Germany). Proton signals were assigned on the basis of the DQF-COSY and TOCSY spectra. 13C signals were assigned on the HSQC spectra on the basis of the assignment of proton signals. The linkage position in the oligosaccharides was determined by detecting inter-ring cross peaks in each HMBC spectrum.

Preparation of 1,2-β-oligoglucans and 1,2-β-glucan for Assay

1,2-β-Oligoglucans [Sopn, n denotes the degree of polymerization (DP) of the oligosaccharide] was synthesized without using expensive Sop2 by incubation of a reaction mixture (20 ml) containing 500 mM Glc1P, 250 mM glucose, and 2.0 mg/ml Lin1839r (pH 7.0 adjusted with HCl) at 30°C for 14 days. After performing electrodialysis on a Microacylizer S1 with an AC-220-10 cartridge (Astom Corp., Tokyo Japan) to remove the salts contained in the reaction mixture, a Toyopearl HW-40F column, as described above, was used to separate the products. Fractions containing Sop3, Sop4, and Sop5 were collected, followed by lyophilization. 1,2-β-glucan was produced by incubation of a reaction mixture (50 ml) containing 750 mM Glc1P, 180 mM glucose, and 80 μg/ml Lin1839r in 375 mM MOPS–NaOH (pH 7.0) at 30°C for 8 days. The product was separated as described above. The collected sample solution was concentrated into approximately 15 ml, and then an equal volume of ethanol was added to the 1,2-β-glucan solution. The first precipitate was separated from the supernatant by centrifugation after incubation of the sample at 20°C for 1 day. The second precipitate generated in the first supernatant after the incubation for an additional 1 day was also collected by centrifugation. The final precipitate was obtained after storage of the second supernatant at −30°C for over 1 year. The precipitates were dried under vacuum. The average DP of 1,2-β-glucan was calculated from the ratio of the peak area of C-6 protons of the internal glucose units appearing near 3.94 ppm against that of the C-2 proton at the non-reducing end appearing near 3.33 ppm.

Kinetic Analysis

The initial velocities of the phosphorolytic reactions with Sopns were determined under the standard conditions with 1.0 μg/ml Lin1839r and a combination of initial concentrations of each substrate and Pi. The kinetic parameters for Sop3 were calculated by curve-fitting the experimental data to the theoretical equation (1) for a sequential Bi Bi mechanism using GraFit version 7.0.3 (Erithacus Software Ltd., London, UK).

Kinetic analysis of the synthetic reactions with suitable acceptors was performed under the standard conditions with 2.0 μg/ml Lin1839r and different concentrations of the acceptor substrate or Glc1P as the donor with 10 mM each opposite substrate. The kinetic parameters were calculated by curve fitting the experimental data to the Michaelis-Menten equation (2) using GraFit version 7.0.3.

Results

L. innocua Clip11262 possesses a single gene encoding the GH94 protein (Lin1839) in its genome (GenBank accession number: AL592022.1). Alignment of the amino acid sequences of GH94 proteins revealed that the common catalytic nucleophile aspartate residue was conserved as D739 in Lin1839. The amino acid sequence of the Lin1839 protein showed no predicted N-terminal signal peptide on the basis of a SignalP 4.0 analysis (http://www.cbs.dtu.dk/services/SignalP/) [23], suggesting that it is a cytosolic protein. Its activity was not predictable from the phylogenetic tree analysis with other characterized GH94 enzymes (Fig. 1).

Figure 1

Phylogenetic tree of GH94 proteins.

Genes are represented with the organism names and GenBank™ accession numbers. Characterized enzymes are categorized in boxes framed with broken lines according to their substrate specificities. In the case of CGSs, only GH94 domains are used for the alignment. The gene cloned in this study is represented with a black background and white letters. GH94 proteins from Listeria are boxed with a bold line.

Substrate Specificity in the Synthetic Reaction

The acceptor specificity of Lin1839r in the synthetic reaction was examined using 10 mM of various putative carbohydrate acceptors in the presence of 10 mM Glc1P as the donor. Lin1839r did not utilize any monosaccharides as the acceptor to significantly liberate inorganic phosphate under the reaction conditions described in Table 1. We noticed that a spot of probable polysaccharide appeared at the origin of the TLC for the reactions with 50 mM monosaccharides and 50 mM Glc1P with higher concentrations of Lin1839r (2 mg/ml) only when glucose was used as the acceptor, but no oligosaccharides were detected (Fig. 2A). After a long incubation period, a series of oligosaccharides were detected on TLC (Fig. 2B). Next, we examined the acceptor specificity for disaccharides at 10 mM. The enzyme showed the highest activity with Sop2 (43 s−1) and a detectable activity with laminaribiose (0.48 s−1) but no activity with other disaccharides (Table 1). The reaction with Sop2 produced a series of oligosaccharides (Fig. 2C). The reaction with laminaribiose as the acceptor produced a polymer initially followed by generation of oligomers (Fig. 2D), a pattern similar to that for glucose. The enzyme exhibited activity only on Glc1P among the sugar 1-phosphates examined (Table 1).

Table 1

Substrate specificity of Lin1839r in the synthetic reaction.

Substrate	Relative activitya (%)
Acceptorb
d-Glucose	N.Dd , e
2-Deoxy-d-glucose	N.D
d-Xylose	N.D
d-Mannose	N.D
d-Galactose	N.D
l-Fucose	N.D
l-Arabinose	N.D
l-Rhammose	N.D
d-Glucosamine	N.D
N-Acetyl-d-glucosamine	N.D
d-Galactosamine	N.D
N-Acetyl-d-galactosamine	N.D
Sucrose	N.D
Maltose	N.D
Lactose	N.D
Sophorose	100 (43f)
Laminaribiose	1.1 (0.48)
Cellobiose	N.D
Gentiobiose	N.D
Donorc
Glc1P	100 (43)
α-d-Galactose 1-phoshate	N.D
α-d-Mannose 1-phoshate	N.D
N-Acetyl-α-d-glucosamine 1-phoshate	N.D

The specific activity of Lin1839r in the presence of 10 mM Sop2 and 10 mM Glc1P was defined as 100% relative activity.

Measured at 10 mM with 10 mM Glc1P as the donor.

Measured at 10 mM with 10 mM Sop2 as the acceptor.

N.D represents not determined owing to <0.2% relative activity.

The specific activity on glucose at 100 mM was 0.095 s−1.

Values in parentheses represent specific activity (s−1).

Figure 2

TLC analysis of reaction products from acceptors and Glc1P.

(A) Reaction products from monosaccharides and Glc1P. Reactions were performed with 2 mg/ml of Lin1839r in the presence of 50 mM monosaccharides and 50 mM Glc1P for 1 h. (B) Time course of the reaction products from glucose and Glc1P. Substrates used were 50 mM glucose and 50 mM Glc1P. The enzyme concentration used was 2 mg/ml. (C) Reaction products after reaction for 2 h using 10 mM Sop2 and 20 mM Glc1P as substrates. (D) Time course of the reaction products from laminaribiose and Glc1P. The substrates used were 10 mM laminaribiose and 10 mM Glc1P. The enzyme concentration used was 0.5 mg/ml. (A, C) Presence and absence of Lin1839r are represented with ‘+’ and ‘−’, respectively. (B, D) M, marker. Numbers under a line represent reaction time.

Basic Properties of Lin1839r

Purified Lin1839r was detected at 120 kDa as a single band on SDS-PAGE, which corresponded to the theoretical molecular mass of Lin1839r with His6 tag (123,817 Da). Size-exclusion chromatography of Lin1839r resulted in elution of Lin1839r as a 110-kDa protein, suggesting that Lin1839r is a monomeric protein. Lin1839r was stable up to 37°C. The remaining activity of Lin1839r after incubation for 20 min drastically decreased at temperatures >45°C. The optimal temperature was 37–45°C (Fig. 3A). Lin1839r was stable in the range of pH 4.5–9.5, and its optimal pH was 7.5–8.0 (Fig. 3B).

Figure 3

Effects of temperature and pH on enzyme activity and stability of Lin1839r.

(A) The temperature optimum and stability are represented by closed and open circles, respectively. (B) The optimum pH and stability are represented by a single line and a dashed line, respectively. The buffers used for reaction and incubation are sodium acetate (pH 4.0–5.5, closed circle), MES–NaOH (pH 5.5–6.5, open circle), BISTRIS (pH 6.0–7.0, closed triangle), MOPS–NaOH (pH 6.5–7.5, open triangle), HEPES–NaOH (pH 7.5–8.5, closed square), 2-hydroxy-3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulphonic acid–NaOH (pH 8.0–8.5, open square), Tris–HCl (pH 7.5–9.0, open rhombic), and glycine–NaOH (pH 8.6–10, closed rhombic).

Analysis of Products of the Synthetic Reaction Catalyzed by Lin1839r

Each oligosaccharide produced in the reaction mixture with Sop2 as the acceptor and Glc1P as the donor was isolated by gel filtration. The trisaccharide and tetrasaccharide produced were identified as Sop3 and Sop4, respectively, on the basis of the NMR spectra (Fig. S1–S2, Table S1–2). The polymer product produced from glucose and Glc1P at the early stage of the reaction was also isolated by gel filtration. Simple 1H and 13C NMR spectra of the compound indicated that it is a homopolymer containing only a 1,2-β-glucosyl linkage and an average DP of 39 (Fig. S3, Table S3).

Preparation of 1,2-β-glucan and Sopns

High concentrations of glucose and Glc1P as substrates were successfully adapted to production of 1,2-β-glucan (at a low concentration of Lin1839r) and Sopns (at a high concentration of Lin1839r for a long reaction term) at a larger scale. No precipitate was generated during the entire reaction period, suggesting that the generated 1,2-β-glucan was very soluble in water. For the production of the polymer, addition of ethanol to the 1,2-β-glucan solution caused gradual precipitation. The amounts of the first, second and third precipitates were 1.3, 0.90 and 0.25 g, respectively. The average DPs of these precipitates were 77, 64 and 39, respectively. The amounts of Sop2, Sop3, Sop4 and Sop5 obtained were 98, 120, 100 and 35 mg, respectively.

Kinetic Analysis of the Reverse and Forward Phosphorolytic Reactions

In the reverse phosphorolytic reaction, the kinetic parameters of Lin1839r for activity on the Sopns as acceptors were determined. The enzyme showed similar values of k cat and K m for Sop2, Sop3, and Sop4, which led to similar k cat/K m values for these oligosaccharides (Table 2). The kinetic parameters for Glc1P were in the same range as those of other inverting phosphorylases [13], [24], [25]. In the phosphorolytic reaction, the kinetic parameters of Lin1839r for the Sopns and 1,2-β-glucan (DP = 77) as a substrate were determined. The enzyme exhibited similar k cat and K m values for Sop3, Sop4, and Sop5 (Table 3). The k cat/K m values for 1,2-β-glucan were much smaller than those for Sop3, Sop4 and Sop5, but the activity was significantly detectable. The enzyme showed only a negligible phosphorolytic activity on Sop2 (Table 3). These results indicated that the enzyme catalyzed reversible phosphorolysis of Sopns with ≥DP 3.

Table 2

Kinetic parameters for Sopns in the synthetic reaction.

Substrate	k cat (s−1)	K m (mM)	k cat/K m (s−1mM−1)
Sop2 a	97±4	8.5±0.6	11±1
Sop3 a	110±10	6.0±0.9	18±1
Sop4 a	90±5	6.8±0.7	13±1
Glc1P b	43±2	1.2±0.2	34±4

Up to 10 mM acceptors were used. 10 mM Glc1P was used as a donor.

Up to 10 mM Glc1P was used. 10 mM Sop2 was used as an acceptor.

Table 3

Kinetic parameters for phosphorolysis of Sopns and 1,2-β-glucan.

Substrate	k cat (s−1)	K m (mM)	k cat/K m (s−1mM−1)
Sop2 a	–c	–	–
Sop3	19±1	1.0±0.1	19±2
Sop4	30±1	1.2±0.1	24±1
Sop5	31±1	1.8±0.1	17±1
1,2-β-Glucan b	–	–	0.40±0.07

Specific activity was <0.2 (s−1) at 10 mM.

Up to 4 mg/ml 1,2-β-glucan (average DP = 77) was used.

–, not determined.

Reaction Mechanism of Phosphorolysis

Double reciprocal plots of initial velocities against various initial concentrations of Sop3 and phosphate yielded a series of lines intersecting at a point (Fig. 4). This result indicated that the phosphorolytic reaction on Sop3 followed a sequential Bi Bi mechanism, same as inverting phoshorylases [24], [26]–[28]. The kinetic parameters of Lin1839r determined by regression data are shown in the Fig. 4 legend. The values of k cat, K mA, and K mB were in the same range as those of other inverting phosphorylases [24], [28]–[30].

Figure 4

Double reciprocal plot for the phosphorolysis of Sop3 with different concentrations of inorganic phosphate.

Concentrations of inorganic phosphate were 0.5(open triangle), 1.0 mM (filled square), 2.0 mM (open square), 3.0 mM (filled circle), and 5.0 mM (open circle). The kinetic parameters are as follows: k cat = 21±1 (s−1), K mA = 0.66±0.14 (mM), K mB = 1.3±0.2 (mM), and K iA = 3.3±0.6 (mM), where A represents Sop3 and B is Pi. Grafit version 7.0.3 was used to perform non-linear regression and calculation of values.

Discussion

Classification of Lin1839 Protein

We found that Lin1839r has phosphorylase activity highly specific to Sopns with ≥DP 3 in phosphorolysis (Fig. 5). The specificity on DP is similar with that of cellodextrin phosphorylase and implies the existence of subsites −1 to +2 in Lin1839 protein. The structural prediction of the architecture of Lin1839 is not possible due to the poor identities (less than 20%) in the amino acid sequence with other GH94 enzymes whose structures have been solved [10], [11], [31], [32].

Figure 5

Reaction scheme for the Lin1839 protein.

The C-terminal GH94 domain of CGS from Brucella abortus possesses phosphorolytic activity on 1,2-β-glucan [18]. However, Lin1839r is clearly different from the CGS in the catalytic reaction with respect to factors described below.

CGS is a fusion enzyme composed of GT84 and GH94 that synthesizes cyclic 1,2-β-glucan (CβG) [33], [34]. CGS synthesizes CβGs through the four enzymatic reactions: (i) initiation (transferring glucose to CGS), (ii) elongation of 1,2-β-glucan, (iii) regulation of DP of 1,2-β-glucan and (iv) cyclization [35]. The GH94 domain is involved in (iii) by controlling the length of the polysaccharide chain produced by the GT domain [18]. The GT domain is able to produce 1,2-β-glucan from UDP-glucose without the GH94 domain. Thus, CGS is virtually a glycosyl transferase, and the GH94 domain is enzymologically just an accessory domain. The phosphorolytic activity of the C-terminal domain (1493–2867) of CGS on linearized CβG was 0.725 U/mg [18], whereas the phosphorolytic activity of Lin1839r on Sop3 was much higher (19 s−1 = 9.2 U/mg). Therefore, Lin1839r is a novel enzyme that should be given a new EC number, and we propose 1,2-β-oligoglucan: phosphate α-glucosyltransferase as the systematic name and 1,2-β-oligoglucan phosphorylase as the short name for this Lin1839 protein. Sophorodextrin phosphorylase is a possible alternative name.

Synthesis of 1,2-β-glucan and Sopns

Lin1839r synthesized a series of Sopns from Sop2 and Glc1P (Fig. 2C). It also produced 1,2-β-glucan from glucose and Glc1P without any detectable oligosaccharides at the early stage of the reaction, and then a series of Sopns accumulated (Fig. 2B). These phenomena can be explained as described below.

At the initial stage, glucose acted as a poor acceptor to generate Sop2 slowly. The resultant Sop2 was preferentially converted into a polymer because the Sop2 and Sopns generated act as actual acceptors much more than glucose (Fig. 6A). Similar generation of polymeric compounds with a monosaccharide as the acceptor has been reported on production of cellulose-like material using cellodextrin phosphorylase [36]. The polymer formation continued until Glc1P/Pi ratio reached nearly equilibrium. Equilibriums between Sopn and Sopn+1 (n≥2) were nearly completed at this stage (Fig. 6B). However, most part of glucose still remained in the reaction mixture, meaning that equilibrium between glucose and Sop2 was not completed (Fig. 6B). Sop2 was still being generated from glucose and Glc1P. The continuous formation of Sop2 increased the number of the Sopn molecules, causing the decrease in the average DP of Sopns. Finally, small oligosaccharides were concomitantly generated with disappearance of the polymer (Fig. 6C). This finding is in clear contrast to the reaction of cellodextrin phosphorylase in which the products immediately precipitated no longer participated in the reaction [36].

Figure 6

Possible mechanism of the production of 1,2-β-glucan and Sopns from glucose and Glc1P.

Possible reaction schemes at the beginning of the reaction (A), after Glc1P/Pi ratio reached equilibrium (B), and at the end of the reaction (C) are shown. Open and slashed circles represent the glucose moiety derived from Glc1P and material glucose, respectively. Lines connecting these circles represent the 1,2-β-glucosyl linkage. Dashed and bold arrows represent reactions with poor and preferential substrates, respectively. Glc1P is drawn with an open circle, and Pi represents inorganic phosphate. Accumulation of 1,2-β-glucan and Sopns are surrounded by a dashed rounded box.

Physiological Role of 1,2-β-oligoglucan Phosphorylase from L. innocua

Most β-1,2-linked glucose polymers have been found as CβG, a homopolymer of β-1,2-linked glucose with DP 17–24 [37]. CβG is distributed in some α-proteobacteria [38]–[40]. After CβG is synthesized by CGS, it is transported into periplasm by an ABC-transporter system involving Cgt protein. CβG is then modified with anionic molecules, such as succinate, in the case of Brucella. Rhizobium phaseoli secretes CβG as an exopolysaccharide [41]. CβG is involved in adaption to hypo-osmotic conditions for Rhizobium meliloti and Agrobacterium tumefaciens [42]. Defects in the cgs genes of these bacteria cause non-motile phenotypes because of defects in the assembly of flagella [43], [44]. Brucella CβG acts in lipid rafts found on host cell membranes to avoid the innate immune system [45]. CβG suppresses plant immune responses in the case of Xanthomonas campestris pv. campestris [46]. However, L. inncoua does not have a CGS homolog or synthetic system of CβG according to genomic information [47], implying that L. inncoua utilizes exogenous 1,2-β-glucan. A gene cluster is formed around the lin1839 gene. A putative ATP-binding protein ABC-type transporter gene (lin1841–lin1843) may be involved in intake of Sopns and/or 1,2-β-glucan. A putative GH3 β-glucosidase gene (lin1840) is thought to degrade the substrates with concerted action of the Lin1839 protein. LacI transcription factor gene (lin1838) may regulate expression of the genes in the gene cluster. Therefore, it is speculated that the gene cluster is involved in metabolism of Sopns and/or 1,2-β-glucan in a specific environmental condition.

Conclusion

Although numerous studies on 1,3- and 1,4-β-glucans have been reported, 1,2-β-glucan has not been further studied. This is probably because of the difficulty in obtaining 1,2-β-glucan and its oligomers. For instance, Sop2 is only available as an expensive reagent, although it has been well known since 1962 as a powerful inducer of cellulase from Trichoderma reesei, the hyper-cellulase-producing fungus [48]. We identified the Lin1839 protein as a novel 1,2-β-oligoglucan phosphorylase requiring a new EC number. This enzyme enables easy preparation of 1,2-β-glucan and its oligomers, including Sop2, and is an important milestone in the development of procedures for study of the functions of 1,2-β-glucan and related compounds.

NMR spectra of Sop3. (A) 1H-NMR, (B) 13C-NMR, (C) DQF-COSY, (D) TOCSY, (E) HSQC and (F) HMBC. I, II, and III denote first, second, and third glucose residues from reducing end, respectively. Letters in parenthesis represent position of hydroxyl group on the anomeric carbon. Arabic numbers shown with roman numbers represent positions of carbons and protons in sugar rings.

(PDF)

Click here for additional data file.

NMR spectra of Sop4. (A) 1H-NMR, (B) 13C-NMR, (C) DQF-COSY, (D) TOCSY, (E) HSQC and (F) HMBC. I, II, and III denote first, second, and third glucose residues from reducing end, respectively. Letters in parenthesis represent position of hydroxyl group on the anomeric carbon. Arabic numbers shown with roman numbers represent positions of carbons and protons in sugar rings.

(PDF)

Click here for additional data file.

NMR spectra of 1,2-β-glucan. (A) 1H-NMR, (B) 13C-NMR. Numbers under chemical shifts and in parenthesis represent positions of protons (A) and carbons (B). Letters in parenthesis represent position of hydroxyl group on the anomeric carbon.

(PDF)

Click here for additional data file.

Chemical shifts in 13C-NMR and 1H-NMR spectra of Sop3.

(PDF)

Click here for additional data file.

Chemical shifts in 13C-NMR and 1H-NMR spectra of Sop4.

(PDF)

Click here for additional data file.

Chemical shifts in 13C-NMR and 1H-NMR spectra of 1,2-β-glucan.

(PDF)

Click here for additional data file.