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2-O-α-D-Glucosylglycerol Phosphorylase from Bacillus selenitireducens MLS10 Possessing Hydrolytic Activity on β-D-Glucose 1-Phosphate

The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel_2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s−1. No carbohydrate acted as acceptor for the reverse phosphorolysis using β-d-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P with a k cat of 2.8 s−1; moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H2 18O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-d-glucosylglycerol (GG) at the rate of 180 s−1. Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a k cat of 95 s−1. We propose 2-O-α-d-glucopyranosylglycerol: phosphate β-d-glucosyltransferase as the systematic name and 2-O-α-d-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates.

Various oligosaccharides have been synthesized by phosphorylases using the reverse phosphorolysis from the corresponding sugar 1-phosphate as a donor substrate and suitable carbohydrate acceptors [1,2,5,6]. In addition, the reversibility allows the practical synthesis of oligosaccharides from the readily available natural sugars by using a single phosphorylase [7,8], by the combined reaction of two phosphorylases that share the same sugar 1-phosphate or utilizing the compatible ones [1,[9][10][11][12][13], or by the combined reaction of two phosphorylases producing different sugar 1-phosphates with additional enzymes to convert the sugar 1-phosphates [14][15][16].
Bacillus selenitireducens is an anaerobic haloalkaliphilic bacterium that is often isolated from the bottom sediments [24]. It grows optimally in a salinity of 1.5 M NaCl. The salt-dependent proteins isolated from this bacterium, such as the halophilic extracellular arsenate reductase that shows its optimal activity at 2.5 M NaCl, can be adapted to function in extreme environments [25]. The genomic sequence of the B. selenitireducens MLS10 strain, which was isolated from the anoxic mud of a saline lake, has been published (GenBank accession No. CP001791.1). B. selenitireducens MLS10 possesses three genes encoding possible inverting phosphorylases belonging to GH65. We have revealed the activities of two of them: one (locus: Bsel_2056) is a maltose phosphorylase that produces derivatives of maltose possessing a branch at position 2 [26], and the other (locus: Bsel_1207) is a potassium iondependent trehalose phosphorylase [27]. In this study, we identified the specificity of the third GH65 protein (locus Bsel_2816, GenBank accession No. ADI00307.1) and found that it is unique among the known phosphorylases.

Amino Acid Sequence Analysis
Multiple alignments of the amino acid sequences of GH65 proteins with known activities with that of Bsel_2816 were performed using the ClustalW version 2.1 on the DDBJ sever (http://clustalw.ddbj.nig.ac.jp/index.php?lang = ja). A phylogenetic tree was drawn from the results obtained with the neighbor-joining method using a TreeView version 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html).

Cloning of bsel_2816
A gene (GenBank ID: ADI00307.1) encoding Bsel_2816 was amplified by PCR from the genomic DNA of B. selenitireducens used as template and KOD-plus DNA polymerase (Toyobo, Osaka, Japan) with the following primers based on the genomic sequence: 59-aaaccatgggccatgaaattggagaacatc-39 as the forward primer containing the NcoI site (underlined) and 59-tttctcgaggcgggacttggtgatgcg-39as the reverse primer containing the XhoI site (underlined). The amplified gene was purified using the FastGene Gel/ PCR Extraction Kit (Nippon Genetics Co., Ltd., Tokyo, Japan), digested by NcoI and XhoI (New England Biolabs, Beverly, MA, USA), and inserted into pET28a (+) (Novagen, Madison, WI, USA) to add a His 6 -tag at the C-terminus of the recombinant protein. The expression plasmid was propagated in Escherichia coli DH5a (Toyobo), purified with the FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.), and verified by sequencing (Operon Biotechnologies, Tokyo, Japan).

Site-directed Mutagenesis
Site-directed mutagenesis was carried out by the method of Braman et al. [28] but using the KOD-plus DNA polymerase. The pET28a plasmid carrying the bsel_2816 gene was used as template for PCR to obtain the DNA fragment containing the mutation. Pairs of the mutagenic primers (59-cagggtcctgatgcataccatgagaacg-39 for E475A, 59-gttcagggtcctgatcaataccatgagaac-39 for E475Q, and the complementary strands) were used. The parental strands in the PCR products were digested with DpnI, which specifically digests methylated and hemimethylated DNA, and the amplified DNA were retained. The nicked plasmid DNA incorporating the desired mutation was transformed into the E. coli DH5a. The expression plasmid for the mutants was purified with the FastGene Plasmid Mini Kit (Nippon Genetics Co., Ltd.), and verified by sequencing (Operon Biotechnologies).

Expression and Purification of Recombinant Proteins
E. coli BL21 (DE3) (Novagen) transformants harboring the expression plasmid for Bsel_2816 or its mutants were grown at 37uC in 200 ml of Luria-Bertani medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) containing 50 mg/ml of kanamycin up to an absorbance (Abs) of 0.6 at 600 nm. The expressions were induced by 0.1 mM isopropyl b-D-thiogalactopyranoside and continued at 18uC for 24 h. Wet cells, harvested by centrifugation at 20,0006g for 20 min, were suspended in 50 mM HEPES-NaOH buffer (pH 7.0) containing 500 mM NaCl (buffer A) and 10% glycerol. The suspended cells were disrupted by sonication (Branson sonifier 250A; Branson Ultrasonics, Emerson Japan, Ltd., Kanagawa, Japan) and the supernatant, collected by centrifugation at 20,0006g for 20 min, was applied to a HisTrap HP column (GE Healthcare, Buckinghamshire, UK) equilibrated with buffer A containing 10 mM imidazole using an Ä KTA prime system (GE Healthcare). After a wash with buffer A containing 22 mM imidazole and the following elution using a 222400 mM imidazole linear gradient in buffer A, the fractions containing the recombinant protein (Bsel_2816) were pooled, dialyzed against 10 mM HEPES-NaOH buffer (pH 7.0), and concentrated (AMICON Ultra-15 filter; Millipore Co., Billerica, MA, USA). The concentration of the wild-type, E475Q mutant, and E475A mutant was determined spectrophotometrically at 280 nm using a theoretical extinction coefficient of e = 124,110 M 21 cm 21 on the basis of amino acid sequences [29]. The molecular masses of the purified proteins was estimated by SDS-PAGE (Mini-PROTEAN Tetra electrophoresis system; Bio-Rad Laboratories, Inc., Hercules, CA, USA) and by gel filtration (HiLoad 26/600 Superdex 200 pg; GE Healthcare) equilibrated with 10 mM HEPES-NaOH buffer (pH 7.0) containing 150 mM NaCl at the flow rate of 0.5 ml/min and using the marker proteins for molecular weight determination on High Pressure Liquid Chromatography (HPLC) (Oriental Yeast Co., Ltd., Tokyo, Japan) as standards.

Thin Layer Chromatography (TLC)
TLC was performed on a TLC plate (Kieselgel 60 F254; Merck, Darmstadt, Germany). After spotting the samples, the plate was developed once with acetonitrile-water (4:1 v/v) or twice with acetonitrile-water (9:1 v/v). The TLC plates were then soaked in 5% sulfuric acid-methanol solution and heated in an oven until the spots of the carbohydrates became sufficiently visible.

Enzymatic Quantification of Glc and bGlc1P
The concentration of Glc was determined by using a glucose oxidase/peroxidase kit, the Glucose C-II Test Wako. Two volumes of the coloring reagent of the kit were added to one volume of the sample. The increase in the Abs 505 was measured after incubation for 60 min at 37uC. The concentration of bGlc1P was determined by using the bPGM/G6PDH method [30] with the working reagent containing 5 U/ml bPGM, 5 U/ml G6PDH, 4 mM thio-NAD + , 20 mg/ml Glc16bP, and 10 mM MgCl 2 in 50 mM MOPS-NaOH buffer (pH 7.0). An aliquot of sample was mixed with the same volume of the working reagent and the increase in the Abs 400 was measured after incubation for 60 min at 37uC.

Cleavage of a-linked Glucobioses
To examine the specificity on a-linked glucobioses, each reaction mixture containing each disaccharide (10 mM; kojibiose, nigerose, maltose, isomaltose, or trehalose), 10 mM inorganic phosphate (Pi), and 29 mM Bsel_2816 in 50 mM MES-NaOH buffer (pH 6.0) was incubated at 30uC for 3 h. The products were analyzed by TLC. The reaction rate for the cleavage of kojibiose was determined by quantifying the Glc liberated in the reaction mixtures containing various concentrations of kojibiose (1-10 mM) and Pi (0-10 mM) in 40 mM sodium acetate buffer (pH 5.5) at 30uC. An aliquot of the reaction mixture was mixed with the same volume of dimethyl sulfoxide to stop the enzymatic reaction, followed by the enzymatic quantification of Glc. The kinetic parameters were calculated by curve-fitting the experimental data to the theoretical equation (i) for sequential Bi Bi mechanism (A = kojibiose, B = Pi) using GraFit version 7.0.2 (Erithacus Software Ltd., London, UK).
Acceptor specificity analysis. To determine the acceptor specificity of Bsel_2816 in the reverse phosphorolysis, each reaction mixture containing 10 mM each acceptor and 10 mM bGlc1P in 50 mM MES-NaOH buffer (pH 6.0) was reacted with Bsel_2816 at 30uC. After the reaction, the products were analyzed by TLC.

Preparation and Structural Determination of the Product from Glycerol
A reaction mixture (5 ml) containing 500 mM bGlc1P (disodium salt buffered at pH 6.0 with HCl), 1 M glycerol, and 0.25 mg/ ml Bsel_2816 was incubated at 30uC for 18 h. After terminating the reaction by boiling, an aliquot (3 ml) was deionized with Amberlite MB-3 (Organo, Tokyo, Japan), applied on an Amberlite IRA400 column (OH 2 type, 40 ml), and eluted with H 2 O to remove the reducing sugars. The fractions containing the product were collected, concentrated to 9 ml, and loaded on a Toyopearl HW40F column (50 mm internal diameter6800 mm; Tosoh, Tokyo, Japan) equilibrated with distilled water. The product was eluted by distilled water at a flow rate of 5 ml/min. The fractions containing the product were collected, concentrated, and lyophilized to be syrup (298 mg). The one-dimensional ( 1 H and 13 C) and two-dimensional [double-quantum-filtered correlation spectroscopy (DQF-COSY), heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond correlation (HMBC)] NMR spectra of the product were recorded in D 2 O with 2-methyl-2-propanol as an internal standard at 298 K using the Bruker Avance 500 spectrometer (Bruker Biospin, Rheinstetten, Germany). The product was determined to be 2-O-a-D-glucosylglycerol (GG).

Measurement of Enzyme Activity
The phosphorolytic activity of GG was determined by quantifying bGlc1P produced during the enzymatic reaction from 10 mM GG and 10 mM Pi in 40 mM HEPES-NaOH buffer (pH 8.0) at 30uC. An aliquot of the reaction mixture was mixed with the same volume of dimethyl sulfoxide to stop the enzymatic reaction, followed by the enzymatic quantification of bGlc1P. The hydrolytic activity on bGlc1P was determined by measuring the increase in Glc released during the enzymatic reaction from 10 mM bGlc1P in 40 mM MES-NaOH buffer (pH 6.0) or 40 mM HEPES-NaOH buffer (pH 7.5) at 30uC. An aliquot of the reaction mixture was mixed with the same volume of dimethyl sulfoxide to stop the enzymatic reaction, followed by the enzymatic quantification of Glc. The synthetic activity of GG was determined by measuring the increase in Pi in the reaction mixture containing 10 mM bGlc1P and 10 mM glycerol in 40 mM HEPES-NaOH buffer (pH 7.5) at 30uC by using the method of Lowry and Lopez [31].

Temperature and pH Profile
The effects of pH on the phosphorolytic, hydrolytic, and synthetic activities of 212 nM Bsel_2816 were measured under the standard conditions by substituting each 40 mM reaction buffer into the following 40 mM buffers: sodium citrate (pH 3.0-5.5), Bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane-HCl (pH 5.5-7.0), HEPES-NaOH (pH 7.0-8.5), and glycine-NaOH (pH 8.5-10.5). The thermal and pH stabilities were evaluated by measuring the residual synthetic activity under the standard conditions after the incubation of Bsel_2816 (7 mM and 11 mM, respectively) at temperatures 30-90uC for 15 min in 10 mM HEPES-NaOH buffer (pH 7.0) containing 0.1% bovine serum albumin and at the various pH values at 4uC for 24 h, respectively.

The Kinetic Parameters of Bsel_2816
A kinetic analysis of the phosphorolytic reaction of GG was performed using the continuous bGlc1P assay. The reaction mixtures (150 ml) were prepared in the wells of a 96-well microtiter plate with various concentrations of GG (1.0-20 mM) and Pi (0.5-10 mM), and 5.2 nM Bsel_2816 in 40 mM HEPES-NaOH buffer (pH 8.0) containing 2.5 U/ml bPGM, 2.5 U/ml G6PDH, 2 mM thio-NAD + , 5 mg/ml Glc16bP, and 5 mM MgCl 2 . The reaction was carried out in a temperature-controlled microplate reader (Multiskan GO, ThermoFisher Scientific, Waltham, MA) at 30uC, and the Abs 400 was continuously monitored at 30-s intervals without stopping the reaction. The kinetic parameters were calculated by curve-fitting the experimental data to the theoretical equation The inhibition of the hydrolysis of bGlc1P by glycerol was examined in the reaction mixture containing 10 mM bGlc1P and various concentrations of glycerol (0-100 mM) in 40 mM HEPES-NaOH buffer (pH 7.5) and the increase in the concentration of Glc was quantified. The rate of the release of Glc was regressed with the concentration of glycerol to determine the dissociation constant of glycerol using the equation (iii) with GraFit version 7.0.2.
Position of Hydrolysis in bGlc1P bGlc1P bis(cyclohexylammonium) salt (46 mg, 0.10 mmol) was dissolved in 1 ml of H 2 O and applied to a Dowex 50 column (H + form, Wako Pure Chemicals) to remove the counter-ion (cyclohexylamine). An appropriate amount of 1 M NaOH was added to the decationized solution containing free bGlc1P (5 ml) adjusting pH to 6.0, followed by concentration to 1 ml by a centrifuge evaporator to obtain 100 mM bGlc1P-Na buffer (pH 6.0). An aliquot of the 100 mM bGlc1P-Na buffer (20 ml) was mixed with 70 ml of H 2 18 O (97%, Sigma-Aldrich) or unlabeled H 2 O as a control. The hydrolysis of bGlc1P was started by adding 10 ml of 29 mM Bsel_2816 in 4 mM MES-NaOH buffer (pH 6.0) to the solution. After the incubation at 30uC for 4 h, bGlc1P was completely hydrolyzed into Glc and Pi. The solution was lyophilized, dissolved in 50% methanol, and subjected to negative and positive ESI-MS analysis by using a Bruker APEX II 70e Fourier transformed cyclotron resonance mass spectrometer (Bruker Daltonics, Billerica, MA, USA).

Specificity on the Anomeric Configuration
The anomeric configuration of the glucose generated during the hydrolysis of bGlc1P was analyzed by HPLC [32], using an HPLC system (Prominence, Shimadzu, Kyoto, Japan) equipped with a refractive index detector, RID10A (Shimadzu). The hydrolysis of bGlc1P was performed in the reaction mixture containing 50 mM bGlc1P and 13.8 mM Bsel_2816 prepared in 20 mM MES-NaOH buffer (pH 6.0) at 30uC. After the reaction for 1.5 and 25 min, aliquots (10 ml each) of the reaction mixtures were immediately injected into the HPLC and separated with a TSK amide-80 column (4.6 mm internal diameter6250 mm; Tosoh, Tokyo, Japan) by isocratic mode with 80% acetonitrile at a flow rate of 1.5 ml/min.

Gene Cloning, Expression, and Purification of Recombinant Bsel_2816
The phylogenetic tree analysis showed that Bsel_2816 is distantly related with the kojibiose phosphorylase and nigerose phosphorylase (Fig. 1). In this study, the bsel_2816 gene cloned from the genomic DNA of B. selenitireducens MLS10 was expressed as a His 6 -tag fusion protein in E. coli BL21 (DE3) to investigate its enzymatic properties. Purified protein (approximately 14 mg) was obtained from the cell lysate of 200 ml culture. The purified Bsel_2816 migrated in SDS-PAGE as a single protein band with an estimated molecular mass of approximately 87 kDa, in agreement with the theoretical molecular weight of 86,259. However, this native molecular mass was determined to be 174 kDa by gel filtration, suggesting that Bsel_2816 was a homodimeric enzyme.

Examination of the Phosphorolytic Activity on the Substrates of the known GH65 Phosphorylases
The phosphorolytic activity of Bsel_2816 on a-linked glucobioses (i.e., kojibiose, nigerose, maltose, isomaltose, and trehalose) including the known substrates of the GH65 enzymes was examined. Bsel_2816 cleaved kojibiose at a slow rate only in the presence of Pi. To our surprise, Glc was detected as the sole product and bGlc1P was not detected at all. The optimum pH for the cleavage of kojibiose was determined to be 5.5. The measurement of the reaction rate with various concentrations of kojibiose and Pi revealed that the reaction proceeded with a sequential Bi Bi mechanism, similarly to other inverting phosphorylases, with the following kinetic parameters: K mA , 8.9 mM; K iA , 41 mM; K mB , 0.19 mM; k cat 0.43 s 21 , where A and B represent kojibiose and Pi, respectively (Fig. 2). The reaction mechanism suggests that Pi was involved in the cleavage of kojibiose even though no bGlc1P was detected in the products.

Position of the Hydrolysis in bGlc1P
The enzymatic hydrolysis of phosphate esters is generally catalyzed by phosphatases that cleave the PO bond of the phosphate ester [33]. To investigate the mechanism of the hydrolysis of bGlc1P by Bsel_2816, the incorporation of an oxygen atom was examined in 18 O-labled water. In negative MS (Fig. S1A) 18 O was incorporated into Glc. It is concluded that the anomeric CO bond was cleaved during the hydrolysis of bGlc1P, indicating that the hydrolysis was a glycosidase-type reaction and not a phosphatase-type reaction (Fig. 3).

The Anomeric Configuration of the Hydrolytic Product of bGlc1P
The anomeric configuration of Glc generated through the hydrolysis of bGlc1P by Bsel_2816 was determined by HPLC (Fig. 4). The a-anomer of Glc was predominant at the initiation of bGlc1P hydrolysis, indicating that Bsel_2816 catalyzes the hydrolysis of bGlc1P with the inversion of the anomeric configuration. This result suggests that the hydrolysis was not due to a phosphatase-type reaction, in which the anomeric configuration of Glc should be retained during the hydrolysis. In addition, the anomeric inversion suggests that water molecules play a role as acceptors in the reverse phosphorolysis causing the hydrolysis.

Determination of the Specificity of Bsel_2816
Because no acceptor was found within sugars, various alcohols (1-butanol, 2-butanol, cyclohexanol, benzylalcohol, hydroquinone, and phenol) and polyols (ethylene glycol, glycerol, erythritol, xylitol D-arabinitol, L-arabinitol, and D-glucitol) were used to identify the suitable acceptor. Only the hydrolysis of bGlc1P was detected with all the alcohols examined (data not shown). Among   polyols, the consumption of bGlc1P was obviously faster with glycerol than with others (Fig. 5). A spot exhibiting slightly different R f value with Glc was detected on TLC. The synthetic reaction gave a single product from glycerol. The NMR spectra are shown in Fig. S2 and the assignment of the signals are summarized in Table 2. The J 1,2 value of the Glc residue (3.9 Hz) indicates an a-D-glucoside. The HMBC cross signals indicate the glycosyl linkage with the position 2 of glycerol. Thus, the product was identified to be GG.
In the presence of glycerol, the synthesis of GG was much faster than the hydrolysis of bGlc1P (Fig. 6). In the presence of 10 mM glycerol, the hydrolysis rate was only 0.63% of that of the synthesis of GG. The hydrolysis was inhibited by glycerol ( Fig. 6) with the K d value of 7.6 mM. In addition, Bsel_2816 exhibited a weak reverse phosphorolysis activity on ethylene glycol, yielding two products (Glc and unknown) on TLC (Fig. 5). We confirmed the phosphorolysis of GG by the enzyme. During the phosphorolysis, the major product was bGlc1P, but a trace amount of Glc was detectable (Fig. 7). Herein, we propose 2-O-a-D-glucopyronosylglycerol: phosphate b-D-glucosyltransferase as the systematic name and 2-O-a-D-glucopyronosylglycerol phosphorylase (GGP) as the short name for Bsel_2816.

Basic Properties of Bsel_2816
Bsel_2816 was stable up to 35uC during 15-min incubation, and it was stable at pH between 5.5210.5 at 4uC for 24 h (data not shown). The optimum pHs for the phosphorolytic, synthetic, and hydrolytic reactions were pH 8.0, pH 7.5, and pH 6.0, respectively (data not shown).

Kinetic Analyses of Bsel_2816
The phosphorolytic reaction of GG was investigated at pH 8.0 at 30uC. Double reciprocal plots of the initial velocities against various initial concentrations of GG and P i presented a series of lines intersecting at a point (Fig. 8), indicating that the phosphorolysis follows a sequential Bi Bi mechanism, as reported with other inverting phosphorylases [1,17,19,20,26,[34][35][36][37][38][39]. The kinetic parameters were calculated to be K mA , 1.1 mM; K iA , 18 mM; K mB , 0.57 mM; and k cat 95 s 21 , where A and B are GG and Pi, respectively. The parameters for GG are in the same ranges of those of the other inverting phosphorylases [1,17,19,20,26,[34][35][36][37][38][39], indicating that GG is the true substrate of Bsel_2816.
The kinetic parameters of the substrates in the synthetic reaction (glycerol and bGlc1P) were determined at pH 7.5. The K m and k cat values with glycerol were 5.7 mM and 163 s 21 , respectively. The K m value of glycerol agreed with the K d value determined by the inhibition of hydrolysis of bGlc1P (7.6 mM). The K m and k cat values of bGlc1P were 0.45 mM and 180 s 21 , respectively.

Phosphorolytic and Hydrolytic Activities of Mutant Bsel_2816
Alignment of the amino acid sequences of GH65 phosphorylases suggests that E475 of Bsel_2816 is the general acid (for phosphorolysis)/base (for reverse phosphorolysis) catalyst [40,41]. The phosphorolytic and hydrolytic activities of two mutants of Figure 5. Screening for the suitable acceptor within polyols in the reverse phosphorolysis by Bsel_2816. The acceptors used are: 1, ethylene glycol; 2, glycerol; 3, erythritol; 4, no acceptor. S1 and S2, Glc and as bGlc1P as standards, respectively. The TLC plate was developed twice with acetonitrile-water (9:1 v/v). doi:10.1371/journal.pone.0086548.g005  Bsel_2816 at E475, E475A and E475Q, were less than 0.01% compared with those of the wild-type Bsel_2816, suggesting that both reactions were catalyzed by the same catalytic residue E475.

Discussion
We have determined that Bsel_2816 is a GGP. The reactions catalyzed by GG are summarized in Fig. 9. GGP is a unique phosphorylase in the following two characteristics.
First, any other phosphorylase reported exhibits its highest phosphorolytic activity on the glycosyl linkages bound with sugar residues (Table 1). Contrarily, GGP phosphorolyzes the glucosyl linkage bound with glycerol. Even though GG can be produced by other phosphorylases such as sucrose phosphorylase [42,43], it is one of their minor substrates. The phosphorylases generally exhibit K m values 1-10 mM with their main acceptors. Structural analyses of various phosphorylases have revealed that these enzymes recognize their acceptor molecules through their pyranose rings (or furanose ring in the case of sucrose phosphorylase) by a number of hydrogen bond interactions to obtain their strict acceptor specificities with the K m values. The K m value of GGP with glycerol was 5.7 mM, similarly to that of other phosphorylases, even though glycerol is a rather small molecule and does not adopt any rigid conformation.
Second, the other feature of GGP is the hydrolysis of bGlc1P. Other inverting phosphorylases do not hydrolyze sugar 1phosphates [2] and this is the first report describing the significant hydrolysis by an inverting phosphorylase.
To investigate the mechanism of the hydrolysis, we determined the linkage cleaved in the hydrolysis, the anomeric configuration of the hydrolytic product, and the catalytic residue of the enzyme. Our results prove that the hydrolysis of bGlc1P occurs through the same catalytic mechanism as the reverse phosphorolysis, not through a phosphatase-type reaction. The hydrolysis is explainable as a glucosyl transfer reaction from bGlc1P to a water molecule with the anomeric inversion. The phosphate-dependent hydrolysis of kojibiose is not considered to be a direct hydrolysis. It is explained as follows: GGP phosphorolyzes kojibiose at a very slow rate to form bGlc1P that is immediately hydrolyzed to generate Glc and Pi by the same enzyme. In this mechanism, the a-anomer of Glc is considered to be generated during the hydrolysis, indicating a retaining hydrolysis. Unfortunately, the anomeric analysis during the hydrolysis of kojibiose was unsuccessful because of its very slow reaction. On the other hand, the hydrolytic reaction was much slower than the phosphorolysis and the synthesis of GG, suggesting that the hydrolytic activity may be negligible in its in vivo role.
Recently, indication of faint hydrolytic activity on a-mannnose 1-phosphate (aMan1P) by UhgbMP belonging to GH130, a b-1,4mannooligosaccharide phosphorylase from unknown human gut bacterium, has been reported [44]. In the case, the enzyme at high concentration produced b-1,4-mannooligosaccharides with high degree of polymerization only from aMan1P, suggesting that the enzyme initially hydrolyzed aMan1P to generate mannose. The resultant mannose was not detected because it readily acted as the acceptor molecule to form b-1,4-mannobiose, which was a better acceptor than mannose to generate the polymer. This enzyme may share the same hydrolytic mechanism with GGP.
GG has been reported as a compatible solute for the control of the cytoplasmic osmotic pressure of cyanobacteria [45,46]. Because B. selenitireducens is a halophilic bacterium, which is often isolated from soil with high concentration of salts [24], GG may possess a similar role in this bacterium. The biosynthetic pathway of the GG of cyanobacteria is explained with the concomitant reactions of glucosylglycerol-phosphate synthase (EC 2.4.1.213) [47] and glucosylglycerol phosphatase (EC 3.1.3.69) [48]. Other possible enzymes that form a-glucosyl linkage at the position 2 of the derivatives of glycerol are glucosyl-3-phosphoglycerate synthase (EC 2.4.1.266) [49] and glucosylglycerate synthase (EC 2.4.1.268) [50]. However, no homologous genes encoding these enzymes were found in the genomic sequence of B. selenitireducens MLS10 (GenBank ID: CP000896). The role of GGP is unclear but it may be involved in the biosynthesis of GG as well as in its cleavage. B. selenitireducens possesses potassium ion-dependent trehalose phosphorylase, even though the strain does not have either intracellular or extracellular biosynthetic pathways of trehalose [27]. B. selenitireducens has one more GH65 enzyme, maltose phosphorylase [26], which is a possible candidate to produce bGlc1P. These three enzymes may relate with the  production of GG and trehalose in the cytoplasm of B. selenitireducens as solutes for the control of the cytoplasmic osmotic pressure. GG is often isolated from the traditional Japanese food, fermented with ''koji'' (Aspergillus oryzae) [51]. It is valued for its application as a moisturizing agent and as external reagent protecting proteins against denaturation [43,52]. The compound is commercially produced from maltose and glycerol using the transglycosylation activity of a-glucosidase as a mixture of aglucosylglycerols [53], or from sucrose and glycerol by the action of sucrose phosphorylase [43,54]. GGP is expected to be an alternative catalyst for the production of GG.