Journal of Applied Glycoscience
Online ISSN : 1880-7291
Print ISSN : 1344-7882
ISSN-L : 1344-7882
Regular Papers
Characterization of Two α-1,3-Glucoside Phosphorylases from Clostridium phytofermentans
Takanori NihiraMamoru NishimotoHiroyuki NakaiKen’ichi OhtsuboMotomitsu Kitaoka
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Supplementary material

2014 Volume 61 Issue 2 Pages 59-66

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Abstract

We characterized two α-1,3-glucoside phosphorylases that belonged to glycoside hydrolase family 65 from Clostridium phytofermentans: Cphy_3313 and Cphy_3314. Cphy_3313 was a typical nigerose phosphorylase that phosphorolyzed nigerose into D-glucose and β-D-glucose 1-phosphate (βGlc1P). Cphy_3314 catalyzed the synthesis of a series of α-1,3-oligoglucans using nigerose as the acceptor and βGlc1P as the donor. Kinetic analyses of their phosphorolytic reactions with α-1,3-oligoglucans (DP = 3 and 4) revealed that Cphy_3314 utilized a typical sequential Bi Bi mechanism, while this enzyme did not exhibit any significant phosphorolytic activity for nigerose. These results suggest that Cphy_3314 is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of α-1,3-oligoglucans with DP of 3 or higher. In this study, we propose 3-O-α-D-oligoglucan: phosphate β-D-glucosyltransferase as the systematic name and α-1,3-oligoglucan phosphorylase as the short name for Cphy_3314.

Abbreviations

βGlc1P, β-D-glucose 1-phosphate; GH, glycoside hydrolase family.

INTRODUCTION

Phosphorylases catalyze the cleavage of glycosyl linkages through substitution with inorganic phosphate (Pi).1) 2) 3) These enzymes reversibly phosphorolyze glycosides to form corresponding monosaccharide 1-phosphates with anomeric retention or inversion. Because all the phosphorylases reported to date have shown strict substrate specificity for phosphorolysis and regioselectivity during reverse phosphorolysis, they are considered useful catalysts for the synthesis of particular glycosides. This reversibility enables the combined reactions of two phosphorylases to produce valuable oligosaccharides from common sugars.4) 5) 6) 7) 8) 9) 10) However, the relatively narrow variations of these phosphorylases limit the use of these enzymes. Thus, discovering novel phosphorylases is desired.

Phosphorylases are classified as members of glycoside hydrolase families (GH) 13, 65, 94, 112, and 130, and glycosyltransferase families 4 and 35 in the Carbohydrate-Active Enzymes database (http://www.cazy.org/) based on their amino acid sequence similarities.11) Among these, GH65 is primarily comprised of phosphorylases that mainly catalyze the reversible phosphorolysis of α-glucosides to form β-D-glucose 1-phosphate (βGlc1P) with inversion of the anomeric configuration. The following phosphorylases are currently categorized into GH65 enzymes: maltose phosphorylase (EC 2.4.1.8),12) trehalose phosphorylase (EC 2.4.1.64),13) trehalose 6-phosphate phosphorylase (EC 2.4.1.216),14) kojibiose phosphorylase (EC 2.4.1.230),15) nigerose phosphorylase (EC 2.4.1.279),16) 3-O-α-D-glucosyl-L-rhamnose phosphorylase (EC 2.4.1.282)17) and 2-O-α-D-glucosylglycerol phosphorylase (EC 2.4.1.-).18) Among GH65 enzymes, the structures of maltose phosphorylase from Lactobacillus brevis19) and kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus20) are available.

Clostridium phytofermentans is an anaerobic mesophilic bacterium found in forest soil.21) Its entire genome has been sequenced (GenBank™ accession no. CP000885), which revealed that it possessed numerous glycoside hydrolases that could degrade various biomass carbohydrates. We noted that C. phytofermentans also possessed a number of genes encoding possible inverting phosphorylases, including four GH65, five GH94, three GH112 and two GH130 proteins. We have characterized all three of the GH112 enzymes and two of four GH65 enzymes and determined the following three unreported activities. One GH112 enzyme was a 4-O-β-D-galactosyl-L-rhamnose phosphorylase.22) Two GH65 enzymes were determined to be a nigerose phosphorylase (Cphy_1874)16) and a 3-O-α-D-glucosyl-L-rhamnose phosphorylase (Cphy_1019).17)

In this study, we report on the characterization of the remaining two GH65 enzymes from C. phytofermentans, Cphy_3313 and Cphy_3314, and identify a previously unreported phosphorylase.

MATERIALS AND METHODS

Amino acid sequence analysis. Multiple alignments of the amino acid sequences of GH65 proteins with known activities were made with those of Cphy_3313 and Cphy_3314 using the ClustalW version 2.1 on the DDBJ server (http://clustalw.ddbj.nig.ac.jp/index.php?lang=ja). A phylogenetic tree was constructed from these results by the neighbor-joining method using the TreeView version 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html).

Cloning, expression and purification. Expression vectors for cphy_3313 and cphy_3314 (GenBank ID: ABX43667 and ABX43668, respectively) were constructed by inserting the corresponding gene between the NdeI and XhoI sites of a pET24a (+) vector (Novagen Inc., Madison, USA) to encode for recombinant proteins with a His6 tag sequence added at the C-terminus of the recombinant protein.

Because cphy_3313 contained an NdeI site and an XhoI site, the expression vector for cphy_3313 was constructed using the joint PCR method23) as described below. The cphy_3313 gene was amplified by PCR using genomic DNA from C. phytofermentas22) as the template with the primer pair: 5′-gatatacatatggactggatgttgacggaa-3′ and 5′-ggtgctcgagaatacacactgcaaaagctt-3′ containing NdeI and XhoI sites (underlined), respectively, and the sequence of the vector at each site (italicized) using KOD-plus DNA polymerase (Toyobo Co., Ltd., Osaka, Japan). A linear vector backbone was amplified by PCR using pET24a (+) as the template with the primer pair: 5′-catccagtccatatgtatatctccttctta-3′ and 5′-tattctcgagcaccaccaccaccaccactg-3′ containing NdeI and XhoI sites (underlined), respectively, and the sequences of cphy_3313 (italicized). The amplified cphy_3313 and the vector backbone were purified using the MinElute Reaction Cleanup Kit (Qiagen GmbH, Hilden, Germany). These fragments were fused by overlap extension PCR using KOD-plus DNA polymerase without adding any primer. The insert-vector fusion for cphy_3313 was introduced into Escherichia coli BL21 (DE3) (Novagen Inc.) to form a circular plasmid.

The cphy_3314 was amplified by PCR using genomic DNA from C. phytofermentans with 5′-gatatacatatggcaaaaatagcagattta-3′ as the forward primer containing an NdeI site (underlined) and 5′-ggtgctcgagacatggtatctcaagtttta-3′ as the reverse primer containing an XhoI site (underlined). The amplified gene was purified using the MinElute Reaction Cleanup Kit, digested with NdeI and XhoI (New England Biolabs Inc., Beverly, USA), and inserted into the NdeI and XhoI sites of a pET24a (+) vector.

Both the expression plasmid for Cphy_3314 and the insert-vector fusion for Cphy_3313 were propagated in E. coli BL21 (DE3), purified using the Illustra PlasmidPrep Mini Spin Kit (GE Healthcare UK Ltd., Little Chalfont, UK), and verified by sequencing (ABI 3730xl Sequencer, Applied Biosystems, Foster City, USA).

Each E. coli BL21 (DE3) transformant that harbored each expression plasmid was grown at 37°C in 200 mL of Luria–Bertani medium (1% tryptone, 0.5% yeast extract and 0.5% NaCl) containing 50 μg/mL of kanamycin until absorbance reached 0.6 at 600 nm. Expression was induced by 0.1 mM isopropyl β-D-thiogalactopyranoside and continued at 18°C for 24 h. Cells were then harvested by centrifugation at 10,000 × G for 20 min and suspended in 50 mM HEPES‒NaOH buffer (pH 7.5) containing 500 mM NaCl (buffer A). Suspended cells were disrupted by sonication (Branson Sonifier 250A; Branson Ultrasonics, Emerson Japan, Ltd, Kanagawa, Japan). The supernatant collected by centrifugation at 20,000 × G for 20 min was applied to a HisTrap FF column (GE Healthcare UK Ltd.), equilibrated with buffer A containing 10 mM imidazole using ÄKTA prime (GE Healthcare UK Ltd.).

After washing with buffer A containing 22 mM imidazole and subsequent elution using a 22‒400 mM imidazole linear gradient in buffer A, fractions containing the target protein were pooled, dialyzed against 10 mM HEPES‒NaOH buffer (pH 7.0), and concentrated (AMICON Ultra-15 filter; Millipore Co., Billerica, USA). Protein concentration was spectrophotometrically determined at 280 nm using a theoretical extinction coefficient of ε = 135,000 cm-1M-1 and 139,010 cm-1M-1 for Cphy_3313 and Cphy_3314, respectively, based on their amino acid sequences.24)

The molecular masses of purified Cphy_3313 and Cphy_3314 were estimated by SDS-PAGE (Mini-PROTEAN Tetra electrophoresis system; Bio-Rad Laboratories, Inc., Hercules, USA) and by gel filtration (HiLoad 26/600 Superdex 200pg; GE Healthcare UK Ltd.) equilibrated with 10 mM HEPES‒NaOH buffer (pH 7.0) containing 150 mM NaCl at a flow rate of 0.5 mL/min. Marker Proteins for molecular Weight Determination on High Pressure Liquid Chromatography (Oriental Yeast Co., Ltd., Tokyo, Japan) were used as standards.

Measurement of phosphorolytic and reverse phosphorolytic activity. Phosphorolytic activity was routinely determined by quantifying βGlc1P released during a phosphorolytic reaction in 40 mM MOPS‒NaOH buffer (pH 7.0) for Cphy_3313 or 40 mM HEPES‒NaOH buffer (pH 8.0) containing 0.5 mg/mL of BSA for Cphy_3314 and 10 mM Pi and a 10 mM sugar substrate at 30°C by the β-phosphoglucomutase/glucose 6-phosphate dehydrogenase method25) as described below. Reaction mixtures (50 μL) were prepared in wells of a 384-well microtiter plate with a sugar substrate (routinely 10 mM), Pi (routinely 10 mM), and Cphy_3313 or Cphy_3314 in buffer solution [40 mM MOPS‒NaOH buffer (pH 7.0) for Cphy_3313 or 40 mM HEPES‒NaOH buffer (pH 8.0) containing 0.5 mg/mL of BSA for Cphy_3314] that contained 2.5 U/mL of β-phosphoglucomutase, 2.5 U/mL of glucose 6-phosphate dehydrogenase, 2 mM thio-NAD+, 5 μg/mL of D-glucose 1,6-bisphoaphate and 5 mM MgCl2. Reactions were carried out in a temperature-controlled microplate reader (MultiScan Go, ThermoFisher Scientific, Waltham, USA) at 30°C. Absorbance at 400 nm was continuously monitored at 30 s intervals without stopping a reaction. One IU of phosphorolytic activity was defined as the amount of enzyme that catalyzed the liberation of 1 μmol of βGlc1P from these substrates per min under the above conditions.

Reverse phosphorolytic activity was routinely determined by measuring the increase in Pi using a reaction mixture containing 10 mM βGlc1P and a 10 mM acceptor substrate in 40 mM MOPS‒NaOH buffer (pH 7.0) for Cphy_3313 or 40 mM MOPS‒NaOH buffer (pH 6.5) containing 0.5 mg/mL of BSA for Cphy_3314 at 30°C, in accordance with the method of Lowry and Lopez26) as described previously.16) One IU of reverse phosphorolytic activity was defined as the amount of enzyme that liberated 1 μmol of Pi per minute under the above conditions.

Acceptor specificity of Cphy_3314. Reaction mixtures containing 530 nM Cphy_3314, 10 mM βGlc1P, 10 mM acceptor in 25 mM MOPS‒NaOH buffer (pH 6.5) for various acceptor candidates were incubated at 30°C for 2 h. An aliquot (1 μL) of each reaction mixture was spotted on a TLC plate (Kieselgel 60 F254; Merck KGaA, Darmstadt, Germany), and the plate was developed with a mobile phase of 80% acetonitrile in water. TLC plates were soaked in a 5% sulfuric acid‒methanol solution and heated in an oven until the bands were visible to detect the products generated.

Preparation and structural determinations of Cphy_3314 reaction products. Reaction products for structural determinations were generated in 500 μL of a reaction mixture containing Cphy_3314 (960 nM), 50 mM βGlc1P and 50 mM nigerose in 100 mM MOPS‒NaOH buffer (pH 7.0). This reaction mixture was incubated at 30°C for 20 h, followed by desalting using Amberlite MB3 (Organo Co., Tokyo, Japan). The reaction products were purified using a Toyopearl HW40F column (26 mm internal diameter × 320 mm; Tosoh Co., Tokyo, Japan) equilibrated with distilled water at a flow rate of 0.5 mL/min. Fractions containing each single product were collected and those containing two products were chromatographed again. The pooled fractions were desalted again with Amberlite MB3, followed by lyophilization. The amounts of trisacharide and tetrasaccharide obtained were 3.8 and 3.4 mg, respectively.

One-dimensional (1H and 13C) 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 products were acquired in D2O with 2-methyl-2-propanol as an internal standard using a Bruker Avance 500 or Bruker Avance 800 spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany). Proton signals were assigned based on DQF-COSY spectra. 13C signals were assigned using HSQC spectra, based on the assignments of proton signals. The linkage position of each disaccharide was determined by detecting the inter-ring cross peaks in each HMBC spectrum.

To characterize the phosphorolytic activity of Cphy_3314, α-1,3-oligoglucans (DP = 3 and 4: nigerotriose and nigerotetraose, respectively) were synthesized in a 10 mL reaction mixture containing 960 nM Cphy_3314, 500 mM βGlc1P (buffered at pH 7.0 with HCl), and 500 mM nigerose. After incubation at 30°C for 24 h, an aliquot of the reaction mixture (2 mL) was desalted using Amberlite MB-3 and loaded onto the Toyopearl HW40S column (50 mm internal diameter × 950 mm; Tosoh Corporation) equilibrated with distilled water at a flow rate of 1 mL/min. Fractions containing nigerotriose and nigerotetraose were collected, concentrated, and lyophilized. These sugars were further purified with an HPLC system (Prominence; Shimadzu Corporation, Kyoto, Japan) equipped with a Shodex Asahipak NH2P-50 4E column (4.6 mm internal diameter × 25 cm; Showa Denko KK, Tokyo, Japan) and the RID-10A detector (Shimadzu Corporation) at 30°C under a constant flow (1.0 mL/min) of 60% (by volume) acetonitrile/water as the mobile phase. Fractions containing the products were collected, evaporated to remove acetonitrile, and finally lyophilized to yield 16 and 11 mg of nigerotriose and nigerotetraose, respectively.

Temperature and pH profiles. The effects of pH on the phosphorolytic and synthetic activities using 190 nM Cphy_3313 or 290 nM Cphy_3314 were measured under the standard conditions described above by substituting the buffer solution with the following 50 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). Thermal and pH stabilities were evaluated by measuring residual synthetic activity under the standard conditions after incubating Cphy_3313 or Cphy_3314 (570 nM or 950 nM, respectively) in the temperature range of 25‒80°C for 10 min in 25 mM MOPS‒NaOH buffer (pH 7.0) or 25 mM MOPS‒NaOH buffer (pH 6.5) and at the various pH values at 30°C for 30 min, respectively.

Kinetic analysis. The initial velocities of the phosphorolytic reactions with a substrate were determined using the standard conditions used for the continuous βGlc1P assay with 29 nM Cphy_3313 or 290 nM Cphy_3314 and combinations of initial concentrations of substrates (0.5‒5.0 mM nigerose for Cphy_3313, or 0.5‒5.0 mM nigerotriose or 1.0‒10 mM nigerotetraose for Cphy_3314) and Pi (0.5‒5.0 mM for Cphy_3313 or 1.0‒25 mM for Cphy_3314). Kinetic parameters were determined by fitting the experimental data to the following theoretical equation for a sequential bi-bi mechanism using GraFit version 7.0.2 (Erithacus Software Ltd., London, UK).

v = kcat[E]0[A][B]/(KiAKmB + KmA [B] + KmB [A] + [A][B])

(A = substrate, B = Pi)

Kinetic analysis of a synthetic reaction with an acceptor substrate was performed using standard conditions with Cphy_3313 (107 nM for D-glucose, 2.1 μM for methyl-α-D-glucoside, 430 nM for the other acceptors) or Cphy_3314 (220 nM for nigerose and 580 nM for nigerotriose, and 1.1 μM for the other acceptors) by substituting a 10 mM acceptor substrate with various concentrations of acceptors (1.0‒70 mM) and 10 mM βGlc1P with various concentration of βGlc1P (0.25‒20 mM). The kinetic parameters for acceptor substrates were determined by fitting the experimental data to the Michaelis‒Menten equation: {v = kcat [E]0 [S]/(Km + [S])}, using GraFit version 7.0.2.

RESULTS

Gene cloning, expression and purification.

Cphy_3313 and Cphy_3314 were not predicted to possess N-terminal signal peptides according to the SignalP 4.0 server (http://www.cbs.dtu.dk/services/SignalP/),27) which suggested that these enzymes were located in the cytoplasm. A phylogenetic tree analysis showed that Cphy_3313 was relatively homologous to nigerose phosphorylase of the same strain (Cphy_1874)16) (Fig. 1). They had 52% identity and 70% similarity in their amino acid sequences. In contrast, Cphy_3314 appeared between the clades of maltose phosphorylase and trehalose phosphorylase.

Fig. 1.

Phylogenetic tree for characterized GH65 enzymes.

Locus tags are given for the proteins from Clostridium phytofermentans. Sequences used were: 1, Cphy_3313 of C. phytofermentans ISDg (GenBank ID: ABX43667.1); 2, Cphy_1874 of C. phytofermentans ISDg (ABX42243.1); 3, Cphy_3314 of C. phytofermentans ISDg (ABX43668.1); 4, Bsel_2056 of Bacillus selenitireducens MLS10 (ADH99560.1); 5, MapA of Paenibacillus sp. SH-55 (BAD97810.1); 6, MPase of Bacillus sp. RK-1 (BAC54904.1); 7, EF0957 of Enterococcus faecalis V583 (AAO80764.1); 8, maltose phosphorylase of Lactobacillus sanfranciscensis DSM 20451 (CAA11905.1); 9, maltose phosphorylase of Lactobacillus brevis ATCC 8287 (UniProt ID: Q7SIE1); 10, LBA1870 of Lactobacillus acidophilus NCFM (AAV43670.1); 11, TreP of Thermoanaerobacter brockii ATCC 35047 (AAE18727.1); 12, Bsel_1207 of B. selenitireducens MLS10 (ADH98720.1); 13, TPase of Geobacillus stearothermophilus SK-1 (BAC20640.1); 14, TrePP of Lactococcus lactis subsp. lactis Il1403 (AAK04526.1); 15, TreA of Aspergillus nidulans FGSCA4 (EAA66407.1); 16, Atm1 of Metarhizium acridum CQMA102 (ABB51158.1); 17, Atc1 of Candida albicans (AAV05390.1); 18, Ath1 of Saccharomyces cerevisiae S288c (CAA58961.1); 19, Cphy_1019 of C. phytofermentans ISDg (ABX41399.1); 20, Csac_0439 of Caldicellulosiruptor saccharolyticus DSM 8903 (ABP66077.1); 21, KojP of T. brockii ATCC 35047 (AAE30762.1); 22, All1058 of Nostoc sp. PCC 7120 (BAB73015.1); 23, All4989 of Nostoc sp. PCC 7120 (BAB76688.1); 24, Bsel_2816 of B. selenitireducens MLS10 (ADI00307.1).

Recombinant Cphy_3313 and Cphy_3314 proteins were purified to yields of approximately 2 and 5 mg, respectively, using cell lysates from 200 mL cultures. Purified Cphy_3313 and Cphy_3314 migrated in SDS-PAGE as single protein bands with estimated sizes of approximately 85 and 95 kDa, respectively, in agreement with their respective theoretical molecular masses of 86,176, and 93,977. Gel filtration analysis revealed molecular masses for Cphy_3313 and Cphy_3314 of approximately 197 and 196 kDa, respectively, which suggested that these enzymes were homodimers in solution.

Characterization of Cphy_3313.

Cphy_3313 phosphorolyzed nigerose into D-glucose and βGlc1P, suggesting that it was a nigerose phosphorylase, as predicted from its sequence analysis. This reaction followed a sequential Bi Bi mechanism with the following kinetic parameters: kcat = 31 s-1, KmA = 1.2 mM, KmB = 0.49 mM and KiA = 2.0 mM, where A and B represent nigerose and phosphate, respectively (Fig. 2). These values were similar to those for another nigerose phosphorylase, Cphy_1874 (kcat = 67 s-1, KmA = 1.7 mM, KmB = 0.20 mM and KiA = 6.4 mM).16) The acceptor specificity of Cphy_3313 during reverse phosphorolysis was again similar to that of Cphy_187416) as shown in Table 1. The kinetic parameters for major acceptors and donors are summarized in Table 2.

Fig. 2.

Double reciprocal plots for nigerose phosphorolysis by Cphy_3313 at different Pi concentrations.

Pi concentrations are: Open circles, 0.5 mM; closed circles, 1.0 mM; open squares, 2.0 mM; closed squares, 3.0 mM; open triangles, 5.0 mM.

Table 1.

Acceptor specificity of Cphy_3313 compared with the other nigerose phosphorylase.

Following sugars did not show acceptor activity: D-mannose, D-allose, D-fructose, D-lyxose, D-arabinose, L-arabinose, D-glucosamine, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, L-fucose, L-rhamnose, D-glucal, 2-deoxy-D-glucose, α-D-glucose 1-phosphate, α-D-galactose 1-phosphate, D-glucose 6-phosphate, methyl β-D-glucoside, 3-O-methyl-D-glucose, trehalose, kojibiose, nigerose, maltose, isomaltose, sophorose, laminaribiose, cellobiose, gentiobiose, xylobiose, melibiose, lactose, sucrose. aAdopted from Ref. 16). bValues in parenthesis represent relative activities for which specific activities for D-glucose were defined as 100%.

Table 2.

Kinetic parameters for reverse phosphorolysis catalyzed by Cphy_3313.

aβGlc1P (10 mM) was used as the donor. bD-Glucose (10 mM) was used as the acceptor.

Characterization of Cphy_3314.

Cphy_3314 did not significantly phosphorolyze α-linked glucobioses, such as trehalose, kojibiose, nigerose, and maltose that are typical substrates for GH65 phosphorylases. The acceptor specificity of Cphy_3314 was investigated by TLC as shown in Fig. 3. Formation of oligosaccharides was detected when nigerose and maltose were used as acceptors, which suggested that these two disaccharides were good acceptors. The acceptor specificity with nigerose and maltose were determined to be 4.0 and 1.4 IU/mg, respectively. Carbohydrate spots at the TLC origin were detected with the following acceptors (see Fig. 3): D-glucose, D-galactose; D-xylose; D-mannose; trehalose; kojibiose; isomaltose; sucrose; L-arabinose; methyl-α-D-glucoside; methyl-β-D-glucoside; 2-deoxy-D-glucose; 1,5-anhydro-D-glucitol; laminaribiose. The formation of polysaccharides suggested that these sugars were poor acceptors for Cphy_3314 and that their initial glucosyl-transferred products were much better acceptors. Similar phenomena have been reported for phosphorylases specific for DP of ≥ 3 in their phosphorolysis, such as cellodextrin phosphorylase28) and sophorooligosaccharide phosphorylase29) with D-glucose as the acceptor. The kinetic parameters of Cphy_3314 for these acceptors and those for donor βGlc1P are given in Table 3.

Table 3.

Kinetic parameters for reverse phosphorolysis catalyzed by Cphy_3314.

aβGlc1P (10 mM) was used as the donor. bNigerose (10 mM) was used as the acceptor. cDetermined from the slopes of linear s‒v plots.

Reverse phosphorolysis with nigerose as the acceptor resulted in a series of oligosaccharides (Fig. 3). By NMR analysis, the resulting trisaccharide and tetrasaccharide were determined to be 3-O-α-D-glucopyranosyl-3-O-α-D-glucopyranosyl-D-glucose (nigerotriose, J-STAGE Electric Supplentary Material, Fig. S1) and 3-O-α-D-glucopyranosyl-3-O-α-D-glucopyranosyl-3-O-α-D-glucopyranosyl-D-glucose (nigerotetraose, J-STAGE Electric Supplentary Material, Fig. S2), respectively.

Fig 3.

TLC analysis for Cphy_3314 acceptor specificity.

“−” and “+” indicate in the absence and presence of Cphy_3314, respectively. Arrows show the spots of βGlc1P. Acceptors used were: 1, D-glucose; 2, D-galactose; 3, D-xylose; 4, D-mannose; 5 N-acetyl-D-glucosamine; 6, N-acetyl-D-galactosamine; 7, D-fructose; 8, D-galactose 6-phosphate; 9, L-rhamnose; 10, D-allose; 11, D-arabinose; 12, trehalose; 13, kojibiose; 14, maltose; 15, isomaltose; 16, melibiose; 17, sucrose; 18, D-lyxose; 19, L-arabinose; 20, L-fucose; 21, D-glucal; 22, D-glucosamine; 23, methyl-α-D-glucoside; 24, methyl-β-D-glucoside; 25, 3-O-methyl-D-glucose; 26, 2-deoxy-D-glucose; 27, 1,5-anhydro-D-glucitol; 28, L-rhamnose; 29, xylobiose; 30, cellobiose; 31, sophorose; 32, laminaribiose; 33, gentiobiose; 34, lactose; 35, nigerose.

During the phosphorolytic reaction, Cphy_3314 phosphorolyzed nigerotriose and nigerotetraose with inversion of the anomeric configuration and releasing βGlc1P in the presence of Pi. In addition, Cphy_3314 did not cleave nigerotriose and nigerotetraose in the absence of Pi. Double reciprocal plots of the initial velocities against various initial concentrations of nigerotriose or nigerotetraose and phosphate gave a series of lines that intersected at a point (Fig. 4). Kinetic parameters for activities on nigerotriose and nigerotetraose in the equation for the sequential Bi Bi mechanism were determined to be: kcat = 11 and 8.5 s−1, KmA = 3.2 and 2.9 mM, KmB = 11 and 2.6 mM, and KiA = 5.8 and 32 mM, respectively, where A and B represent α-1,3-oligoglucans and phosphate, respectively.

Fig 4.

Double reciprocal plots of the Cphy_3314 phosphorolytic reaction at different Pi concentrations.

(A) Nigerotriose, (B) nigerotetraose. Pi concentrations are: Open circles, 1.0 mM; closed circles, 2.5 mM; open squares, 5.0 mM; closed squares, 10 mM; open triangles, 25 mM.

Phosphorolytic activities on α-1,3-oligoglucans, except for nigerose, have not been previously reported, which suggests that Cphy_3314 is an unreported phosphorylase. Therefore, we propose 3-O-α-D-oligoglucan: phosphate β-D-glucosyltransferase as the systematic name and α-1,3-oligoglucan phosphorylase as the short name for this Cphy_3314.

Basic properties of Cphy_3313 and Cphy_3314.

Cphy_3313 and Cphy_3314 were stable up to 40 and 35°C, respectively, during a 10 min incubation. Cphy_3313 and Cphy_3314 were stable at 30°C for 30 min in the pH ranges of 5.5‒9.0 and 5.5‒9.5, respectively, and exhibited their highest apparent phosphorolytic activity for nigerose and nigerotriose at pH 7.0 and 8.0, respectively. The synthetic reactions using βGlc1P as the donor and D-glucose and nigerose as the acceptors had optimum pH values of 7.0 and 6.5, respectively.

DISCUSSION

We report that Cphy_3313 and Cphy_3314 are a nigerose phosphorylase and an α-1,3-oligoglucan phosphorylase from C. phytofermentans, both of which are considered to be involved in the metabolism of α-1,3-glucans, such as nigeran and pseudonigeran. We previously reported another nigerose phosphorylase, Cphy_1874, encoded for in a gene cluster for an α-1,3-glucan metabolic pathway.16) The presence of three phosphorylases related to the metabolism of α-1,3-glucans suggests that α-1,3-glucans may be important carbon sources for C. phytofermentans. α-1,3-Glucans are often produced by fungi and yeast,30) which may be rich in forest soil where C. phytofermentans inhabits.

Genes encoding for components of an ATP binding cassette (ABC) transporter (cphy_3315 to cphy_3317) are found upstream of cphy_3313 and cphy_3314. It has often been reported that genes encoding for an ABC transporter specific for a phosphorylase substrate are located close to the gene encoding for the phosphorylase.31) 32) 33) Therefore, Cphy_3313 and Cphy_3314 probably utilize α-1,3-oligoglucans that are transported into the cytosol. α-1,3-Oligoglucans may be generated outside of a cell with the degradation of α-1,3-glucans by extracellular enzymes encoded around the other nigerose phosphorylase. A gene encoding for β-phosphoglucomutase, an enzyme that converts βGlc1P into D-glucose 6-phosphate, cphy_3311, is also found close to cphy_3313 and cphy_3314. It should be noted that the other two genes of C. phytofermentans that encode for GH65 phosphorylases (cphy_1019 and cphy_1874) are also surrounded by genes for an ABC transporter (cphy_1013 to cphy_1015 and cphy_1879 to cphy_1881, respectively) and β-phosphoglucomutase (cphy_1021 and cphy_1875, respectively).

C. phytofermentans possesses two phosphorylases for utilizing α-1,3-oligoglucans. Nigerose phosphorylase (Cphy_3313 and Cphy_1874) is strictly specific for disaccharides and α-1,3-oligoglucan phosphorylase is specific for those oligosaccharides with DP values of 3 or higher. Such combinations of two phosphorlyases, one strictly specific for DP = 2 and the other specific for DP ≥ 3, have been reported for the metabolism of cellooligosaccharides by Clostridium thermocellum,34) Clostridium stercorarium35) and Ruminococcus albus36) (cellobiose phosphorylase and cellodextrin phosphorylase), and in the metabolism of β-1,4-mannooligosaccharides by R. albus (4-O-β-D-mannosyl-D-glucose phosphorylase and β-1,4-mannooligosaccharide phosphorylase).37)

Considering the utilization of polysaccharides, such a system makes the number of glycosyl linkages to be phosphorolyzed greater than that by a disaccharide phosphorylase alone. Because phosphorolysis generates a sugar 1-phosphate that directly enters the glycolytic pathway without consuming ATP, a two phosphorylase system is effective for the utilization of polysaccharides by these microorganisms.

In conclusion, we found an unreported phosphorylase, α-1,3-oligoglucan phosphorylase, that requires a new EC number.

ACKNOWLEDGMENTS

We thank the staff members of the Instrumental Analysis Center for Food Chemistry of the National Food Research Institute for recording NMR spectra. We also thank Enago (http://www.enago.jp) for the English language review. This work was supported in part by a grant from Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries and Food Industry and MEXT’s program ‘Promotion of Environmental Improvement for Independence of Young Researchers’ under the Special Coordination Funds for Promoting Science and Technology.

REFERENCES
 
© 2014 by The Japanese Society of Applied Glycoscience
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