Characterization of Two Α-1, 3-Glucoside Phosphorylases From

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J. Appl. Glycosci., 61, 59‒66 (2014) doi: 10.5458/jag.jag. JAG-2013_013 ©2014 The Japanese Society of Applied Glycoscience Regular Paper Characterization of Two α-1,3-Glucoside Phosphorylases from Clostridium phytofermentans (Received January 22, 2014; Accepted February 17, 2014) (J-STAGE Advance Published Date: February 26, 2014) Takanori Nihira,1,2 Mamoru Nishimoto,1 Hiroyuki Nakai,2 Ken’ichi Ohtsubo2 and Motomitsu Kitaoka1,＊ 1National Food Research Institute, National Agriculture and Food Research Organization (2‒1‒12 Kannondai, Tsukuba, Ibaraki 305‒8642, Japan) 2Graduate School of Science and Technology, Niigata University (8050 Ikarashi 2-no-cho, Nishi-ku, Niigata, Niigata 950‒2181, Japan)

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.

Key words: α-1,3-oligoglucan phosphorylase, α-1,3-oligoglucan, nigerose, nigerose phosphorylase, glycoside hydrolase family 65, Clostridium phytofermentans

INTRODUCTION phosphorylase (EC 2.4.1.8),12) trehalose phosphorylase (EC 2.4.1.64),13) trehalose 6-phosphate phosphorylase (EC Phosphorylases catalyze the cleavage of glycosyl linkages 2.4.1.216),14) kojibiose phosphorylase (EC 2.4.1.230),15) through substitution with inorganic phosphate (Pi).1‒3) These nigerose phosphorylase (EC 2.4.1.279),16) 3-O-α-D-glucosyl- enzymes reversibly phosphorolyze glycosides to form L-rhamnose phosphorylase (EC 2.4.1.282)17) and 2-O-α- corresponding monosaccharide 1-phosphates with anomeric D-glucosylglycerol phosphorylase (EC 2.4.1.-).18) Among retention or inversion. Because all the phosphorylases GH65 enzymes, the structures of maltose phosphorylase reported to date have shown strict substrate specificity for from Lactobacillus brevis19) and kojibiose phosphorylase phosphorolysis and regioselectivity during reverse phospho- from Caldicellulosiruptor saccharolyticus20) are available. rolysis, they are considered useful catalysts for the synthesis Clostridium phytofermentans is an anaerobic mesophilic of particular glycosides. This reversibility enables the bacterium found in forest soil.21) Its entire genome has been combined reactions of two phosphorylases to produce sequenced (GenBank™ accession no. CP000885), which valuable oligosaccharides from common sugars.4‒10) revealed that it possessed numerous glycoside hydrolases However, the relatively narrow variations of these phosphor- that could degrade various biomass carbohydrates. We noted ylases limit the use of these enzymes. Thus, discovering that C. phytofermentans also possessed a number of genes novel phosphorylases is desired. encoding possible inverting phosphorylases, including four Phosphorylases are classified as members of glycoside GH65, five GH94, three GH112 and two GH130 proteins. hydrolase families (GH) 13, 65, 94, 112, and 130, and We have characterized all three of the GH112 enzymes and glycosyltransferase families 4 and 35 in the Carbohydrate- two of four GH65 enzymes and determined the following Active Enzymes database (http://www.cazy.org/) based on three unreported activities. One GH112 enzyme was a their amino acid sequence similarities.11) Among these, 4-O-β-D-galactosyl-L-rhamnose phosphorylase.22) Two GH65 is primarily comprised of phosphorylases that mainly GH65 enzymes were determined to be a nigerose phosphor- catalyze the reversible phosphorolysis of α-glucosides to ylase (Cphy_1874)16) and a 3-O-α-D-glucosyl-L-rhamnose form β-D-glucose 1-phosphate (βGlc1P) with inversion of phosphorylase (Cphy_1019).17) the anomeric configuration. The following phosphorylases In this study, we report on the characterization of the are currently categorized into GH65 enzymes: maltose remaining two GH65 enzymes from C. phytofermentans, Cphy_3313 and Cphy_3314, and identify a previously ＊ Corresponding author (Tel. +81‒29‒838‒8071; Fax. +81‒29‒838‒7321; unreported phosphorylase. E-mail: [email protected]). Abbreviations: βGlc1P, β-D-glucose 1-phosphate; GH, glycoside hydrolase family. 60 J. Appl. Glycosci., Vol. 61, No. 2 (2014)

MATERIALS AND METHODS 10,000 × G for 20 min and suspended in 50 mM HEPES‒ NaOH buffer (pH 7.5) containing 500 mM NaCl (buffer A). Amino acid sequence analysis. Multiple alignments of the Suspended cells were disrupted by sonication (Branson amino acid sequences of GH65 proteins with known activi- Sonifier 250A; Branson Ultrasonics, Emerson Japan, Ltd, ties were made with those of Cphy_3313 and Cphy_3314 Kanagawa, Japan). The supernatant collected by centrifuga- using the ClustalW version 2.1 on the DDBJ server (http:// tion at 20,000 × G for 20 min was applied to a HisTrap FF clustalw.ddbj.nig.ac.jp/index.php?lang=ja). A phylogenetic column (GE Healthcare UK Ltd.), equilibrated with buffer A tree was constructed from these results by the neighbor- containing 10 mM imidazole using ÄKTA prime (GE joining method using the TreeView version 1.6.6 (http:// Healthcare UK Ltd.). taxonomy.zoology.gla.ac.uk/rod/rod.html). After washing with buffer A containing 22 mM imidazole Cloning, expression and purification. Expression vectors and subsequent elution using a 22‒400 mM imidazole linear for cphy_3313 and cphy_3314 (GenBank ID: ABX43667 gradient in buffer A, fractions containing the target protein and ABX43668, respectively) were constructed by inserting were pooled, dialyzed against 10 mM HEPES‒NaOH buffer the corresponding gene between the NdeI and XhoI sites of (pH 7.0), and concentrated (AMICON Ultra-15 filter; a pET24a (+) vector (Novagen Inc., Madison, USA) to Millipore Co., Billerica, USA). Protein concentration was encode for recombinant proteins with a His6 tag sequence spectrophotometrically determined at 280 nm using a added at the C-terminus of the recombinant protein. theoretical extinction coefficient of ε = 135,000 cm－1M－1 Because cphy_3313 contained an NdeI site and an XhoI and 139,010 cm－1M－1 for Cphy_3313 and Cphy_3314, site, the expression vector for cphy_3313 was constructed respectively, based on their amino acid sequences.24) using the joint PCR method23) as described below. The The molecular masses of purified Cphy_3313 and cphy_3313 gene was amplified by PCR using genomic DNA Cphy_3314 were estimated by SDS-PAGE (Mini-PROTEAN from C. phytofermentas22) as the template with the primer Tetra electrophoresis system; Bio-Rad Laboratories, Inc., pair: 5′-gatatacatatggactggatgttgacggaa-3′ and 5′-ggtgctcgag Hercules, USA) and by gel filtration (HiLoad 26/600 aatacacactgcaaaagctt-3′ containing NdeI and XhoI sites Superdex 200pg; GE Healthcare UK Ltd.) equilibrated with (underlined), respectively, and the sequence of the vector at 10 mM HEPES‒NaOH buffer (pH 7.0) containing 150 mM each site (italicized) using KOD-plus DNA polymerase NaCl at a flow rate of 0.5 mL/min. Marker Proteins for (Toyobo Co., Ltd., Osaka, Japan). A linear vector backbone molecular Weight Determination on High Pressure Liquid was amplified by PCR using pET24a (+) as the template Chromatography (Oriental Yeast Co., Ltd., Tokyo, Japan) with the primer pair: 5′-catccagtccatatgtatatctccttctta-3′ and were used as standards. 5′-tattctcgagcaccaccaccaccaccactg-3′ containing NdeI and Measurement of phosphorolytic and reverse phosphoro- XhoI sites (underlined), respectively, and the sequences of lytic activity. Phosphorolytic activity was routinely cphy_3313 (italicized). The amplified cphy_3313 and the determined by quantifying βGlc1P released during a vector backbone were purified using the MinElute Reaction phosphorolytic reaction in 40 mM MOPS‒NaOH buffer (pH Cleanup Kit (Qiagen GmbH, Hilden, Germany). These 7.0) for Cphy_3313 or 40 mM HEPES‒NaOH buffer (pH fragments were fused by overlap extension PCR using 8.0) containing 0.5 mg/mL of BSA for Cphy_3314 and KOD-plus DNA polymerase without adding any primer. The 10 mM Pi and a 10 mM sugar substrate at 30°C by the insert-vector fusion for cphy_3313 was introduced into β-phosphoglucomutase/glucose 6-phosphate dehydrogenase Escherichia coli BL21 (DE3) (Novagen Inc.) to form a method25) as described below. Reaction mixtures (50 µL) circular plasmid. were prepared in wells of a 384-well microtiter plate with a The cphy_3314 was amplified by PCR using genomic sugar substrate (routinely 10 mM), Pi (routinely 10 mM), DNA from C. phytofermentans with 5′-gatatacatatggcaaaaa and Cphy_3313 or Cphy_3314 in buffer solution [40 mM tagcagattta-3′ as the forward primer containing an NdeI site MOPS‒NaOH buffer (pH 7.0) for Cphy_3313 or 40 mM (underlined) and 5′-ggtgctcgagacatggtatctcaagtttta-3′ as the HEPES‒NaOH buffer (pH 8.0) containing 0.5 mg/mL of reverse primer containing an XhoI site (underlined). The BSA for Cphy_3314] that contained 2.5 U/mL of amplified gene was purified using the MinElute Reaction β-phosphoglucomutase, 2.5 U/mL of glucose 6-phosphate Cleanup Kit, digested with NdeI and XhoI (New England dehydrogenase, 2 mM thio-NAD+, 5 µg/mL of D-glucose Biolabs Inc., Beverly, USA), and inserted into the NdeI and 1,6-bisphoaphate and 5 mM MgCl2. Reactions were carried XhoI sites of a pET24a (+) vector. out in a temperature-controlled microplate reader (MultiScan Both the expression plasmid for Cphy_3314 and the Go, ThermoFisher Scientific, Waltham, USA) at 30°C. insert-vector fusion for Cphy_3313 were propagated in E. Absorbance at 400 nm was continuously monitored at 30 s coli BL21 (DE3), purified using the Illustra PlasmidPrep intervals without stopping a reaction. One IU of phosphoro- Mini Spin Kit (GE Healthcare UK Ltd., Little Chalfont, lytic activity was defined as the amount of enzyme that UK), and verified by sequencing (ABI 3730xl Sequencer, catalyzed the liberation of 1 µmol of βGlc1P from these Applied Biosystems, Foster City, USA). substrates per min under the above conditions. Each E. coli BL21 (DE3) transformant that harbored each Reverse phosphorolytic activity was routinely determined expression plasmid was grown at 37°C in 200 mL of Luria– by measuring the increase in Pi using a reaction mixture Bertani medium (1% tryptone, 0.5% yeast extract and 0.5% containing 10 mM βGlc1P and a 10 mM acceptor substrate NaCl) containing 50 µg/mL of kanamycin until absorbance in 40 mM MOPS‒NaOH buffer (pH 7.0) for Cphy_3313 or reached 0.6 at 600 nm. Expression was induced by 0.1 mM 40 mM MOPS‒NaOH buffer (pH 6.5) containing 0.5 mg/ isopropyl β-D-thiogalactopyranoside and continued at 18°C mL of BSA for Cphy_3314 at 30°C, in accordance with the for 24 h. Cells were then harvested by centrifugation at method of Lowry and Lopez26) as described previously.16) Nihira et al.: α-1,3-Oligoglucan Phosphorylase 61

One IU of reverse phosphorolytic activity was defined as the collected, evaporated to remove acetonitrile, and finally amount of enzyme that liberated 1 µmol of Pi per minute lyophilized to yield 16 and 11 mg of nigerotriose and nigero- under the above conditions. tetraose, respectively. Acceptor specificity of Cphy_3314. Reaction mixtures Temperature and pH profiles. The effects of pH on the containing 530 nM Cphy_3314, 10 mM βGlc1P, 10 mM phosphorolytic and synthetic activities using 190 nM acceptor in 25 mM MOPS‒NaOH buffer (pH 6.5) for various Cphy_3313 or 290 nM Cphy_3314 were measured under the acceptor candidates were incubated at 30°C for 2 h. An standard conditions described above by substituting the aliquot (1 µL) of each reaction mixture was spotted on a buffer solution with the following 50 mM buffers: sodium TLC plate (Kieselgel 60 F254; Merck KGaA, Darmstadt, citrate (pH 3.0‒5.5); bis(2-hydroxyethyl)iminotris (hydroxy- Germany), and the plate was developed with a mobile phase methyl)methane‒HCl (pH 5.5‒7.0); HEPES‒NaOH (pH of 80% acetonitrile in water. TLC plates were soaked in a 7.0‒8.5); and glycine‒NaOH (pH 8.5‒10.5). Thermal and 5% sulfuric acid‒methanol solution and heated in an oven pH stabilities were evaluated by measuring residual synthet- until the bands were visible to detect the products generated. ic activity under the standard conditions after incubating Preparation and structural determinations of Cphy_3314 Cphy_3313 or Cphy_3314 (570 nM or 950 nM, respective- reaction products. Reaction products for structural ly) in the temperature range of 25‒80°C for 10 min in 25 mM determinations were generated in 500 µL of a reaction MOPS‒NaOH buffer (pH 7.0) or 25 mM MOPS‒NaOH mixture containing Cphy_3314 (960 nM), 50 mM βGlc1P buffer (pH 6.5) and at the various pH values at 30°C for 30 and 50 mM nigerose in 100 mM MOPS‒NaOH buffer (pH min, respectively. 7.0). This reaction mixture was incubated at 30°C for 20 h, Kinetic analysis. The initial velocities of the phosphoro- followed by desalting using Amberlite MB3 (Organo Co., lytic reactions with a substrate were determined using the Tokyo, Japan). The reaction products were purified using a standard conditions used for the continuous βGlc1P assay Toyopearl HW40F column (26 mm internal diameter × with 29 nM Cphy_3313 or 290 nM Cphy_3314 and combina- 320 mm; Tosoh Co., Tokyo, Japan) equilibrated with distilled tions of initial concentrations of substrates (0.5‒5.0 mM water at a flow rate of 0.5 mL/min. Fractions containing each nigerose for Cphy_3313, or 0.5‒5.0 mM nigerotriose or single product were collected and those containing two 1.0‒10 mM nigerotetraose for Cphy_3314) and Pi (0.5‒ products were chromatographed again. The pooled fractions 5.0 mM for Cphy_3313 or 1.0‒25 mM for Cphy_3314). were desalted again with Amberlite MB3, followed by Kinetic parameters were determined by fitting the experi- lyophilization. The amounts of trisacharide and tetrasaccha- mental data to the following theoretical equation for a ride obtained were 3.8 and 3.4 mg, respectively. sequential bi-bi mechanism using GraFit version 7.0.2 One-dimensional (1H and 13C) and two-dimensional (Erithacus Software Ltd., London, UK). [double-quantum-filtered correlation spectroscopy (DQF- v = kcat[E]0[A][B]/(KiAKmB + KmA [B] + KmB [A] + [A][B]) COSY), heteronuclear single-quantum coherence (HSQC), (A = substrate, B = Pi) and heteronuclear multiple-bond correlation (HMBC)] NMR spectra of the products were acquired in D2O with Kinetic analysis of a synthetic reaction with an acceptor 2-methyl-2-propanol as an internal standard using a Bruker substrate was performed using standard conditions with Avance 500 or Bruker Avance 800 spectrometer (Bruker Cphy_3313 (107 nM for D-glucose, 2.1 µM for methyl-α- Biospin GmbH, Rheinstetten, Germany). Proton signals D-glucoside, 430 nM for the other acceptors) or Cphy_3314 were assigned based on DQF-COSY spectra. 13C signals (220 nM for nigerose and 580 nM for nigerotriose, and were assigned using HSQC spectra, based on the assign- 1.1 µM for the other acceptors) by substituting a 10 mM ments of proton signals. The linkage position of each acceptor substrate with various concentrations of acceptors disaccharide was determined by detecting the inter-ring (1.0‒70 mM) and 10 mM βGlc1P with various concentra- cross peaks in each HMBC spectrum. tion of βGlc1P (0.25‒20 mM). The kinetic parameters for To characterize the phosphorolytic activity of Cphy_3314, acceptor substrates were determined by fitting the experi- α-1,3-oligoglucans (DP = 3 and 4: nigerotriose and nigerote- mental data to the Michaelis‒Menten equation: {v = kcat [E]0 traose, respectively) were synthesized in a 10 mL reaction [S]/(Km + [S])}, using GraFit version 7.0.2. mixture containing 960 nM Cphy_3314, 500 mM βGlc1P (buffered at pH 7.0 with HCl), and 500 mM nigerose. After RESULTS incubation at 30°C for 24 h, an aliquot of the reaction mixture (2 mL) was desalted using Amberlite MB-3 and loaded onto Gene cloning, expression and purification. the Toyopearl HW40S column (50 mm internal diameter × Cphy_3313 and Cphy_3314 were not predicted to possess 950 mm; Tosoh Corporation) equilibrated with distilled N-terminal signal peptides according to the SignalP 4.0 water at a flow rate of 1 mL/min. Fractions containing server (http://www.cbs.dtu.dk/services/SignalP/),27) which nigerotriose and nigerotetraose were collected, concentrat- suggested that these enzymes were located in the cytoplasm. ed, and lyophilized. These sugars were further purified with A phylogenetic tree analysis showed that Cphy_3313 was an HPLC system (Prominence; Shimadzu Corporation, relatively homologous to nigerose phosphorylase of the Kyoto, Japan) equipped with a Shodex Asahipak NH2P-50 same strain (Cphy_1874)16) (Fig. 1). They had 52% identity 4E column (4.6 mm internal diameter × 25 cm; Showa and 70% similarity in their amino acid sequences. In contrast, Denko KK, Tokyo, Japan) and the RID-10A detector Cphy_3314 appeared between the clades of maltose (Shimadzu Corporation) at 30°C under a constant flow phosphorylase and trehalose phosphorylase. (1.0 mL/min) of 60% (by volume) acetonitrile/water as the Recombinant Cphy_3313 and Cphy_3314 proteins were mobile phase. Fractions containing the products were purified to yields of approximately 2 and 5 mg, respectively, 62 J. Appl. Glycosci., Vol. 61, No. 2 (2014)

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). 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 Fig. 2. Double reciprocal plots for nigerose phosphorolysis by Cphy_3313 at different Pi concentrations. parameters: kcat = 31 s－1, KmA = 1.2 mM, KmB = 0.49 mM Pi concentrations are: Open circles, 0.5 mM; closed circles, 1.0 mM; and KiA = 2.0 mM, where A and B represent nigerose and open squares, 2.0 mM; closed squares, 3.0 mM; open triangles, phosphate, respectively (Fig. 2). These values were similar 5.0 mM. 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 Cphy_187416) as shown in Table 1. The kinetic parameters reverse phosphorolysis was again similar to that of for major acceptors and donors are summarized in Table 2. Nihira et al.: α-1,3-Oligoglucan Phosphorylase 63

Table 1. Acceptor speciﬁcity of Cphy_3313 compared with the other Table 3. Kinetic parameters for reverse phosphorolysis catalyzed by nigerose phosphorylase. Cphy_3314.

Specific activity (IU/mg) kcat /Km Km (mM) kcat (s－1) Acceptor (mM－1 s－1) Cphy_3313 Cphy_1874a Acceptora b D-Glucose 29 (100) 54 (100) Nigerose 3.1 8.7 2.8 D-Galactose 6.2 (21) 4.6 (8.5) Trehalose ― ― 0.0062c D-Xylose 11 (38) 9.3 (17) Kojibiose 50 3.5 0.069 1,5-Anhydro-D-glucitol 7.5 (26) 8.1 (15) Maltose 3.8 2.7 0.70 Methyl-α-D-glucoside 0.62 (2.1) 0.2 (0.3) Isomaltose ― ― 0.012c Sucrose ― ― 0.0042c Following sugars did not show acceptor activity: D-mannose, Laminaribiose ― ― 0.023c D-allose, D-fructose, D-lyxose, D-arabinose, L-arabinose, D-glucos- D-Glucose ― ― 0.0059c amine, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, L-fucose, Methyl-α-D-glucoside ― ― 0.026c L-rhamnose, D-glucal, 2-deoxy-D-glucose, α-D-glucose 1-phosphate, Nigerotriose 12 17 1.4 α-D-galactose 1-phosphate, D-glucose 6-phosphate, methyl β-D- Donorb glucoside, 3-O-methyl-D-glucose, trehalose, kojibiose, nigerose, βGlc1P 5.1 8.3 1.6 maltose, isomaltose, sophorose, laminaribiose, cellobiose, gentiobi- a ose, xylobiose, melibiose, lactose, sucrose. Adopted from Ref. 16). aβGlc1P (10 mM) was used as the donor. bNigerose (10 mM) was b Values in parenthesis represent relative activities for which specific used as the acceptor. cDetermined from the slopes of linear s‒v plots. activities for D-glucose were defined as 100%.

analysis, the resulting trisaccharide and tetrasaccharide were Table 2. Kinetic parameters for reverse phosphorolysis catalyzed by determined to be 3-O-α-D-glucopyranosyl-3-O-α-D- Cphy_3313. glucopyranosyl-D-glucose (nigerotriose, J-STAGE Electric kcat /Km Supplentary Material, Fig. S1) and 3-O-α-D-glucopyranosyl- Substrate Km (mM) kcat (s－1) (mM－1 s－1) 3-O-α-D-glucopyranosyl-3-O-α-D-glucopyranosyl-D- Acceptora glucose (nigerotetraose, J-STAGE Electric Supplentary D-Glucose 2.1 52 24 Material, Fig. S2), respectively. D-Galactose 20 26 1.3 During the phosphorolytic reaction, Cphy_3314 phospho- D-Xylose 17 40 2.4 rolyzed nigerotriose and nigerotetraose with inversion of the 1,5-Anhydro-D-glucitol 17 30 1.8 anomeric configuration and releasing βGlc1P in the presence Methyl-α-D-glucoside 79 8.3 0.11 Donorb of Pi. In addition, Cphy_3314 did not cleave nigerotriose βGlc1P 1.2 58 49 and nigerotetraose in the absence of Pi. Double reciprocal plots of the initial velocities against various initial concen- aβGlc1P (10 mM) was used as the donor. bD-Glucose (10 mM) was used as the acceptor. trations 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 Characterization of Cphy_3314. in the equation for the sequential Bi Bi mechanism were Cphy_3314 did not significantly phosphorolyze α-linked determined to be: kcat = 11 and 8.5 s－1, KmA = 3.2 and glucobioses, such as trehalose, kojibiose, nigerose, and 2.9 mM, KmB = 11 and 2.6 mM, and KiA = 5.8 and 32 mM, maltose that are typical substrates for GH65 phosphorylases. respectively, where A and B represent α-1,3-oligoglucans The acceptor specificity of Cphy_3314 was investigated by and phosphate, respectively. TLC as shown in Fig. 3. Formation of oligosaccharides was Phosphorolytic activities on α-1,3-oligoglucans, except detected when nigerose and maltose were used as acceptors, for nigerose, have not been previously reported, which which suggested that these two disaccharides were good suggests that Cphy_3314 is an unreported phosphorylase. acceptors. The acceptor specificity with nigerose and maltose Therefore, we propose 3-O-α-D-oligoglucan: phosphate were determined to be 4.0 and 1.4 IU/mg, respectively. β-D-glucosyltransferase as the systematic name and Carbohydrate spots at the TLC origin were detected with the α-1,3-oligoglucan phosphorylase as the short name for this following acceptors (see Fig. 3): D-glucose, D-galactose; Cphy_3314. D-xylose; D-mannose; trehalose; kojibiose; isomaltose; sucrose; L-arabinose; methyl-α-D-glucoside; methyl-β-D- Basic properties of Cphy_3313 and Cphy_3314. glucoside; 2-deoxy-D-glucose; 1,5-anhydro-D-glucitol; Cphy_3313 and Cphy_3314 were stable up to 40 and laminaribiose. The formation of polysaccharides suggested 35°C, respectively, during a 10 min incubation. Cphy_3313 that these sugars were poor acceptors for Cphy_3314 and and Cphy_3314 were stable at 30°C for 30 min in the pH that their initial glucosyl-transferred products were much ranges of 5.5‒9.0 and 5.5‒9.5, respectively, and exhibited better acceptors. Similar phenomena have been reported for their highest apparent phosphorolytic activity for nigerose phosphorylases specific for DP of ≥ 3 in their phosphoroly- and nigerotriose at pH 7.0 and 8.0, respectively. The synthet- sis, such as cellodextrin phosphorylase28) and sophorooligo- ic reactions using βGlc1P as the donor and D-glucose and saccharide phosphorylase29) with D-glucose as the acceptor. nigerose as the acceptors had optimum pH values of 7.0 and The kinetic parameters of Cphy_3314 for these acceptors 6.5, respectively. and those for donor βGlc1P are given in Table 3. Reverse phosphorolysis with nigerose as the acceptor resulted in a series of oligosaccharides (Fig. 3). By NMR 64 J. Appl. Glycosci., Vol. 61, No. 2 (2014)

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.

DISCUSSION Cphy_3313 and Cphy_3314 probably utilize α-1,3- oligoglucans that are transported into the cytosol. α-1,3-Oligoglucans We report that Cphy_3313 and Cphy_3314 are a nigerose may be generated outside of a cell with the degradation of phosphorylase and an α-1,3-oligoglucan phosphorylase α-1,3-glucans by extracellular enzymes encoded around the from C. phytofermentans, both of which are considered to be other nigerose phosphorylase. A gene encoding for involved in the metabolism of α-1,3-glucans, such as nigeran β-phosphoglucomutase, an enzyme that converts βGlc1P and pseudonigeran. We previously reported another nigerose into D-glucose 6-phosphate, cphy_3311, is also found close phosphorylase, Cphy_1874, encoded for in a gene cluster for to cphy_3313 and cphy_3314. It should be noted that the an α-1,3-glucan metabolic pathway.16) The presence of three other two genes of C. phytofermentans that encode for GH65 phosphorylases related to the metabolism of α-1,3-glucans phosphorylases (cphy_1019 and cphy_1874) are also suggests that α-1,3-glucans may be important carbon sources surrounded by genes for an ABC transporter (cphy_1013 to for C. phytofermentans. α-1,3-Glucans are often produced cphy_1015 and cphy_1879 to cphy_1881, respectively) and by fungi and yeast,30) which may be rich in forest soil where β-phosphoglucomutase (cphy_1021 and cphy_1875, respec- C. phytofermentans inhabits. tively). Genes encoding for components of an ATP binding C. phytofermentans possesses two phosphorylases for cassette (ABC) transporter (cphy_3315 to cphy_3317) are utilizing α-1,3-oligoglucans. Nigerose phosphorylase found upstream of cphy_3313 and cphy_3314. It has often (Cphy_3313 and Cphy_1874) is strictly specific for been reported that genes encoding for an ABC transporter disaccharides and α-1,3-oligoglucan phosphorylase is specific for a phosphorylase substrate are located close to the specific for those oligosaccharides with DP values of 3 or gene encoding for the phosphorylase.31‒33) Therefore, higher. Such combinations of two phosphorlyases, one Nihira et al.: α-1,3-Oligoglucan Phosphorylase 65

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