Discovery of Two β-1,2-Mannoside Phosphorylases Showing Different Chain-Length Specificities from Thermoanaerobacter sp. X-514

We characterized Teth514_1788 and Teth514_1789, belonging to glycoside hydrolase family 130, from Thermoanaerobacter sp. X-514. These two enzymes catalyzed the synthesis of 1,2-β-oligomannan using β-1,2-mannobiose and d-mannose as the optimal acceptors, respectively, in the presence of the donor α-d-mannose 1-phosphate. Kinetic analysis of the phosphorolytic reaction toward 1,2-β-oligomannan revealed that these enzymes followed a typical sequential Bi Bi mechanism. The kinetic parameters of the phosphorolysis of 1,2-β-oligomannan indicate that Teth514_1788 and Teth514_1789 prefer 1,2-β-oligomannans containing a DP ≥3 and β-1,2-Man2, respectively. These results indicate that the two enzymes are novel inverting phosphorylases that exhibit distinct chain-length specificities toward 1,2-β-oligomannan. Here, we propose 1,2-β-oligomannan:phosphate α-d-mannosyltransferase as the systematic name and 1,2-β-oligomannan phosphorylase as the short name for Teth514_1788 and β-1,2-mannobiose:phosphate α-d-mannosyltransferase as the systematic name and β-1,2-mannobiose phosphorylase as the short name for Teth514_1789.


Introduction
Glycoside phosphorylases catalyze the cleavage of glycosyl linkages via a substitution with inorganic phosphate [1][2][3][4].These enzymes phosphorolyze particular glycosides to form corresponding sugar 1-phosphates with retention or inversion of the anomeric configuration [1][2][3][4].Because the phosphorylase reactions are reversible, various oligosaccharides have been synthesized via reverse phosphorolysis using the corresponding sugar 1-phosphate as a donor substrate and suitable carbohydrate acceptors [3,4].In addition, these reversible catalytic reactions are well suited for the practical synthesis of oligosaccharides from abundantly available natural sugars without using costly sugar 1-phosphate as the starting material by using a single phosphorylase [1,3,5] or by combined reaction with two phosphorylases that share the same sugar 1-phosphate [6][7][8] or that produce different sugar 1-phosphates with additional enzymes to convert the sugar 1-phosphates [8][9][10].However, the relatively narrow range of variations of phosphorylases limits the application of these phosphorylases.Therefore, the discovery of a novel phosphorylase showing unreported substrate specificity and regioselectivity is desired to expand the number of synthesizable oligosaccharides.
Currently, all of the reported GH130 phosphorylases have originated from anaerobes and are considered to be involved in the catabolism of b-mannosides under anaerobic conditions.In Ruminococcus albus, a 4-O-b-mannosyl-D-glucose phosphorylase and a b-1,4-mannooligosaccharide phosphorylase have been proposed to be involved in the degradation of the hemicellulosic b-1,4-mannan in place cells, together with GH26 endo-and/or exo-b-mannanases (EC 3.2.1.78and EC 3.2.1.-)and a cellobiose 2-epimerase (EC 5.1.3.11)[14].In Bacteroides thetaiotaomicron, a GH130 1,4-b-mannosyl-N-acetyl-D-glucosamine phosphorylase releases a-Man1P from a common core disaccharide of N-glycans that have been liberated by sequential glycoside hydrolase-catalyzed reactions from a complex-type N-glycan [10].The resultant a-Man1P is converted into D-fructose 6-phosphate from Man6P via the sequential reaction of phosphomannomutase (EC 5.4.2.8) and mannose-6-phosphate isomerase (EC 5.3.1.8)and enters glycolysis.The catabolic pathways that include GH130 phosphorylases that enable anaerobes to produce a-Man1P directly without consuming ATP are energetically efficient when compared with the conventional catabolic pathway that contains ATP-dependent carbohydrate kinase because only three molecules of ATP are available via the glycolytic pathway from glucose 6-phosphate.
In this study, we noticed that the anaerobic thermophile Thermoanaerobacter sp.X-514 possesses two genes encoding two GH130 proteins, Teth514_1788 and Teth514_1789, which have unknown functions in the genome.Here, we describe two novel GH130 phosphorylases that show unique substrate specificity toward 1,2-b-oligomannan.Interestingly, our results suggest that both phosphorylases are involved in GDP-D-mannose biosynthesis in this anaerobic bacterium.

Cloning, Expression, and Purification
The two genes encoding Teth514_1788 and Teth514_1789 (GenBank accession numbers ABY93074 and ABY93073, respectively) were amplified from the genomic DNA of Thermoanaerobacter sp.X-514 via a PCR performed using KODplus DNA polymerase (Toyobo, Osaka, Japan) together with the following oligonucleotides, which were designed based on the genomic sequence (GenBank accession number CP000923) [20]: 59-ggaattccatatgataaaattaaagagatt-39 as the forward primer containing an NdeI site (underlined) and 59-tttctcgagaaatttgatatctttcatctc-39 as the reverse primer containing an XhoI site (underlined) for Teth514_1788; and 59-ggaattccatatgttcaggctaacaagact-39 as the forward primer containing an NdeI site (underlined) and 59-tttctcgagaaattttactttttctttttc-39 as the reverse primer containing an XhoI site (underlined) for Teth514_1789.The amplified genes were purified using a FastGene Gel/PCR Extraction Kit (Nippon Genetics Co, Tokyo, Japan), digested with NdeI and XhoI (New England Biolabs, Beverly, MA, USA), and inserted into pET24a (+) (Novagen, Madison, WI, USA) to encode a His 6 -tagged fusion at the C-terminus of each recombinant protein.The expression plasmids were propagated in Escherichia coli DH5a (Toyobo), purified using a FastGene Plasmid Mini Kit (Nippon Genetics Co.), and verified via sequencing (Operon Biotechnologies, Tokyo, Japan).An E. coli Rosetta 2 (DE3) (Novagen) transformant harboring each of the expression plasmids was grown at 37 ˚C in 200 mL of Luria-Bertani medium (1% tryptone, 0.5% yeast extract, and 0.5% NaCl) containing 50 mg?mL 21kanamycin and 30 mg?mL 21  chloramphenicol until the absorbance reached 0.6 at 600 nm.The expression was then induced using 0.1 mM isopropyl-b-D-thiogalactopyranoside and continued at 18 ˚C for 24 h.The cells were harvested via centrifugation at 10,0006g for 20 min and suspended in 50 mM HEPES-NaOH buffer (pH 7.0) containing 500 mM NaCl (buffer A).The suspended cells were disrupted via sonication (Branson Sonifier 250A; Branson Ultrasonics Division of Emerson Japan, Kanagawa, Japan), and the supernatant was collected via centrifugation at 20,0006g for 20 min.The supernatant was applied to a HisTrap FF column (GE Healthcare, Buckinghamshire, UK) and equilibrated with buffer A containing 10 mM imidazole using an A ¨KTA Prime (GE Healthcare).After washing with buffer A containing 22 mM imidazole and subsequently eluting the proteins with a 22-400 mM imidazole linear gradient in buffer A, the fractions containing the recombinant protein were pooled, dialyzed against 10 mM HEPES-NaOH buffer (pH 7.0), and concentrated (AMICON Ultra-15 filter; Millipore, Billerica, MA, USA).The protein concentrations were determined spectrophotometrically at 280 nm using the theoretical extinction coefficients of e565,320 and 78,840 M 21 cm 21 , which are based on the amino acid sequences of Teth514_1788 and Teth514_1789, respectively [21].The molecular masses of the proteins were estimated via SDS-PAGE (Mini-PROTEAN Tetra electrophoresis system using 4-15% MiniPROTEAN TGX Precast Polyacrylamide Gels; Bio-Rad Laboratories, Inc., Hercules, CA, USA) using BLUE Star Prestained Protein-Ladder (Nippon Genetics Co.) as standard and gel filtration chromatography (HiLoad 26/600 Superdex 200 pg; GE Healthcare), which was performed using a column equilibrated with 10 mM HEPES-NaOH buffer (pH 7.0) containing 150 mM NaCl; the flow rate was 0.5 ml/min and Marker Proteins for Molecular Weight Determination on High Pressure Liquid Chromatography (Oriental Yeast, Tokyo, Japan) were used as standards.

Measurement of Synthetic Activity
The synthetic activity was routinely determined by measuring the increase in inorganic phosphate (P i ) using a reaction mixture containing 10 mM a-Man1P (a-Man1P bis(cyclohexylammonium) salt, which was synthesized from D-mannose and ATP using N-acetylhexosamine 1-kinase [22,23]) and 10 mM D-mannose in 40 mM sodium acetate buffer (pH 5.0 for Teth514_1788 or pH 5.5 for Teth514_1789) at 30 ˚C, following the method created by Lowry and Lopez [24] as described previously [10].

Donor Specificity Analysis
To investigate the donor specificities of Teth514_1788 and Teth514_1789, the synthetic reactions were examined under the aforementioned standard conditions, and we substituted a-Man1P with the following sugar-phosphate derivatives: b-Lfucose 1-phosphate, a-D-galactose 1-phosphate, a-D-glucosamine 1-phosphate, a-D-glucose 1-phosphate, and a-D-xylose 1-phosphate (Sigma-Aldrich).The reactions contained 120 mM Teth514_1788 or 22 mM Teth514_1789 and were performed for 2 h at 30 ˚C.The reaction products were analyzed using TLC as described in the preceding subsection.

Structure Determination
The reaction products used for the structural studies were generated in 500-mL reaction mixtures (pH 5.0 for Teth514_1788; pH 5.5 for Teth514_1789) containing 50 mM a-Man1P plus 50 mM of each acceptor carbohydrate and Teth514_1788 (530 nM for D-mannose and D-fructose and 72 nM for b-1,2mannobiose (b-1,2-Man 2 )) or Teth514_1789 (64 nM for D-mannose and Dfructose).The reaction mixtures were incubated at 30 ˚C for 24 h and then desalted using Amberlite MB-4 (Organo, Tokyo, Japan).The reaction products were purified using an HPLC system (Prominence; Shimadzu, Kyoto, Japan) equipped with a Shodex Asahipak NH2P-50 4E column (4.6-mm internal diameter 625 cm, 5 mm; Showa Denko K.K., Tokyo, Japan) at 30 ˚C under a constant flow (1.0 ml?min 21 ) of 70% acetonitrile in water as the mobile phase.The fractions containing the reaction products were collected and lyophilized.The molecular masses of the products were determined using electrospray ionization MS (ESI-MS).The ESI-MS spectra were recorded in the positive-ion mode on a time-of-flight (TOF)-MS system (JMS-T100 LP AccuTOF LC-Plus; JEOL Co., Tokyo, Japan) equipped with an ESI source (JEOL Co.).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 products were acquired in D 2 O, using 2-methyl-2-propanol as an internal standard (d H 1.23 and d C 31.2), using a Bruker DMX 600 spectrometer (Bruker Biospin, Rheinstetten, Germany) or a Bruker Avance 800 spectrometer (Bruker Biospin).The proton signals were assigned based on the DQF-COSY spectra.The 13 C signals were assigned using the HSQC spectra based on the assignment of the proton signals.The linkage position in each product was determined by detecting the inter-ring cross-peaks in each HMBC spectrum.The anomeric configuration was confirmed based on the 1 J C1-H1 coupling constant that was extracted from the non-decoupled HSQC spectrum.

Measurement of Phosphorolytic Activity
The phosphorolysis substrates of Teth514_1788 and Teth514_1789 were generated in a 4-mL reaction mixture (pH 5.0) containing 14 mM Teth514_1788, 500 mM a-Man1P, and 500 mM D-mannose.After incubation at 30 ˚C for 72 h, the reaction mixture was desalted using Amberlite MB-4.The reaction products were purified using an HPLC system equipped with a Shodex Asahipak NH2P-50 10E column (10-mm internal diameter 625 cm; Showa Denko K.K.) at 30 ˚C under a constant flow (3.0 ml?min 21 ) of 65% acetonitrile in water (the mobile phase).The fractions containing the products were collected and lyophilized.The phosphorolytic activity was routinely determined by quantifying the a-Man1P released during a phosphorolytic reaction in 40 mM MES-NaOH buffer (pH 6.0) containing 10 mM substrate and 10 mM P i at 30 ˚C using the previously described colorimetric method [25].

Temperature and pH Profile
The effects of pH on the phosphorolytic and synthetic activities of Teth514_1788 (44 nM) and Teth514_1789 (32 nM) were measured under the standard conditions described above and the following 40 mM buffers were used: 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 activities (under the standard conditions) after incubating Teth514_1788 (360 nM) and Teth514_1789 (320 nM) at 30-90 ˚C for 30 min or under various pH conditions at 4 ˚C for 24 h, respectively.
We analyzed the kinetics of the synthetic reactions using suitable acceptors and the standard conditions described above.We used Teth514_1788 (290 nM for

Preparation of the Recombinant Teth514_1788 and Teth514_1789
Recombinant Teth514_1788 and Teth514_1789 fused with a His 6 -tag at the Cterminus were purified using nickel-chelate affinity chromatography, with yields of 18 and 3.0 mg, respectively, from lysates prepared using 400 mL of cell culture.The purified Teth514_1788 and Teth514_1789 migrated in the SDS-PAGE as single protein bands with an estimated size of 34 kDa, which agrees with the theoretical molecular masses of 33,997 and 36,175, respectively.Furthermore, based on the gel filtration chromatography, the molecular masses of Teth514_1788 and Teth514_1789 were estimated to be 50 and 27 kDa, respectively.These results indicate that Teth514_1788 and Teth514_1789 exist as a homodimer and monomer in solution, respectively.In contrast, B. thetaiotaomicron GH130 b-1,4-D-mannosyl-N-acetyl-D-glucosamine phosphorylase [10] and R. albus GH130 b-1,4-mannooligosaccharide phosphorylase [14] have been reported to exist as a homotetramer and homohexamer, respectively.

Acceptor and Donor Specificities of Teth514_1788 and Teth514_1789 in Synthetic Reactions
The acceptor specificity in a synthetic reaction was examined using various carbohydrate acceptor candidates (see ''Materials and Methods'') together with a-Man1P as the donor.Teth514_1789 used D-mannose and D-fructose as the suitable acceptors.The synthetic reaction performed using D-mannose generated products 1 and 2 (Fig. 2A), and their structures were determined using 1 H and 13 C NMR spectroscopy.The results of the HMBC experiments showed that each Fig. 1.The phylogenetic tree of the GH130 proteins.The multiple alignments were performed using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/ clustalw2/).A phylogenetic tree was constructed using TreeView Version 1.6.6 (http://taxonomy.zoology.gla.ac.uk/rod/rod.html).The genes encoding the GH130 proteins (http://www.cazy.org/GH130.html) are represented according to the organism names and GenBank accession numbers.The genes cloned in this study are shown in white letters on a black background.The enzyme names of the characterized phosphorylases are shown in bold.doi:10.1371/journal.pone.0114882.g001product exhibited correlation cross-peaks between the C2 of the D-mannose residues on the acceptor side and the H-1 of the D-mannosyl residues on the donor side.The 1 J C1-H1 coupling constant (160,162 Hz) of the D-mannosyl residues on the donor side were consistent with b-linkages [26].The assignments of the 1 H and 13 C NMR chemical shifts are provided in S1 Table .Based on these results, we identified products 1 and , respectively.The synthetic reaction performed using D-fructose also generated two products (products 4 and 5, Fig. 2B).In the  Teth514_1788.The mixtures were incubated at 30˚C for 24 h and then desalted using Amberlite MB-4.The reaction products were analyzed using an HPLC system equipped with a Shodex Asahipak NH2P-50 4E column (4.6-mm internal diameter 625 cm, 5 mm) at 30˚C under a constant flow (1.0 ml?min 21 ) of 70% acetonitrile in water (mobile phase).The product numbers described in the text are in bold.The fractions containing the reaction products were collected and lyophilized.The amounts of products 1 and 2 obtained from D-mannose (A and C) were 1.5 and 1.2 mg (Teth514_1789) and 0.8 and 1.0 mg (Teth514_1788), respectively.The amounts of products 4 and 5 obtained from D-fructose (B and D) were 1.4 and 2.3 mg (Teth514_1789) and 1.0 and 0.5 mg (Teth514_1788), respectively.The amounts of products 2 and 3 obtained from b-1,2-Man 2 (E) using Teth514_1788 were 1.0, and 1.3 mg, respectively.The masses were as follows: products 1, 2, 3, 4, and 5 were m/z 365.preference can be explained by the ring conformations and the orientations of the substituting groups of D-mannose and D-fructose.When the hydroxyl groups at the linkage positions of D-mannopyranose and b-D-fructopyranose are aligned, the orientations of the hydroxyl groups at C2, C3, C4, and C6 of D-mannopyranose and at C5, C4, C3, and C1 of b-D-fructopyranose are conserved (Fig. 3).Therefore, the b-1,5-linkage on b-D-fructopyranose and the b-1,2-linkage on D-mannopyranose will be formed.Although D-mannose and D-fructose also acted as acceptors with Teth514_1788, the formation of oligosaccharides containing a degree of polymerization (DP) between 2-5 were detected (Fig. 2C  and 3D).The production of oligosaccharides with a DP $4 by Teth514_1789 was not detected under the reaction conditions (Fig. 2A and 2B).The 1 H NMR spectra of the oligosaccharides that were generated from D-mannose and Dfructose and containing a DP between 2-3 were identical to the spectra of products 1 and 2 from D-mannose and products 4 and 5 from D-fructose with Teth514_1789 (S1 Figure ).Furthermore, we confirmed that Teth514_1788 used product 1 (b-1,2-Man 2 ) (Fig. 3E) as the acceptor, and this resulted in the synthesis of oligosaccharides with DP 3 (product 2), DP4 (product 3), and DP5.

Kinetic Analysis of the Synthetic and Phosphorolytic Reactions
We determined the kinetic parameters of the four acceptors (D-mannose, D-fructose, b-1,2-Man 2 , and b-1,2-Man 3 ) used by Teth514_1788 and Teth514_1789 to investigate the acceptor preference in the presence of the a-Man1P donor (Table 1).The k cat /K m value of D-mannose for Teth514_1789 was highest among the four tested acceptors.The k cat /K m value of b-1,2-Man 2 was 6 times lower than that of D-mannose, and no obvious activity was observed against b-1,2-Man 3 .These results indicate that D-mannose is the most effective acceptor for Teth514_1789.In regards to Teth514_1788, the k cat /K m values of b-1,2-Man 2 and b-1,2-Man 3 were 7-8 times greater than that of D-mannose.These kinetic parameters indicate that Teth514_1788 and Teth514_1789 exhibit distinct chainlength specificities toward 1,2-b-oligomannan in synthetic reactions.The kinetic parameters measured for a-Man1P are similar to other inverting phosphorylases for their specific donors [3,10,13,14,16,[28][29][30].
Based on the above information, we propose 1,2-b-oligomannan:phosphate a-D-mannosyltransferase as the systematic name and 1,2-b-oligomannan phosphorylase as the short name for Teth514_1788 and b-1,2-mannobiose:phosphate a-D-mannosyltransferase as the systematic name and b-1,2-mannobiose phosphorylase as the short name for Teth514_1789.

Basic Properties of Teth514_1788 and Teth514_1789
Teth514_1788 and Teth514_1789 were stable up to 55 and 75 ˚C, respectively, during a 30 min incubation (Fig. 5A) and were stable in pH ranges between 4.0-9.5 and 5.5-9.5, respectively, at 4 ˚C for 24 h (Fig. 5B).Furthermore, both Teth514_1788 and Teth514_1789 exhibited the highest apparent phosphorolytic activity at pH 6.0 (Fig. 5C and 5D), whereas the optimal pH values for the synthetic reactions were 5.0 and 5.5, respectively (Fig. 5C and 5D).
doi:10.1371/journal.pone.0114882.g006phosphate guanylyltransferase (Teth514_1787).In this pathway, Teth514_1788 and Teth514_1789, which exhibit distinct chain-length specificities, play a key role in the efficient supply of a-Man1P, which is the precursor of GDP-D-mannose.This is the first report of a salvage pathway for GDP-D-mannose biosynthesis in which phosphorylases participate.One notable feature of this new GDP-Dmannose biosynthetic pathway is that it allows anaerobes such as Thermoanaerobacter to use the energy from ATP more efficiently than via conventional de novo and salvage pathways where ATP-dependent hexokinase participates; this is because D-mannose can be phosphorylated without the consumption of ATP.We further suggest that this salvage pathway for GDP-Dmannose biosynthesis is a common pathway in Thermoanaerobacter (Fig. 6).

Fig. 4 .
Fig. 4. Double-reciprocal plots of the phosphorolysis catalyzed by Teth514_1788 (A, B, and C) and Teth514_1789 (D and E).We measured the initial velocities for the phosphorolysis of 1,2-b-oligomannan at various concentrations of 1,2-b-oligomannan and P i .Open circle, 0.1 mM P i ; closed circle, 0.2 mM P i ; open square, 0.3 mM P i ; closed square, 0.5 mM P i ; open triangle, 1 mM P i ; and closed triangle, 2 mM P i .doi:10.1371/journal.pone.0114882.g004

Table 1 .
The kinetic parameters for the synthetic reactions catalyzed by Teth514_1788 and Teth514_1789.

Table 2 .
The kinetic parameters for the phosphorolytic reactions catalyzed by Teth514_1788 and Teth514_1789.kcat (s 21 ) K mA (mM) K mB (mM) K iA (mM) k cat/ K mA (s 21 mM 21 )