Discovery of Cellobionic Acid Phosphorylase in Cellulolytic Bacteria and Fungi

FEBS Letters 587 (2013) 3556–3561

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Discovery of cellobionic acid phosphorylase in cellulolytic bacteria and fungi

Takanori Nihira a, Yuka Saito a, Mamoru Nishimoto b, Motomitsu Kitaoka b, Kiyohiko Igarashi c, ⇑ Ken’ichi Ohtsubo a, Hiroyuki Nakai a, a Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan b National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki 305-8642, Japan c Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan article info abstract

Article history: A novel phosphorylase was characterized as new member of glycoside hydrolase family 94 from the Received 20 August 2013 cellulolytic bacterium Xanthomonas campestris and the fungus Neurospora crassa. The enzyme cat- Revised 9 September 2013 alyzed reversible phosphorolysis of cellobionic acid. We propose 4-O-b-D-glucopyranosyl-D-gluconic Accepted 10 September 2013 acid: phosphate a-D-glucosyltransferase as the systematic name and cellobionic acid phosphorylase Available online 19 September 2013 as the short names for the novel enzyme. Several cellulolytic fungi of the phylum Ascomycota also Edited by Judit Ovádi possess homologous proteins. We, therefore, suggest that the enzyme plays a crucial role in cellulose degradation where cellobionic acid as oxidized cellulolytic product is converted into a-D-glucose 1- phosphate and D-gluconic acid to enter glycolysis and the pentose phosphate pathway, respectively. Keywords: Cellobionic acid phosphorylase Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Glycoside hydrolase family 94 Xanthomonas campestris Neurospora crassa

1. Introduction polysaccharide monooxygenase and cellobiohydrolase [5–7]. The resultant cellobionic acid has been believed to be hydrolyzed by Cellulose degradation is one of the most crucial bioprocesses in intracellular or extracellular b-glucosidase to form D-glucose and the natural carbon cycle, because cellulose is an abundant organic D-gluconic acid at the beginning of the discovery of cellobiose compound on Earth. The hydrolytic enzymes, generally called cel- dehydrogenase [3], whereas cellobionic acid is a worse substrate lulase including endo-glucanase (EC 3.2.1.4), cellobiohydrolase (EC than cellobiose as typical substrate for b-glucosidase [11]. This is 3.2.1.91 or EC 3.2.1.176), and b-glucosidase (EC 3.2.1.4), have major because the hydrolysate D-gluconic acid is a strong non-competi- roles in the process [1,2]. The contribution of oxidative enzymes tive inhibitor of b-glucosidase [12], clearly suggesting another met- such as cellobiose dehydrogenase (EC 1.1.99.18) [3,4] and lytic abolic scheme should be applied for the cellobionate metabolism. polysaccharide monooxygenase [5–7] has lately attracted consid- Phosphorylases are members of enzymes involved in the intra- erable attention in enzymatic saccharification of cellulosic biomass cellular catabolism of particular glycosides [13–15]. These en- [8–10]. Although the oxidized cellulolytic products often occur zymes reversibly phosphorolyze glycosides, to produce sugar 1- during cellulose degradation, their metabolic pathway has not phosphates with strict substrate specificities. The catabolic path- been elucidated yet because the process is not simply connected way, including the phosphorolysis that enables direct production to glycolysis as they are a mixture of neutral and oxidized sugars. of phosphorylated sugars without consuming ATP, is energetically Cellobionic acid (4-O-b-D-glucopyranosyl-D-gluconic acid) is an efficient. However, there is little variation among phosphorylases intermediate in mycotic cellulose degradation [1,2]. It is obtained with only 21 known activities [15]. It is, thus, desired to identify by spontaneous hydrolysis of cellobiono-1,5-lactone, which is phosphorylases with previously unreported substrate specificities. extracellularly converted from cellobiose by cellobiose dehydroge- Phosphorylases have been classified as members of glycoside nase [3,4], as well as from cellulose by the combined action of lytic hydrolase families (GH) 13, 65, 94, 112, and 130, or glycosyltransferase families 4 or 35 in the Carbohydrate-Active Enzymes database (http://www.cazy.org/) based on amino acid sequence Abbreviations: GH, glycoside hydrolase family; aGlc1P, a-D-glucose 1-phosphate; PAGE, polyacrylamide-gel electrophoresis similarity [16]. Among these families, GH94 is primarily comprised ⇑ Corresponding author. Fax: +81 25 262 6692. of phosphorylases that catalyze reversible phosphorolysis of E-mail address: [email protected] (H. Nakai). b-D-glucosides to form a-D-glucose 1-phosphate (aGlc1P) with

0014-5793/$36.00 Ó 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.febslet.2013.09.014 T. Nihira et al. / FEBS Letters 587 (2013) 3556–3561 3557 inversion of the anomeric conﬁguration. At present, cellobiose 2.3. Measurement of enzymatic activity phosphorylase (EC 2.4.1.20), cellodextrin phosphorylase (EC 2.4.1.49), laminaribiose phosphorylase (EC 2.4.1.31), N,N0-diacetyl- The phosphorolytic activity was routinely determined by quan- chitobiose phosphorylase (EC 2.4.1.280), and cyclic b-1,2-glucan tifying aGlc1P released during the reaction in 40 mM HEPES–NaOH synthase (EC 2.4.1.–) are categorized into GH94. (pH 7.0) or 40 mM MOPS–NaOH (pH 7.0) for XCC4077 or In this study, we discovered cellobionic acid phosphorylases NCU09425, respectively, containing 10 mM substrate and 10 mM

(XCC4077 and NCU09425) from the plant pathogenic bacterium inorganic phosphate (Pi)at30°C by using the phosphoglucomu- Xanthomonas campestris and red bread mold of the phylum Asco- tase-glucose 6-phosphate dehydrogenase method [18] as de- mycota Neurospora crassa, respectively, as new members of scribed previously [19]. GH94. We propose a new metabolic pathway of cellobionic acid The synthetic activity was routinely determined by measuring that it is converted by energetically-efﬁcient phosphorolysis in the increase in Pi using a reaction mixture containing 10 mM the cytoplasm of cellulolytic organisms into aGlc1P and D-gluconic aGlc1P (a-D-glucose 1-phosphate disodium salt hydrate; Sigma– acid to enter the glycolysis and pentose phosphate pathway, Aldrich, St. Louis, MO, USA) and 10 mM D-gluconic acid (gluconic respectively. acid sodium salt; Nacalai Tesque, Kyoto, Japan) in 40 mM MES– NaOH (pH 6.0) or 40 mM MOPS–NaOH (pH 6.5) for XCC4077 or NCU09425, respectively, at 30 °C by following the method of Lowry 2. Materials and methods and Lopez [20] as described previously [21].

2.1. Construction of expression plasmids 2.4. Temperature and pH proﬁles

Two genes encoding XCC4077 and NCU09425 (GenBank ID: The effects of pH on the phosphorolytic and synthetic activities AAM43298.1 and EAA28929.1, respectively) were amplified by using 9.9 nM XCC4077 or 7.9 nM NCU09425 were measured under PCR from genomic DNA of Xanthomonas campestris pv. campestris the standard conditions described above, using the following ATCC33913 and Neurospora crassa OR74A, respectively, using 40 mM buffers: sodium citrate (pH 3.0–5.5), Bis–Tris–HCl (pH KOD-plus DNA polymerase (Toyobo, Osaka, Japan) with 5.5–7.0), HEPES–NaOH (pH 7.0–8.5), and glycine–NaOH (pH 8.5– oligonucleotide primers (Table S1) constructed based on the gen- 10.5). The thermal and pH stabilities were evaluated by measuring ome sequences (GenBank ID: AE008922 and AABX020000076, the residual synthetic activity under the standard conditions after respectively). Each amplified gene was inserted into pET24a (+) incubation of 3.9 lM XCC4077 or 6.6 lM NCU09425 at a tempera- (Novagen, Madison, WI, USA) with the restriction endonuclease ture range of 30–90 °C for 15 min and at various pH values at 4 °C sites (Table S1) using Ligation high Ver.2 (Toyobo). Each expression for 24 h, respectively. plasmid was propagated in Escherichia coli DH5a (Toyobo), purified by a FastGene Plasmid Mini Kit (Nippon Genetics Co., Tokyo, Japan), 2.5. Substrate specificity analysis and verified by sequencing (Operon Biotechnologies, Tokyo, Japan). The phosphorolytic activities of XCC4077 (78 lM) on b-linked 2.2. Recombinant enzyme preparation glucobioses (cellobiose, sophorose, laminaribiose, gentiobiose) and N,N0-diacetylchitobiose were examined under the standard E. coli BL21 (DE3) (Novagen) transformant harboring the conditions described above. expression plasmid was grown at 37 °C in 200 ml of Luria–Bertani To investigate the acceptor specificities of XCC4077 (78 nM), medium containing 50 lg/ml kanamycin, until the absorbance synthetic reactions were performed under the standard conditions reached 0.6 at 600 nm. The expression was induced by 0.1 mM iso- described above, by substituting D-gluconic acid with carbohydrate propyl b-D-thiogalactopyranoside and continued at 18 °C for 24 h. acceptor candidates (Table S2). Cells were harvested by centrifugation at 20000g for 20 min and suspended in 50 mM HEPES–NaOH (pH 7.0) containing 2.6. Structural determination of reaction product 500 mM NaCl (buffer A). The suspended cells were disrupted by sonication (Branson sonifier 250A, Branson Ultrasonics, Emerson Reaction products for structural determination were generated Japan, Kanagawa, Japan). The supernatant collected by centrifuga- in 1 ml of reaction mixture (pH 6.0) containing 500 mM aGlc1P tion at 20000g for 20 min was applied to a HisTrap FF column (GE and 500 mM D-gluconic acid or D-glucuronic acid with 20 lM Healthcare, Buckinghamshire, UK), equilibrated with buffer A con- XCC4077. The reaction mixtures were incubated at 30 °C for 24 h, taining 10 mM imidazole, by using an ÄKTA prime (GE Healthcare). followed by treatment with a-D-glucose 1-phosphatase from E. coli After washing with buffer A containing 22 mM imidazole and at 30 °C for 24 h, as described previously [19]. The reaction subsequent elution by using a 22–400 mM imidazole linear gradi- products were separated on a Toyopearl HW-40S column (26 mm ent in buffer A, fractions containing the target proteins were internal diameter 700 mm; Tosoh, Tokyo, Japan), equilibrated pooled, dialyzed against 10 mM HEPES–NaOH buffer (pH 7.0), with distilled water, at a flow rate of 1.0 ml/min. Fractions contain- and concentrated (AMICON Ultra; Millipore, Billerica, MA, USA). ing the reaction products were collected and lyophilized. The The protein concentration was determined spectrophotometrically amount of product obtained was 58 and 39 mg from D-gluconic at 280 nm using theoretical extinction coefficients of e = 168,110 acid and from D-glucuronic acid, respectively. The structures of and 161,120 cm1 M1, based on the amino acid sequences of the products were identified by comparing their 1H and 13CNMR

XCC4077 and NCU09425, respectively [17]. The molecular masses spectra acquired in D2O with 2-methyl-2-propanol as an internal of purified proteins were estimated by SDS–polyacrylamide-gel standard using a Bruker Avance 800 spectrometer (Bruker Biospin, electrophoresis (SDS–PAGE, Mini-PROTEAN Tetra electrophoresis Rheinstetten, Germany) with those of authentic data. system; Bio-Rad Laboratories, Hercules, CA, USA) and by gel filtration (HiLoad 26/600 Superdex 200 pg; GE Healthcare) equilibrated 2.7. Kinetic analysis with 10 mM HEPES–NaOH buffer (pH 7.0) containing 150 mM NaCl at a flow rate of 0.5 ml/min, using Marker Proteins for molecular The initial velocities of the phosphorolytic reactions were deter- Weight Determination on High Pressure Liquid Chromatography mined under the standard conditions with 54 nM XCC4077 or (Oriental Yeast Co., Tokyo, Japan) as standards. 2.0 nM NCU09425 and a combination of initial concentrations of 3558 T. Nihira et al. / FEBS Letters 587 (2013) 3556–3561

cellobionic acid (0.5–10 mM) and Pi (0.1–2.0 mM). The kinetic cell lysate of a 200 ml culture. Purified XCC4077 migrated in parameters were calculated by curve-fitting the experimental data SDS–PAGE as a single protein band with an estimated size of to the theoretical Eq. (1) for a sequential bi–bi mechanism using approximately 84 kDa (Fig. S1A), which is in agreement with the GraFit version 7.0.2 (Erithacus Software Ltd., London, UK). theoretical molecular weight of 89,700. However, the molecular mass of XCC4077 was determined by gel filtration to be 179 kDa m ¼ V ½A½B=ðK K þ K ½AþK ½Bþ½A½BÞ max iA mB mB mA (Fig. S1B), indicating that this protein is a homodimer in solution. ðA ¼ cellobionic acid; B ¼ phosphateÞð1Þ

Kinetic analysis of the synthetic reaction was performed at 3.2. Specificity analysis of XCC4077 in phosphorolysis and synthetic 30 °C under the standard conditions with 3.7 nM XCC4077 or reactions 9.9 nM NCU09425 and various concentrations of D-gluconic acid (0.5–20 mM), D-glucuronic acid (0.5–20 mM) as the acceptor, or The phosphorolytic activity of XCC4077 on b-linked glucobioses aGlc1P (0.3–5.0 mM for XCC4077 or 0.1–10 mM for NCU09425) (cellobiose, sophorose, laminaribiose, gentiobiose) and N,N0-diace- as the donor with 10 mM each opposite substrate. The kinetic tylchitobiose including known substrates for GH94 enzymes were parameters were calculated by curve fitting the experimental data examined. No disaccharides tested were phosphorolyzed to the Michaelis–Menten equation {v = kcat [E]0[S]/(Km + [S])} (Table S3). Therefore, the synthetic activity was examined using using GraFit version 7.0.2. various sugars as the acceptor (Table S2), together with aGlc1P as the donor. XCC4077 was a novel disaccharide-specif ic phos- 3. Results and discussion phorylase showing unique specificity; it catalyzed the syntheses of single disaccharides from D-gluconic acid and D-glucuronic acid 3.1. Production and purification of recombinant XCC4077 as the acceptor in the presence of aGlc1P as the donor. The NMR spectra of the products from D-gluconic acid and D-glucuronic acid We noticed that XCC4077 encoded in the genomic DNA of were identical with those of authentic 4-O-b-D-glucopyranosyl-D- Xanthomonas campestris pv. campestris ATCC33913 exhibits low gluconic acid (cellobionic acid) [26] and 3-O-b-D-glucopyranosyl- sequence identity (21–24%) with GH94 b-d-glycoside phosphoryl- D-glucuronic acid [19], respectively. Kinetic parameters for the ases characterized as cellobiose [22], cellodextrin [23], laminaribi- synthetic reactions were determined to investigate the accepter ose [19], and N,N’-diacetylchitobiose phosphorylases [24] and it preferences of XCC4077 (Table 1). The Km value of D-gluconic acid could not be categorized into any of the known GH94 enzymes was in the millimolar range and the kcat/Km value of D-gluconic acid based on phylogenetic tree analysis (Fig. 1). SignalP 4.0 analysis was around 5 times higher than that of D-glucuronic acid. These (http://www.cbs.dtu.dk/services/SignalP/) [25] of the amino acid kinetic parameters indicate that D-gluconic acid is the most effec- sequence of XCC4077 showed no predicted N-terminal signal pep- tive acceptor for XCC4077. Noticeably, the acceptor preference tide, suggesting that this protein is involved in a certain carbohy- can be explained by the steric conformations and orientations of drate metabolism in the cytoplasm as reported for a variety of substituting groups of D-gluconic acid and D-glucuronic acid. When carbohydrate phosphorylases [13–15]. In this study, recombinant the hydroxyl groups of D-gluconic acid and D-glucuronic acid at the enzyme with an additional His6-tag sequence at the C-terminus linkage positions are aligned, the steric conformations of both was produced in E. coli BL21 (DE3) to investigate its enzymatic acceptor molecules with the carboxyl groups at C1 of D-gluconic property and physiological role in the cellulolytic bacterium. acid and C6 of D-glucuronic acid are conserved (Fig. 2). In addition, Approximately 16 mg of purified XCC4077 was obtained from the the orientations of C3 and C4 hydroxyl groups of D-gluconic acid

Fig. 1. Phylogenetic tree of GH94 enzymes characterized. 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 GH94 enzymes characterized (http:// www.cazy.org/GH94.html) are represented by the organism names and GenBankTM accession numbers and are categorized in boxes that are framed with broken lines according to their substrate speciﬁcities. The genes cloned in this study are represented with a gray background. T. Nihira et al. / FEBS Letters 587 (2013) 3556–3561 3559

Table 1 a cellobionic acid phosphorylase homologous protein (Table S4)by Kinetic parameters for the synthetic reactions catalyzed by XCC4077. using of BLAST search (http://www.ncbi.nlm.nih.gov/blast/). It 1 1 1 Substrate Km (mM) kcat (s ) kcat/Km (mM s ) should be noted that the homologous genes are found only in Asco- Acceptor mycota, but not Basidiomycota. According to SignalP 4.0 analysis, a a D-gluconic acid 2.0 ± 0.4 173 ± 10 87 homologous protein (NCU09425) from Neurospora crassa OR74A a D-glucuronic acid 2.4 ± 0.2 43 ± 1 18 was predicted to be located in the cytoplasm; this protein Donor exhibited 57% sequence identity with XCC4077 derived from the aGlc1P b 0.21 ± 0.06 127 ± 7 605 cellulolytic bacterium. In addition, phylogenetic tree analysis also

a The range of acceptor concentration was 0.5–20 mM. categorized NCU09425 as a cellobionic acid phosphorylase b The range of donor concentration was 0.3–5 mM. (Fig. 1). These results suggest that NCU09425 is involved in the metabolism of cellobionic acid in the cytoplasm and motivated are the same as those of C4 and C3 hydroxyl groups D-glucuronic the detailed characterization of NCU09425. acid, respectively. Moreover, the fact that D-galacturonic acid did As done for XCC4077, approximately 5.4 mg of purified not act as acceptor in the synthetic reaction also suggests that NCU09425 was obtained as His6-tag fusion protein. NCU09425 mi- the orientation of C3 hydroxyl group of D-gluconic acid, corre- grated in SDS–PAGE as a single protein band with an estimated size sponding to C4 hydroxyl group of D-glucuronic acid, is important of approximately 87 kDa (Fig. S2A), which is in agreement with the for acceptor recognition of XCC4077. On the basis of the acceptor theoretical molecular weight of 90769, whereas the molecular specificity and considering that the kinetic parameters of aGlc1P mass was determined by gel filtration to be 171 kDa (Fig. S2B). are in the same range as those for other inverting phosphorylases These results indicate that NCU09425 is a homodimeric enzyme, belonging to GH94 [13], we here propose 4-O-b-D-glucopyrano- as same with XCC4077. NCU09425 catalyzed reversible phospho- syl-D-gluconic acid: phosphate a-D-glucosyltransferase as the sys- rolysis of cellobionic acid with inversion of the anomeric configu- tematic name and cellobionic acid phosphorylase as the short ration. The phosphorolytic reaction followed a sequential bi-bi name for XCC4077 (Fig. 2A). mechanism (Fig. 3B) and the kinetic parameters were calculated 1 We confirmed that XCC4077 phosphorolyzed cellobionic acid to be kcat=306 ± 15 s , KmA = 1.0 ± 0.2 mM, KmB = 0.10 ± 0.03 mM, with inversion of the anomeric configuration. Double reciprocal and KiA = 7.3 ± 3 mM, where A and B represent cellobionic acid plots of the initial velocities against various initial concentrations and Pi, respectively. In addition, the kinetic parameters that appear of cellobionic acid and Pi gave a series of lines intersecting at a in the Michaelis–Menten equation were determined for the syn- point (Fig. 3A). The result indicates that the phosphorolytic reac- thetic reaction. The Km and kcat values of D-gluconic acid were tion follows a sequential bi–bi mechanism, similar to inverting 3.4 ± 0.7 mM and 146 ± 12 s1, respectively The kinetic parameters phosphorylases [13–15]. The kinetic parameters were calculated of NCU09425 for the phosphorolysis and synthesis of cellobionic 1 to be kcat=114 ± 3 s , KmA = 0.19 ± 0.05 mM, KmB = 0.12 ± 0.02 mM, acid are in the same range as those of XCC4077, indicating that and KiA = 0.68 ± 0.3 mM, where A and B represent cellobionic acid NCU09425 is another cellobionic acid phosphorylase which is in and Pi, respectively. The parameters of cellobionic acid are in the agreement with the results of sequence analysis. same ranges with those of the other inverting phosphorylases [13], indicating that cellobionic acid is the true substrate of 3.4. Basic properties of XCC4077 and NCU09425 XCC4077. XCC4077 and NCU09425 were stable up to 35 °C during 15 min 3.3. Production, purification, and characterization of recombinant of incubation (Fig. S3A) and in the pH ranges of 5.0–10.5 and 5.5– NCU09425 9.0, respectively, at 4 °C for 24 h (Fig. S3B). The optimum pH for the phosphorolytic and synthetic reactions catalyzed by XCC4077 or We noticed that several cellulolytic fungi of the phylum Asco- NCU09425 were pH 7.0 and 6.0 or pH 7.0 and 6.5, respectively mycota, represented by Neurospora crassa, possess a gene encoding (Fig. S3C).

Fig. 2. Schematic representation of the reversible phosphorolysis of cellobionic acid (A) and 3-O-b-D-glucopyranosyl-D-glucuronic acid (B) catalyzed by XCC4077. 3560 T. Nihira et al. / FEBS Letters 587 (2013) 3556–3561

First, cellulose is degraded by endo-glucanase and cellobiohydrolase. The resultant cellobiose as the main component is oxidized by cellobiose dehydrogenase to produce cellobiono-1,5-lactone, which reacts spontaneously with water to form cellobionic acid. In addition, cellobionic acid is released from cellulose by the combined action of lytic polysaccharide monooxygenase and cellobiohydrolase. The cellobionic acid is transported into the cytoplasm, followed by phosphorolysis by cellobionic acid phosphorylase (NCU09425) to produce aGlc1P and D-gluconic acid. The released aGlc1P is converted into D-glucose 6-phosphate by a-phosphoglu- comutase (EC 2.7.5.1) to enter the glycolysis. The released D-gluconic acid is converted into ribulose 5-phosphate via 6- phosphogluconate by the sequential reaction of gluconokinase (EC 2.7.1.12) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44) to enter the pentose phosphate pathway. Thus, NCU09425 plays a crucial role in the catabolism of cellobionic acid, allowing it to be an alternative to b-glucosidase. One of the notable features of the new oxidative metabolic pathway for cellulose is that the fungus uses ATP-derived energy efficiently, because it is possible to phosphorylate a D-glucose residue of cellobionic acid directly without consuming ATP by ATP-dependent carbohydrate kinase. In addition, it should be noted that a variety of cellulolytic fungi of the phylum Ascomycota also possess a cellobionic acid phosphorylase homologous protein (Table S4), according to sequence analyses by using BLAST (http://www.ncbi.nlm.nih.gov/ blast/) and WoLF PSORT (http://psort.hgc.jp/) search. These find- ings suggest that the energetically efficient catabolism of cellobionic acid generally occurs in the oxidative cellulose metabolism by the fungi of the phylum Ascomycota possess. Though bacteria do not possess cellobiose dehydrogenase, the presence of cellobionic acid has been reported in the culture media of cellulolytic bacteria [27]. The bacterial oxidation of oligosaccharides could be catalyzed by soluble aldose dehydrogenase (EC 1.1.1.121) possessing a wide substrate specificity, initially reported as YliI gene from E. coli [28]. X. campestris pv. campestris ATCC 33913 also possesses a homologous gene (XCC3813). Its deduced amino acid sequence shows 36% identity and 54% homology with that of YliI. In addition, we have confirmed that all of the 14 gen- ome-sequenced strains of Xanthomonas possess cellobionic acid phosphorylase and gluconokinase homologous genes (Table S5). There is another possibility that cellobionic acid is produced by the combined action of the bacterial lytic polysaccharide monooxygenase and cellobiohydrolase [6]. The presence of bacterial cellobionic acid phosphorylase is thus reasonable to explain the Fig. 3. Double reciprocal plots of the phosphorolysis of cellobionic acid catalyzed by bacterial oxidative degradation of cellulose. However, it should XCC4077 (A) and NCU09425 (B). The initial velocities on the phosphorolytic reaction were determined with a combination of initial concentrations of cellob- be noted that X. campestris pv. campestris ATCC 33913 possesses ionic acid (0.5, 1.0, 2.0, 3.0, 5.0, and 10 mM) and Pi (open circles, 0.1 mM; filled no gene homologous to those encoding known bacterial lytic poly- circles, 0.2 mM; open squares, 0.3 mM; filled squares, 0.5 mM; open triangles, saccharide monooxygenases. 1.0 mM; filled triangles, 2.0 mM). Xanthomonas is a notorious bacterial genus that includes various plant pathogenic species causing damages in agricultural crops [29]. Further understanding of the infection is thus desired. 3.5. Physiological role of cellobionic acid phosphorylase Cellobionic acid phosphorylase present in infection-causing bacteria that inhabit plant tissues would play an important role Cellobionic acid had been considered to be metabolized by because it allows the bacteria to utilize cellulose provided by the b-glucosidase [3]. However, cellobionic acid is a worse substrate hosts. than cellobiose as typical substrate for b-glucosidase because the hydrolysate D-gluconic acid is a non-competitive inhibitor of b-glucosidase [11,12]. Therefore, there is a real need for a better Acknowledgments understanding of the metabolism of cellobionic acid. In this study, cellobionic acid phosphorylases (XCC4077 and NCU09425) were This work was supported in part by a program ‘‘Promotion of identified from X. campestris and N. crassa, respectively, suggesting Environmental Improvement for Independence of Young Research- the existence of a metabolic pathway for cellobionic acid contain- ers’’ under the Special Coordination Funds for Promoting Science ing these phosphorylases in the cellulolytic organisms. and Technology to H.N., and by a Grant-in-Aid for Innovative Areas A possible oxidative metabolism for cellulose by red bread mold (No. 24114001 and 24114008 to K.I.) from the Japanese Ministry of of the phylum Ascomycota N. crassa OR74A would be as follows. Education, Culture, Sports, and Technology (MEXT). T. Nihira et al. / FEBS Letters 587 (2013) 3556–3561 3561

Appendix A. Supplementary data [15] Nakai, H., Kitaoka, M., Svensson, B. and Ohtsubo, K. (2013) Recent development of phosphorylases possessing large potential for oligosaccharide synthesis. Curr. Opin. Chem. Biol. 17, 301–309. Supplementary data associated with this article can be found, [16] Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V. and in the online version, at http://dx.doi.org/10.1016/j.febslet.2013. Henrissat, B. (2009) The Carbohydrate-Active EnZymes database (CAZy): an 09.014. expert resource for glycogenomics. Nucleic Acids Res. 37, D233–238. [17] Pace, C.N., Vajdos, F., Fee, L., Grimsley, G. and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 4, 2411– References 2423. [18] Michal, G. (1984) D-Glucose 1-phosphate in: Method of Enzymatic Analysis [1] Lynd, L.R., Weimer, P.J., van Zyl, W.H. and Pretorius, I.S. (2002) Microbial (Bergmeyer, H.U., Bergmeyer, J. and Grassl, M., Eds.), 3rd ed, pp. 185–191, cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Verlag Chemie, Weinheim. Vol. 6. Rev. 66, 506–577. [19] Nihira, T., Saito, Y., Kitaoka, M., Otsubo, K. and Nakai, H. (2012) [2] Baldrian, P. and Valásková, V. (2008) Degradation of cellulose by Characterization of a laminaribiose phosphorylase from Acholeplasma basidiomycetous fungi. FEMS Microbiol. Rev. 32, 501–521. laidlawii PG-8A and production of 1,3-b-D-glucosyl disaccharides. Carbohydr. [3] Westermark, U. and Eriksson, K.E. (1974) Carbohydrate-dependent enzyme Res. 361, 49–54. quinone reduction during lignin degradation. Acta Chem. Scand. Ser. B 28, [20] Lowry, O.H. and Lopez, J.A. (1946) The determination of inorganic phosphate 204–208. in the presence of labile phosphate esters. J. Biol. Chem. 162, 421–428. [4] Henriksson, G., Johansson, G. and Pettersson, G. (2000) A critical review of [21] Nihira, T., Nakai, H., Chiku, K. and Kitaoka, M. (2012) Discovery of nigerose cellobiose dehydrogenases. J. Biotechnol. 78, 93–113. phosphorylase from Clostridium phytofermentans. Appl. Microbiol. Biotechnol. [5] Vaaje-Kolstad, G., Westereng, B., Horn, S.J., Liu, Z., Zhai, H., Sørlie, M. and 93, 1513–1522. Eijsink, V.G. (2010) An oxidative enzyme boosting the enzymatic conversion of [22] Liu, A., Tomita, H., Li, H., Miyaki, H., Aoyagi, C., Kaneko, S. and Hayashi, K. recalcitrant polysaccharides. Science 330, 219–222. (1998) Cloning, sequencing and expression of cellobiose phosphorylase gene [6] Forsberg, Z., Vaaje-Kolstad, G., Westereng, B., Bunæs, A.C., Stenstrøm, Y., from Cellvibrio gilvus. J. Ferment. Bioeng. 85, 511–513. MacKenzie, A., Sørlie, M., Horn, S.J. and Eijsink, V.G. (2011) Cleavage of [23] Sawano, T., Saburi, W., Hamura, K., Matsui, H. and Mori, H. () Characterization cellulose by a CBM33 protein. Protein Sci. 20, 1479–1483. of Ruminococcus albus cellodextrin phosphorylase and identification of a key [7] Quinlan, R.J., Sweeney, M.D., Lo Leggio, L., Otten, H., Poulsen, J.C., Johansen, phenylalanine residue for acceptor specificity and affinity to the phosphate K.S., Krogh, K.B., Jørgensen, C.I., Tovborg, M., Anthonsen, A., Tryfona, T., Walter, group. FEBS J., http://dx.doi.org/10.1111/febs.12408. C.P., Dupree, P., Xu, F., Davies, G.J. and Walton, P.H. (2011) Insights into the [24] Honda, Y., Kitaoka, M. and Hayashi, K. (2004) Reaction mechanism of oxidative degradation of cellulose by a copper metalloenzyme that exploits chitobiose phosphorylase from Vibrio proteolyticus: identification of family biomass components. Proc. Natl. Acad. Sci. USA 108, 15079–15084. 36 glycosyltransferase in Vibrio. Biochem. J. 377, 225–232. [8] Zˇifcˇáková, L. and Baldrian, P. (2012) Fungal polysaccharide monooxygenases: [25] Nielsen, H., Engelbrecht, J., Brunak, S. and von Heijne, G. (1997) Identification new players in the decomposition of cellulose. Fungal Ecol. 5, 481–489. of prokaryotic and eukaryotic signal peptides and prediction of their cleavage [9] Phillips, C.M., Beeson, W.T., Cate, J.H. and Marletta, M.A. (2011) Cellobiose sites. Protein Eng. 10, 1–6. dehydrogenase and a copper-dependent polysaccharide monooxygenase [26] Moe, S.T., Holen, A.K. and Schult, T. (2002) 4-O-b-D-Glucopyranosyl-D-gluconic potentiate cellulose degradation by Neurospora crassa. ACS Chem. Biol. 6, acid (cellobionic acid) produced by ozonation of cellobiose: isolation by HPLC 1399–1406. and assignment on NMR shifts. J. Carbohydr. Chem. 21, 513–520. ´ [10] Horn, S.J., Vaaje-Kolstad, G., Westereng, B. and Eijsink, V.G. (2012) Novel [27] Nouaille, R., Matulova, M., Pätoprsty, V., Delort, A.M. and Forano, E. (2009) enzymes for the degradation of cellulose. Biotechnol. Biofuels 5, 45. Production of oligosaccharides and cellobionic acid by Fibrobacter succinogenes [11] Cannella, D., Hsieh, C.W., Felby, C. and Jørgensen, H. (2012) Production and S85 growing on sugars, cellulose and wheat straw. Appl. Microbiol. Biotechnol. effect of aldonic acids during enzymatic hydrolysis of lignocellulose at high 83, 425–433. dry matter content. Biotechnol. Biofuels 5, 26. [28] Southall, S.M., Doel, J.J., Richardson, D.J. and Oubrie, A. (2006) Soluble aldose [12] Dale, M., Ensley, H., Kern, K., Sastry, K. and Byers, L. (1985) Reversible sugar dehydrogenase from Escherichia coli: a highly exposed active site inhibitors of b-glucosidase. Biochemistry 24, 3530–3539. conferring broad substrate specificity. J. Biol. Chem. 281, 30650–30659. [13] Kitaoka, M. and Hayashi, K. (2002) Carbohydrate-processing phosphorolytic [29] Williams, P.H. (1980) Black rot: a continuing threat to world crucifers. Plant enzymes. Trends Glycosci. Glycotechnol. 14, 35–50. Dis. 64, 736–742. [14] Luley-Goedl, C. and Nidetzky, B. (2010) Carbohydrate synthesis by disaccharide phosphorylases: reactions, catalytic mechanisms and application in the glycosciences. Biotechnol. J. 5, 1324–1338.