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An Exo-��-1,3-Galactanase Having a Novel ��-1,3-Galactan BindingModule from Phanerochaete chrysosporiumHitomi Ichinose¶, Makoto Yoshida‡, Toshihisa Kotake‡‡, Atsushi Kuno§,

Kiyohiko Igarashi‡, Yoichi Tsumuraya‡‡, Masahiro Samejima‡,Jun Hirabayashi§, Hideyuki Kobayashi¶, and Satoshi Kaneko*¶

From the ¶Biological Function Division, National Food Research Institute, 2-1-12Kannondai, Tsukuba, Ibaraki 305-8642, Japan, ‡Graduate School of Agricultural andLife Sciences, Department of Biomaterials Sciences, The University of Tokyo, 1-1-1Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, ‡‡Faculty of Science, Saitama University,

255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan, §Research Center forGlycoscience, National Institute of Advanced Industrial Science and Technology (AIST),

AIST Tsukuba Central 6, 1-1-1 Higashi Tsukuba, Ibaraki 305-8566, JapanRunning Title: exo-�-1,3-galactanase from Phanerochaete chrysosporium

* To whom correspondence should be addressed.Dr. Satoshi Kaneko, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki305-8642, Japan, Phone. +81-29-838-8022; FAX. +81-29-838-7996; E-mail:sakaneko@nfri.affrc.go.jp

An exo-�� -1,3-galactanase genefrom Phanerochaete chrysosporium hasbeen cloned, sequenced and expressedin Pichia pastoris. The complete aminoacid sequence of the exo-�� -1,3-galactanase indicated that the enzymeconsists of a N-terminal catalyticmodule with similarity to glycosidehydrolase family 43 (GH43) and anadditional unknown functional domainsimilar to carbohydrate bindingmodule family 6 (CBM6) in the C-terminal region. The molecular mass ofthe recombinant enzyme was estimatedas 55 kDa based on SDS-PAGE. Theoptimum pH and temperature of thisenzyme were pH 4.5 and 50°C,respectively. The enzyme showedreactivity toward only �� -1,3-linkedgalactosyl oligosaccharides andpolysaccharide as substrates, but didnot hydrolyze �� -1,4-linkedgalactooligosaccharides, �� -1,6-linkedgalactooligosaccharides, pecticgalactan, larch arabinogalactan,arabinan, gum arabic, debranchedarabinan, laminarin, solublebirchwood xylan or soluble oat speltxylan. The enzyme also did nothydrolyze �� -1,3-galactosylgalactosaminide, �� -1,3-galactosylglucosaminide or �� -1,3-galactosylarabinofuranoside, suggesting that it

specifically cleaves the internal �� -1,3-linkage of two galactosyl residues.High-performance liquidchromatographic analysis of thehydrolysis products showed that theenzyme produced galactose from �� -1,3-galactan in an exo-acting manner.However, no activity towards p-nitrophenyl �� -galactopyranoside wasdetected. When incubated witharabinogalactan proteins, the enzymeproduced oligosaccharides togetherwith galactose, suggesting that it isable to bypass �� -1,6-linked galactosylside chains. The C-terminal CBM6 didnot show any affinity for knownsubstrates of CBM6 such as xylan,cellulose and �� -1,3-glucan, though itbound �� -1,3-galactan when analyzedby affinity electrophoresis. Frontalaffinity chromatography for the CBM6moiety using several kinds of terminalgalactose-containing oligosaccharidesas the analytes clearly indicated thatthe CBM6 specifically interacted witholigosaccharides containing a �� -1,3-galactobiose moiety. When the degreeof polymerization of galactoseoligomers was increased, the bindingaffinity of the CBM6 showed nomarked change.

JBC Papers in Press. Published on May 2, 2005 as Manuscript M501024200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

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Arabinogalactan proteins(AGPs) are a family of complexproteoglycans widely distributed inplants. They are found in extracellularmatrix associated with the plasmamembrane and cell wall [1,2]. Althoughtheir precise functions are unknown,AGPs have been implicated in diversedevelopmental roles, includingdifferentiation, cell-cell recognition, andembryogenesis [3-5]. AGPs arecharacterized by large amounts ofcarbohydrate components rich ingalactose (sugars in the present study areD series unless otherwise designated) andL-arabinose, and protein components richin hydroxyproline, serine, threonine,alanine and glycine [1]. The carbohydratemoieties of AGPs have a commonstructure consisting of a �-1,3-galactosylbackbone to which side chains of �-1,6-galactosyl residues are attached throughO-6. L-Arabinose and lesser amounts ofother auxiliary sugars, such as glucuronicacid, L-rhamnose and L-fucose, areattached to the side chains, usually atnon-reducing terminals [1]. Because ofthe large number of putative proteincores and the complex structure of thecarbohydrate moieties, it is difficult toseparate particular AGP molecules fromother AGP species in plant tissues, andconsequently it difficult to elucidate theprecise characteristics of individualAGPs.

Galactanases that hydrolyze �-1,3- or �-1,6-galactosyl linkages wouldbe useful tools to analyze the structureand function of the carbohydrate moietiesof AGPs, but little research has beendone in this area. However, we recentlysucceeded in the molecular cloning ofendo-�-1,6-galactanase fromTrichoderma viride, and we clarified indetail the substrate specificity of therecombinant enzyme [6]. In the presentwork, we focused on exo-�-1,3-galactanases (EC 3.2.1.145) that cleavethe main-chain �-1,3-linkage of thecarbohydrate moiety of AGPs. So far, �-1,3-galactanase has been purified from

only two organisms, Irpex lacteus(Il1,3Gal) [7] and Aspergillus niger(An1,3Gal) [8]. The amino acid sequenceof exo-�-1,3-galactanase is unknown. Inthe present study, we succeeded incloning an exo-�-1,3-galactanase genefrom Phanerochaete chrysosporium(Pc1,3Gal43A) and found that theenzyme contains domains resemblingglycoside hydrolase (GH) family 43 andcarbohydrate binding module (CBM)family 6. As far as we know, this is thefirst report of the cloning of an exo-�-1,3-galactanase and of a CBM that binds�-1,3-galactan.

MATERIALS AND METHODS

Substrates—Arabinan wasprepared from sugar beet by the methoddescribed previously [9]. p-Nitrophenyl�-galactopyranoside (PNP-�-Galp), p-nitrophenyl �-galactopyranoside (PNP-�-Galp), p-nitrophenyl �-glucopyranoside(PNP-�-Glcp), p-nitrophenyl �-L-arabinopyranoside (PNP-�-L-Arap), p-nitrophenyl �-xylopyranoside (PNP-�-Xylp), p-nitrophenyl �-mannnopyranoside (PNP-�-Manp), p-nitrophenyl �-L-fucopyranoside (PNP-�-L-Fucp), larch arabinogalactan, laminarin,birchwood xylan, oat spelt xylan, �-1,3-galactosyl L-arabinofuranoside and �-1,3-galactosyl glucosaminide werepurchased from Sigma ChemicalCompany (St. Louis, MO, U.S.A.).Debranched arabinan, pectic galactanfrom lupin and potato galactan were fromMegazyme International Ltd. (Wicklow,Ireland). �-1,3-Galactosylgalactosaminide was from BioCarb (Lund,Sweden). Gum arabic and hydroxyethylcellulose were from Nacalai tesque Inc.(Kyoto, Japan). CM-curdlan was fromWako Pure Chemical Industries, Ltd.(Osaka, Japan). �-1,3-Galactan, nativeand �-L-arabinofuranosidase-treatedAGPs from radish and �-1,3-, �-1,4-, and�-1,6-galacto-oligosaccharides wereprepared as described previously [7]. �-1,3-�-1,6-Galactan from Prototheca

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zopfii was prepared as reportedpreviously [10]. Methyl �-glycoside of �-1,3-galactotetraose and -pentaose werekind gifts from Dr. Kovác of the NationalInstitutes of Health, NIDDK (NationalInstitute of Diabetes and Digestive andKidney Diseases, Bethesda, MD, U.S.A.).�-1,3-Xylan was a kind gift from Dr. Y.Kitamura of the National Food ResearchInstitute (Tsukuba, Japan).

cDNA cloning of exo-�-1,3-galactanase gene—An exo-�-1,3-galactanase was purified from acommercial enzyme preparation(Driselase, Kyowa Hakko Kogyo Co.,Ltd. Tokyo, Japan), which is the culturesupernatant of I. lacteus , according to themethod described previously [7]. PurifiedIl1,3Gal was separated by sodiumdodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAGE) [11], andelectroblotted onto a polyvinylidenedifluoride (PVDF) membrane (MilliporeCorp., Bedford, MA, U.S.A.). The N-terminal amino acid sequence wasanalyzed on an HP G105A proteinsequencer (Hewlett Packard, Palo Alto,CA, U.S.A.) and the sequence up to 24amino acids was determined: peptide 1,ETQFVSGAAWTDTSGNVFQAHGGG.To determine internal sequences, peptidefragments were prepared. In-gel proteasedigestion [12] was carried out byincubating purified exo-�-1,3-galactanase (3 μg) and V8 protease (0.6μg, Wako Pure Chemical Industries, Ltd.,Osaka, Japan) for 15 min at roomtemperature at the bottom of the stackinggel of SDS-PAGE. The resulting peptidefragments were separated by SDS-PAGE(12% acrylamide gel), electroblotted ontoPVDF membranes, and sequenced asdescribed above. The following aminoacid sequence was obtained: peptide 2,GLFQDDDSAGTAYLLYASDNNQQFKKSRLD. The P. chrysosporium genomedatabase was searched for a genecorresponding to the determined aminoacid sequences of Il1,3Gal using thetblastn algorithm (BLOSUM 62 matrix,e-value = 10) on the DOE Joint Genome

Institute website (http://genome.jgi-psf.org/whiterot1/whiterot1.home.html),and a sequence, designated pc.71.18.1,was identified. Based on the obtainednucleotide sequence, primers weredesigned as follows: F1 (5’-CAA AACCAG ATC GTC TCT GG-3’), R1 (5’-GAG GCG CGA GAT CTT GAA-3’), F2(5’-CGT TTG CCC GAG GGT CA-3’),R2 (5’-CCG ACA GAA TTG TCG AGGATC-3’), and F3 (5’-AAC GTC CAGGCA GGG A-3’).

Total RNA was extracted from P.chrysosporium grown in Kremer andWood medium [13] containing 2%cellulose powder, Whatman CF11(Whatman Inc., Clifton, NJ, U.S.A.), at37°C during 3 days. The first-strandcDNA from the total RNA wassynthesized using a Ready-To-Go

TMYou-

Prime First-strand Beads kit (AmershamBiosciences Corp., Piscataway, NJ,U.S.A.) with a 3’ RACE primer(Invitrogen Corp., Carlsbad, CA, U.S.A.).Polymerase chain reaction (PCR) wasperformed with the following primerpairs: F1 and R1 for first PCR, F2 andR2 for second PCR, and F3 and theoligonucleotide (complementing 3’RACE primer; 5’-GGC CAC GCG TCGACT AGT AC-3’) for 3’ rapidamplification of cDNA ends (RACE). AllPCR products were subcloned into thepGEM-T Easy vector (Promega, Madison,WI, U.S.A.). The nucleotide sequenceswere determined with an ABI PRISM 310Genetic Analyzer (Applied Biosystems,Foster City, CA, U.S.A.) according to themanufacturer’s instructions, andassembled for completion of the openreading frame encoding the exo-�-1,3-galactanase.

Expression of Pc1,3Gal43A andPcCBM6 genes— PCR was carried outusing two oligonucleotide primers basedon the nucleotide sequence of the matureprotein (5’ primer; 5’-GAA TTC CAAAAT CAG ATC GTC TCT GG-3’ and 3’primer; 5’- GCG GCC GCT CAG TACACA ATG ATC TTG T-3’). The productwas digested with EcoRI and NotI

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(underlined), and then ligated into thecorresponding site of the Pichiaexpression vector pPICZ�A (Invitrogen).To express family 6 CBM fromPc1,3Gal43A (PcCBM6), the followingprimers were designed: (5’ primer; 5’-CCA TGG CAG GGA CGT ACT CGGTCG CC-3’ and 3’ primer; 5’-AAG CTTTCA GTA CAC AAT GAT CTT GTC-3’).PCR was carried out using the primers,and the product was subcloned into thepGEM-T Easy vector. The vector wasdigested with EcoRI, and the fragmentwas ligated into the EcoRI site of thepPICZ�A. Approximately 10 �g of therecombinant plasmid(pPICZ�A/Pc1,3Gal43A orpPICZ�A/PcCBM6) was linearized withBpu1102I (Takara Bio Inc., Otsu, Japan)prior to transformation of Pichia pastorisstrain KM71H (Mut

S) cells.

Electroporation and selection oftransformants were carried out accordingto the instruction manual of theEasySelect

TMPichia expression kit

(version G; Invitrogen). The selectedclone (KM71H/Pc1,3Gal43A orKM71H/PcCBM6) was cultured in YPGmedium consisting of 1% (w/v) yeastextract, 2% (w/v) peptone, and 1% (v/v)glycerol at 30°C for 3 days. The cellswere harvested by centrifugation at 2,000x g for 5 min, then suspended in YPMmedium containing 1% (w/v) yeastextract, 2% (w/v) peptone, and 1% (v/v)methanol as an inducer, and cultivated at30°C for 2 days with constant shaking.Methanol was supplemented at 1%concentration every 24 h during theperiod of induction.

Purification of recombinantPc1,3Gal43A and PcCBM6—Therecombinant enzyme was purified fromthe culture supernatant of the P. pastoristransformant carrying the Pc1,3Gal43Agene. The crude enzyme preparation wasdialyzed against 10 mM acetate buffer,pH 5.0, and applied to a CM-SepharoseFast Flow (Amersham Biosciences)column (5 x 50 mm) at the flow rate of 1ml per min. The column was washed with

the same buffer to remove unboundmaterials, then the bound proteins wereeluted with a linear gradient of sodiumchloride (0-1 M). The eluate wasfractionated into 1 ml portions. Thefractions with exo-�-1,3-galactanaseactivity (fraction tubes 5-9) werecombined and dialyzed against deionizedwater. The final preparation thusobtained was used as purified enzyme.The purification procedure wasperformed in triplicate.

PcCBM6 was purified from theculture supernatant of P. pastoristransformant KM71H/PcCBM6 using aDEAE-Sepharose Fast Flow (AmershamBiosciences) column (5 x 50 mm). Thesupernatant was dialyzed against 20 mMTris-HCl buffer, pH 8.0, and applied tothe column at the flow rate of 1 ml permin, and the eluate was fractionated into1 ml portions. PcCBM6 was eluted in theunadsorbed fractions. The purity ofPcCBM6 was determined by SDS-PAGEand the fractions containing purePcCBM6 were combined and dialyzedagainst deionized water. The finalpreparation thus obtained was used aspurified PcCBM6.

Enzyme assay and measurementof protein—Activity of exo-�-1,3-galactanase was determined by theSomogyi-Nelson method [14]. Thereaction mixture consists of 20 μl ofMcIlvaine buffer (0.2 M Na2HPO4-0.1 Mcitric acid), pH 4.5 and 25 μl of 1% (w/v)�-1,3-galactan. After 5 min of pre-incubation at 37°C, 5 μl of enzyme wasadded to the solution, and the mixturewas incubated at 37°C for 15 min. Thereactions were terminated by heating at100°C for 5 min (standard condition).One unit of enzyme activity is defined asthe amount of enzyme that released 1μmol of galactose per min. Proteinamounts of the enzyme was measured byusing the BCA protein assay reagent kit(Pierce Biotechnology Inc., Rockford, IL,U.S.A.) with bovine serum albumin(BSA) as a standard.

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Enzymic properties—The effectsof pH on the activity and stability ofPc1,3Gal43A were investigated with aseries of glycine-HCl buffers from pH2.0 to 2.6, McIlvaine buffers from pH 2.6to 7.6 and Atkins-Pantin buffers (0.2 Mboric acid / 0.2 M KCl / 0.2 M Na2CO3)from pH 7.6 to 11.0. The activity of theexo-�-1,3-galactanase was assayed underthe standard conditions described above.For determination of the pH stability ofPc1,3Gal43A, the enzyme was pre-incubated at various pH values with 0.1%(w/v) BSA in the absence of substrate at30°C for 1 h, and the residual activitywas assayed under the standardconditions. The effect of temperature onthe activity of Pc1,3Gal43A wasdetermined by incubation at differenttempratures. With the exception oftemperature, the assay conditions werethe same as described for the standardmethod. For the temperature-stabilitymeasurement of Pc1,3Gal43A, theenzyme was pre-incubated at varioustemperatures at pH 4.5 with 0.1% (w/v)BSA for 1 h and the residual activity wasdetermined under the standard conditions.

Substrate specificity—Thesubstrate specificity of Pc1,3Gal43Atoward various PNP-glycosides wasdetermined. Each assay mixturecontained 25 μl of a 2 mM PNP-glycoside solution, 20 μl of McIlvainebuffer, pH 4.5 and 5 μl of enzymesolution. The reactions were performed at37°C for 10 min and terminated byadding 100 μl of 0.2 M Na2CO3. Theamount of PNP released was calculatedfrom the absorbance at 400 nm.

The substrate specificity ofPc1,3Gal43A toward polysaccharideswas determined using �-1,3-galactan,gum arabic, larch arabinogalactan,debranched arabinan, arabinan, solubleoat spelt xylan, soluble birchwood xylan,pectic galactan, lupin galactan, CM-curdlan, laminarin, �-1,3-�-1,6-galactanand native and �-L-arabinofuranosidase-treated AGPs from radish as substrates.The reactions were performed in

McIlvaine buffer, pH 4.5, containing0.5% (w/v) substrate and 6.9 nM enzyme.

The substrate specificity ofPc1,3Gal43A toward oligosaccharideswas determined using �-1,3-, �-1,4-, and�-1,6-linked galactosyl oligosaccharides,�-1,3-galactosyl galactosaminide, �-1,3-galactosyl glucosaminide and �-1,3-galactosyl L-arabinofuranoside as thesubstrate. The reactions were performedin the same buffer, containing 5 mMsubstrate and 23 nM enzyme. Afterincubation at 37°C for the appropriatereaction time, the activity wasdetermined by the Somogyi-Nelsonmethod [14].

The kinetic parameters ofPc1,3Gal43A toward �-1,3-galactan weredetermined. The reactions wereperformed in McIlvaine buffer, pH 4.5,containing 0.1-0.8% (w/v) substrates and6.9 nM enzyme at 37°C. The reducingpower of released galactose wasdetermined by the Somogyi-Nelsonmethod [14]. The molecular weight of �-1,3-galactan was estimated as follows.Trifluoroacetic acid (TFA; 2 M) wasadded to �-1,3-galactan, and heated at121°C for 1 h. The 2 M TFA wasevaporated under a stream of air. Thenisopropyl alcohol was added andevaporated to remove the TFAcompletely. The reducing power wascompared between untreated and TFA-treated �-1,3-galactans, and the degree ofpolymerization (dp) of galactose wascalculated. The value (dp 50) was used todetermine kcat for �-1,3-galactan.

To evaluate the catalyticefficiency of the enzyme toward �-1,3-galactooligosaccharides such as �-1,3-galactobiose, �-1,3-galactotriose, methyl�-1,3-galactotetraoside and methyl �-1,3-galactopentaoside, 1.6-1.8 nM enzymewas incubated with 10 μM substrate inMcIlvaine buffer, pH 4.5 for up to 120min at 37°C. At regular intervals, analiquot (100 μl) of the reaction mixturewas taken and the reaction wasterminated by heating at 100°C for 5 min.The samples were quantified by high-

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performance anion-exchangechromatography with a pulsedamperometric detection (HPAEC-PAD)system using L-fucose as an internalstandard. The samples were analyzedusing a CarboPac

TMPA1 column (4 x 250

mm, Dionex Corp., Sunnyvale, CA,U.S.A.) eluted with 0.1 M NaOH (0-5min), followed by a linear gradient (5-20min) of sodium acetate (0-0.1 M) at theflow rate of 1 ml per min. The reactioncurves of galactooligosaccharides wereused to determine the kcat/Km of thereaction by applying the followingequation as described by Matsui et al .[15],

k•t = ln([S0]/[St])where k = (kcat/Km) [E], t represents time,and [S0] and [St] represent substrateconcentrations at time 0 and t ,respectively. This relationship is onlyvalid when [E] << [S] << Km.

For the hydrolysis of �-1,3-galactotriose and methyl �-1,3-galactopentaoside, the reactions wereperformed in McIlvaine buffer, pH 4.5,containing 5 mM substrate and 6.9 nMenzyme. For the hydrolysis of �-1,3-galactan, gum arabic after twice-repeatedSmith degradation and �-L-arabinofuranosidase-treated radish rootAGP by Pc1,3Gal43A, the reactions wereperformed in same buffer, containing0.5% (w/v) substrate, and 6.9 nM enzyme,for up to 3 h at 37°C. At regular intervals,an aliquot (10 μl) of the reaction mixturewas taken and the reaction wasterminated by heating at 100°C for 5 min.The samples were analyzed by HPAEC-PAD as described above.

Affinity gel electrophoresis andfrontal affinity chromatography—Theaffinity of PcCBM6 for a range of solublepolysaccharides was determined byaffinity gel electrophoresis. The methodwas essentially as described by Tommeet al. [16], using polysaccharide ligands.Affinity electrophoresis was carried outfor 1 h using native 7% (w/v)polyacrylamide gel containing 0.3%

(w/v) various polysaccharides. BSA wasused as the nonbinding negative control.

The affinity of PcCBM6 for arange of oligosaccharides wasdetermined by frontal affinitychromatography (FAC). The method wasessentially as described by Hirabayashi etal. [17,18], using pyridylaminated (PA)oligosaccharides containing a terminalgalactose residue as shown in Figure 6B.PA-lactose (Gal-�-1,4-Glc), PA-Gb3(Gal-�-1,4-Gal-�-1,4-Glc), PA-GA1(Gal-�-1,3-GalNAc-�-1,4-Gal-�-1,4-Glc),PA-LNT (Gal-�-1,3-GlcNAc-�-1,3-Gal-�-1,4-Glc), PA-LNnT (Gal-�-1,4-GlcNAc-�-1,3-Gal-�-1,4-Glc), PA-Galilipentasaccharide (Gal-�-1,3-Gal-�-1,4-GlcNAc-�-1,3-Gal-�-1,4-Glc), and PA-rhamnose were purchased from TaKaRa(Kyoto, Japan). Non-labeled �GalLac(Gal-�-1,3-Gal-�-1,4-Glc) and �Gal2Lac(Gal-�-1,3-Gal-�-1,3-Gal-�-1,4-Glc)were gifts from Dr. T. Urashima (ObihiroUniversity of Agriculture and VeterinaryMedicine) and were labeled with PAusing GlycoTAG (TaKaRa).

The total amount of immobilizedPcCBM6 in the column, Bt was firstdetermined with �1,3Gal3-ABEE (Gal-�-1,3-Gal-�-1,3-Gal-p-aminobenzoic acidethyl ester) using the equation describedpreviously,

[ �A �] �0 • (Vf – V0) = Bt � - Kd • (Vf –V0)

( �E �q �. � 1 �) �where Kd is the dissociation constantbetween interacting biomolecules, Bt isthe total amount of immobilized ligand,[ �A �] �0 is the initial concentration of thePA-oligosaccharide of interest (A), Vf isthe elution volume of A, and V0 is theelution volume of PA-rhamnose, whichhas no affinity for PcCBM6. In theassays employed in this study, [ �A �] �0, theinitial concentration of the PA-oligosaccharides, was 10 nM, which isnegligibly small compared with Kd. Thus,Vf approached the maximum value, Vf ,which is independent of [ �A �] �0, and so weused the following equation to obtain the

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values of Kd of PcCBM6 toward terminalgalactose-containing oligosaccharidederivatives.

Kd = �B t �/ (Vf – V0)

( �E �q �. � 2 �)

RESULTS

Nucleotide sequence of the exo-�-1,3-galactanase—Although exo-�-1,3-galactanase has been purified from I.lacteus and A. niger, no information isavailable about the amino acid sequenceof the enzyme. Therefore, exo-�-1,3-galactanase was purified from I. lacteusand N-terminal and internal amino acidsequences were determined. Based onthese partial amino acid sequences ofIl1,3Gal (Figure 1A, underlined), full-length cDNA for Pc1,3Gal43A wasisolated by reverse-transcription PCR.The sequence, with the deduced aminoacid sequence, is shown in Figure 1A.The cDNA sequence contained an openreading frame (1344 bp) encoding 448-amino-acid protein (Figure 1A). Thesequence contained a putative signalsequence (20 amino acid residues)preceding the putative mature enzyme(428 amino acids, molecular mass of45.632 kDa). Three putative N-glycosylation sites were found in thededuced protein sequence (Figure 1A,double underlined). The deduced aminoacid sequence of Pc1,3Gal43A wascompared with sequences in the proteinand nucleic acid database BLAST(National Center for BiotechnologyInformation,http://www.ncbi.nlm.nih.gov/BLAST/).Pc1,3Gal43A resembled the followinghypothetical proteins, in order ofdecreasing similarity: hypotheticalprotein FG11366.1 from Gibberella zeaePH-1 (GenBank accession numberXP_391542.1); hypothetical proteinMG03844 from Magnaporthe grisea 70-15 (accession number XP_361370.1);hypothetical protein from Neurosporacrassa (accession number XP_327147.1);

hypothetical protein AN7313.2 fromAspergillus nidulans FGSC A4(accession number XP_411450.1).Pc1,3Gal43A also shared low similaritywith the following GH family 43enzymes: �-xylosidase from Clostridiumstercorarium (21.3%, accessionnumber BAC87941.1), �-L-arabinofuranosidase from Streptomyceschartreusis (19.4%, accession numberBAA90772), endo-�-1,4-xylanase fromPaenibacillus polymyxa (19.3%,accession number CAA40378) and endo-�-L-1,5-arabinanase from Cellvibriojaponicus (17.6%, accession numberCAA71485), suggesting thatPc1,3Gal43A is a distantly related toGH43. The BLAST search ofPc1,3Gal43A also revealed that theenzyme has a C-terminal region (from289 to 448) with similarity to CBM6(Figure 1C).

Three putative GH43 enzymes,gx.41.43.1, pc.158.1.1 and pc.71.18.1,were identified in the P. chrysosporiumgenome [19]. The sequence ofPc1,3Gal43A corresponded to that ofpc.71.18.1 and the deduced amino acidsequence of Pc1,3Gal43A shared 43.5%similarity with gx.41.43.1 and 66.7%with pc.158.1.1. BLAST searches ofgx.41.43.1 and pc.158.1.1 suggested thatthese sequences probably encode putative�-xylosidase and endo-arabinanase,respectively.

Expression of recombinant exo-�-1,3-galactanase—The cDNA fragmentencoding the mature enzyme ofPc1,3Gal43A was amplified by PCR,then subcloned into the expression vectorpPICZ�A to obtainpPICZ��/Pc1,3Gal43A. Therecombinant protein was expressed in P.pastoris strain KM71H as a secretedform with the aid of �-factor, which is asecretion signal of yeast. The crudeenzyme showed activity toward �-1,3-galactan, and the recombinant enzymewas purified from culture supernatant ofP. pastoris. The purification ofrecombinant Pc1,3Gal43A is summarized

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in Table 1. Approximately 300 mg perliter of recombinant enzyme wasexpressed. The purified Pc1,3Gal43Agave a single band on SDS-PAGE whenvisualized by staining with CoomassieBrilliant Blue (CBB) R-250 (Figure 2A).The molecular mass of Pc1,3Gal43A wasestimated to be 55 kDa by SDS-PAGE.The specific activity of the recombinantenzyme was 99.8 units/mg, which issomewhat higher than that of purifiedIl1,3Gal (87.8 units/mg) [7] and thenative enzyme purified from culturefiltrate of P. chrysosporium (94.8units/mg), suggesting that the folding ofthe recombinant enzyme was similar tothat of the native enzyme.

The effects of pH andtemperature on purified Pc1,3Gal43Awere investigated. The recombinantPc1,3Gal43A exhibited maximal activityat pH 4.5 (data not shown). The enzymewas stable between pH 3.0 and 6.0,where >80% of the activity was retained(data not shown). The enzyme exhibitedmaximal activity at 50°C (data notshown). Thermostability was analyzed atpH 4.5 after incubation at varioustemperatures for 1 h. More than 80%activity was retained below 55°C (datanot shown). The optimum pH ofPc1,3Gal43A was very similar to that ofIl1,3Gal (pH 4.6). The range of pHstability was rather narrow comparedwith that of Il1,3Gal (pH 3.5-7.5), thoughPc1,3Gal43A was more thermostablethan Il1,3Gal (up to 48°C) [7].

Substrate specificity—The GH43family includes �-xylosidase (EC3.2.1.37), �-L-arabinofuranosidase (EC3.2.1.55), endo-�-L-1,5-arabinanase (EC3.2.1.99) and �-1,4-xylanase (EC 3.2.1.8).Therefore, the substrate specificity ofPc1,3Gal43A was investigated. WhenPc1,3Gal43A was incubated withpolysaccharides, the enzyme showedactivity toward only �-1,3-galactan, butnot gum arabic, larch arabinogalactan,debranched arabinan, arabinan, solubleoat spelt xylan or soluble birchwoodxylan (Table 2). The Km and kcat values of

Pc1,3Gal43A for �-1,3-galactan were4.8±0.2 mg/ml and 8437±235 min

-1,

respectively. The substrate specificitytoward PNP-glycosides such as PNP-�-Galp, PNP-�-Galp, PNP-�-Glcp , PNP-�-L-Arap , PNP-�-Xylp , PNP-�-Manp andPNP-�-L-Fucp was also investigated. Theenzyme did not hydrolyze any PNP-glycoside, not even PNP-�-Galp (datanot shown). The recombinantPc1,3Gal43A did not demonstrate any ofthe activities reported for GH43,suggesting that the enzyme is a novelmember of the GH43 family.

The substrate specificity of theenzyme toward other polysaccharides isshown in Table 2. The enzyme did nothydrolyze �-1,4-linked galactose-containing polysaccharides, such aspectic galactan and arabinofuranosidase-treated galactan from lupin. The enzymedid not act on laminarin, CM-curdlan or�-1,3-xylan, each of which consists of �-1,3-glycan. These data suggest thatPc1,3Gal43A hydrolyzes only �-1,3-linked galactose chains. This isconsistent with the results of the activitytoward oligosaccharides (Table 3).Pc1,3Gal43A specifically hydrolyzed �-1,3-linked galactooligosaccharides, butnot �-1,4-linked or �-1,6-linkedgalactooligosaccharide. It should benoted that Pc1,3Gal43A could nothydrolyze the �-1,3-galactosyl linkage of�-1,3-galactosyl galactosaminide, �-1,3-galactosyl glucosaminide or �-1,3-galactosyl L-arabinofuranoside (Table 3).It appears that Pc1,3Gal43A specificallycleaves the internal �-1,3-linkage of twogalactosyl residues.

Mode of action—The catalyticefficiency of Pc1,3Gal43A toward �-1,3-galactooligosaccharides with different dpis shown in Table 4. The catalyticefficiencies for galactobiose togalactopentaose were the almost the same,suggesting that the number of majorsubsites of Pc1,3Gal43A is two. Thevalue of log(kcat/Km) slightly increasedfrom dp 2 up to dp 4, suggesting the

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enzyme might recognize thetetrasaccharide.

The hydrolysis products of of �-1,3-galactooligosaccharides andpolysaccharides generated byPc1,3Gal43A were analyzed by HPAEC-PAD (Figure 3 and 4). In the hydrolysisof �-1,3-galactotriose, galactose and �-1,3-galactobiose were generated byPc1,3Gal43A (Figure 3B). In the case ofhydrolysis of methyl �-1,3-galactopentaoside, the products wereonly galactose and methyl �-1,3-galactotetraoside from the initial stage ofhydrolysis. These results suggested thatPc1,3Gal43A releases galactose fromnon-reducing ends of the substrates. Thehydrolysis products of �-1,3-galactan,gum arabic after twice-repeated Smithdegradation and �-L-arabinofuranosidase-treated AGP fromradish root produced by Pc1,3Gal43Awere also analyzed by HPAEC-PAD(Figure 4). Only the peak of galactosewas detected when Pc1,3Gal43A wasincubated with �-1,3-galactan (Figure4B). In contrast, several peakscorresponding to oligosaccharide werealso detected along with the peak ofgalactose when Pc1,3Gal43A wasincubated with gum arabic after twice-repeated Smith degradation (Figure 4C)or �-L-arabinofuranosidase-treated rootAGP (Figure 4D). None of the producedoligosaccharides were �-1,3-linkedgalactooligosaccharides, suggesting thatPc1,3Gal43A is capable of bypassing thearabinogalactan side chains in AGPs.

Carbohydrate binding module—To analyze the function of the C-terminalCBM6 of Pc1,3Gal43A, we attempted toexpress the catalytic module and CBM6individually in P. pastoris. Unfortunately,the catalytic module could not beexpressed in P. pastoris, but CBM6 wassuccessfully expressed. The purifiedPcCBM6 appeared as smear bands onSDS-PAGE when visualized by stainingwith CBB R-250, and its molecular masswas somewhat larger than expected(Figure 2B). PcCBM6 has one N-

glycosylation site, so that PcCBM6seems to be expressed as a glycoprotein.After endo-glycosidase H treatment,PcCBM6 appeared as a single band of theexpected size (14.8 kDa) on SDS-PAGE.

Affinity gel electrophoresis wasperformed to investigate the affinity ofPcCBM6 for soluble polysaccharides(Figure 5). Although CBM6 binds tocellulose, xylan and �-1,3-glucan,PcCBM6 did not show any affinity forknown substrates such as hydroxyethylcellulose, soluble birchwood xylan andlaminarin (data not shown). PcCBM6also did not show affinity for native gumarabic (Figure 5B) and larcharabinogalactan (data not shown), whichare �-1,3-�-1,6-galactans, though itshowed significant affinity for gumarabic after several Smith degradationtreatments (Figure 5C-D), suggesting thatPcCBM6 has affinity for �-1,3-galactan.No affinity for potato galactan (�-1,4-galactan) or �-1,3-xylan was seen (datanot shown), suggesting that PcCBM6critically recognizes �-1,3-linkedgalactosyl residues. This is alsosupported by the results of FAC (Figure6). PcCBM6 specifically bound tooligosaccharides containing at least two�-1,3-linked galactosyl residues, such as�GalLac and �Gal2Lac (Figure 6C).However, PcCBM6 did not bind to Lac,Gb3, GA1, LNT, LNnT or Galili pentaeven though these oligosaccharidescontain Gal-�-1,3-GalNAc or Gal-�-1,3-GlcNAc. These data suggest thatPcCBM6 critically recognizes twocontinuous �-1,3-linked galactosylresidues. Using �1,3Gal3-ABEE, Kdvalues for the substrates of PcCBM6were determined. The plot of the FACdata used to determine the affinity ofPcCBM6 for �1,3Gal3-ABEE is shown inFigure 6A. The Kd value of PcCBM6 for�1,3Gal3-ABEE was 265 μM. Then theKd values for �GalLac and �Gal2Lacwere determined from the relativebinding data, based on the Kd value of�1,3Gal3-ABEE (Figure 6C). The Kdvalues for �GalLac and �Gal2Lac were

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82 μM and 217 μM, respectively. Anincrease in the number of �-1,3-linkedgalactosyl residues of theoligosaccharides did not result in higheraffinity for PcCBM6.

DISCUSSION

The glycoside hydrolases arecurrently classified into 99 families thatinclude many kinds of enzymes (seehttp://afmb.cnrs-mrs.fr/CAZY/index.html). �-Galactanhydrolases such as endo-�-galactanasesand �-galactosidases are classified intoGH5 and 53, and GH1, 2, 35 and 42,respectively. GH53 contains �-1,4-galactanases. Recently, we succeeded incloning the gene of an enzyme whichhydrolyzes the �-1,6-galactan side chainsin AGPs, and we concluded that theenzyme is a novel member of GH5. AGH35 �-galactosidase from radish hasbeen reported to cleave both �-1,3- and�-1,6-linkages of galactooligosaccharides,but it does not cleave �-1,4-linkages ofgalactooligosaccharides [20]. Exo-�-1,3-galactanase can apparently bedistinguished from such enzymes, as itspecifically hydrolyzes only �-1,3-linkages of galactan andgalactooligosaccharide [7,8]. However,only two exo-�-1,3-galactanases, from A.niger (An1,3Gal) and I. lacteus(Il1,3Gal), have been reported, and theiramino acid sequences are not known. Inthis work, we cloned an exo-�-1,3-galactanase gene from P. chrysosporiumfor the first time. The deduced aminoacid sequence encoding the Pc1,3Gal43Agene showed similarity with those ofenzymes belonging to GH43, suggestingthat Pc1,3Gal43A is a novel member ofGH43. The GH43 family includesenzymes such as �-xylosidase (EC3.2.1.37), �-L-arabinofuranosidase (EC3.2.1.55), endo-�-L-1,5-arabinanase (EC3.2.1.99) and �-1,4-xylanase (EC 3.2.1.8),and is classified into clan GH-F togetherwith GH62. The catalysis by theseenzymes involves an inverting

mechanism. The crystal structure ofarabinanase A obtained from Cellvibriojaponicus (CjArb43A), belonging toGH43, revealed a five-bladed �-propellerfold [21]. Mutagenesis studies involvingfour kinds of amino acids, includingthree putative catalytic amino acids,Asp38, Asp158 and Glu221, showed thatmutagenesis caused a significantreduction of the activity [21]. When theamino acid sequences of Pc1,3Gal43Aand CjArb43A are compared, onlyGlu221 in CjArb43A was conserved inPc1,3Gal43A (Figure 1B). Since theregion including Glu221 showed highsimilarity to that of Pc1,3Gal43A, it ispossible that Glu208 in Pc1,3Gal43A,corresponding to Glu221 in CjArb43A, isthe catalytic amino acid (Figure 1B,asterisked). Other key amino acids suchas Asp38, Asp158 and Phe114 inCjArb43A are substituted with Ile, Serand Val in Pc1,3Gal43A, respectively,suggesting that the structure of thesubstrate binding cleft of Pc1,3Gal43Awill be very different from that of endo-arabinanase. Thus, it is difficult toevaluate the significance of the structuralfeatures of Pc1,3Gal43A from thestructure of CjArb43A.

When we investigated thesubstrate specificity of Pc1,3Gal43A, theenzyme did not show any activity towardknown substrates of GH43. Pc1,3Gal43Ahas specific reactivity for �-1,3-galactanand the enzyme did not act on �-1,4- or�-1,6-linked galactosyl linkages,indicating that it is an exo-�-1,3-galactanase and differs from known �-galactosidases. Since no activity towardPNP-�-Gal, �-1,3-galactosylglucosaminide or �-1,3-galactosylgalactosaminide was observed, theenzyme likely recognizes the aglyconside of its substrates. As regards thecatalytic efficiency of Pc1,3Gal43A �-1,3-galactooligosaccharides with variousdp values, the value of log(kcat/Km) wasincreased, but only slightly, from dp 2 upto dp 4. Thus, the enzyme appears torecognize disaccharide (subsites -1 and

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+1) and might have two further subsiteson the aglycon side (subsite -1 to subsite+3). When Pc1,3Gal43A hydrolyzedAGPs, the enzyme releasedoligosaccharide, suggesting that it iscapable of bypassing the �-1,6-galactanside chains in AGPs. This uniqueproperty could be related to the numberof subsites.

Pc1,3Gal43A possessed an extraC-terminal domain that resembles CBM6.The module showed affinity for �-1,3-linked galactan orgalactooligosaccharides (Figures 5 and 6),and it recognizes a sequence of at leasttwo successive �-1,3-linked galactoseresidues. As far as we know, this is thefirst report of a �-1,3-galactan bindingmodule. CBM6s are modules consistingof approximately 120 amino acidresidues. To date, cellulose, xylan and/or�-1,3-glucan binding functions have beendemonstrated. The three-dimensionalstructures of five CBM6s have now beensolved [22-27]. The five all have verysimilar �-sandwich structures. TheCBM6 modules contain two clefts (cleftsA and B) that could potentially functionas ligand-binding sites. Cleft B is locatedon the concave surface of one �-sheet,whereas cleft A is found in the loopregion connecting the inner and outer �-sheets of the jellyroll fold. Cleft B is in asimilar location to the ligand bindingsites of CBMs from several otherfamilies, including CBM22, CBM4,CBM29, and CBM17 [28-31], whereascleft A is positioned in the loopsconnecting the two �-sheets andresembles the sugar binding sites oflectins, as has been discussed byBoraston et al. [32]. Very recently, the �-1,3-glucooligosaccharide bindingstructure of CBM6 from a Bacillushalodurans laminarinase (BhCBM6) wassolved [27]. BhCBM6 interactsspecifically with the non-reducing sugar.The unique feature of the BhCBM6binding site is an extended loopcomprising residues Gly907 to Asp912(see Figure 1C, boxed). This loop, and

most notably the side chain of Tyr911,creates a raised platform that follows theU-shaped curvature of thelaminarioligosaccharide up and out ofcleft A and along a surface that isdistinct from the usual cleft A of CBM6s(see Figure 7 in Ref. 27). When theamino acid sequence of PcCBM6 iscompared with those of known CBM6s,the amino acids comprising cleft B,especially aromatic amino acids, werenot conserved, suggesting that cleft B ofPcCBM6 cannot bind sugars. However,as observed for BhCBM6, variation ofthe structure of cleft A might allowbinding of �-1,3-galactan. BecausePcCBM6 did not show affinity for �-1,3-glucan, it might have a distinct cleft Astructure. Indeed, an extra sequencecorresponding to an extended loopcomprising residues Gly907 to Asp912 inBhCBM6 can be observed in the aminoacid sequence of PcCBM6, although thesequence is quite different from that ofBhCBM6 (Figure 1C). Therefore, wespeculate that this loop of PcCBM6 issuitable to bind �-1,3-galactan. In thesugar binding structure of BhCBM6, OHgroups at C-3 and C-4 of the terminalglucose residue were tightly hydrogen-bonded with amino acids (Glu29 andAsn132 in BhCBM6) [27]. These aminoacids are not conserved in PcCBM6.Therefore, PcCBM6 could recognizegalactose via a completely differentbinding mechanism from BhCBM6.

The GH43 family seems to be agood model for investigating structure-function relationships of glycosidehydrolases, as it includes many kinds ofenzymes. The variation of the substratespecificity of CBM6 is also veryinteresting. Further studies on the ofstructure-function relationships of exo-�-1,3-galactanase are in progress.

Acknowledgments— We aregrateful to Dr. Kovác of the NationalInstitutes of Health for supplying themethyl �-glycosides of �-1,3-galactotetraose and -pentaose. We arealso grateful to Dr. Y. Kitamura of

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National Food Research Institute forsupplying �-1,3-xylan, and Dr. K.Mizumoto and R. Takahashi of ToyamaForestry and Forest Products Research

Center, and Dr. Suzuki of MitsubishiRayon Co., Ltd. for supplying larcharabinogalactan.

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11. Laemmli, U. K. (1970) Nature 227 , 680-68512. Hirano, H. (1993) Analysis of protein structure for gene cloning, Tokyo Kagaku

Dohjin, Tokyo, pp 89-92.13. Kremer, S. M., and Wood, P. M. (1992) Eur. J. Biochem. 205 , 133-13814. Somogyi, N. (1952) J. Biol. Chem. 195 , 19-2315. Matsui, I., Ishikawa, K., Matsui, E., Miyairi, S., Fukui, S., and Honda, K. (1991) J.

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Urashima, T., Oka, T., Futai, M., Muller, W. E., Yagi, F., and Kasai, K. (2002)Biochim. Biophys. Acta. 1572 , 232-254

19. Martinez, D., Larrondo, L. F., Putnam, N., Gelpke, M. D., Huang, K., Chapman, J.,Helfenbein, K. G., Ramaiya, P., Detter, J. C., Larimer, F., Coutinho, P. M.,Henrissat, B., Berka, R., Cullen, D., and Rokhsar, D. (2004) Nat. Biotechnol. 22 ,695-700

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21. Nurizzo, D., Turkenburg, J. P., Charnock, S. J., Roberts, S. M., Dodson, E. J.,McKie, V. A., Taylor, E. J., Gilbert, H. J., and Davies, G. J. (2002) Nat. Struct.Biol. 9, 665-668

22. Czjzek, M., Bolam, D. N., Mosbah, A., Allouch, J., Fontes, C. M., Ferreira, L. M.,Bornet, O., Zamboni, V., Darbon, H., Smith, N. L., Black, G. W., Henrissat, B.,and Gilbert, H. J. (2001) J. Biol. Chem. 276, 48580-48587

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25. Boraston, A. B., Notenboom, V., Warren, R. A., Kilburn, D. G., Rose, D. R., andDavies, G. (2003) J. Mol. Biol. 327, 659-669

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FOOTNOTES

The nucleotide sequence depicted in Figure 1 has been submitted to the DDBJnucleotide database under the accession no. AB200390.

The abbreviations used are: Pc1,3Gal43A, Phanerochaete chrysosporium exo-�-1,3-galactanase; GH, glycoside hydrolase family; CBM, carbohydrate binding module;CBM6, carbohydrate binding module family 6; AGPs, arabinogalactan proteins;Il1,3Gal, exo-�-1,3-galactanase from Irpex lacteus; An1,3Gal, exo-�-1,3-galactanasefrom Aspergillus niger; PNP, p-nitrophenyl; SDS-PAGE, sodium dodecyl sulphatepolyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; PCR,polymerase chain reaction; RACE, rapid amplification of cDNA ends; PcCBM6,family 6 CBM from Pc1,3Gal43A; McIlvaine buffer, 0.2 M Na2HPO4-0.1 M citric acidbuffer; BSA, bovine serum albumin; Atkins-Pantin buffer, 0.2 M boric acid / 0.2 MKCl / 0.2 M Na2CO3 buffer; TFA, trifluoroacetic acid; dp, degree of polymerization;HPAEC-PAD, high-performance anion-exchange chromatography with pulsedamperometric detection; FAC, frontal affinity chromatography; PA, pyridylaminated;CBB, Coomassie Brilliant Blue; Gb3, Gal-�-1,4-Gal-�-1,4-Glc; GA1, Gal-�-1,3-GalNAc-�-1,4-Gal-�-1,4-Glc; LNT, Gal-�-1,3-GlcNAc-�-1,3-Gal-�-1,4-Glc; LNnT,Gal-�-1,4-GlcNAc-�-1,3-Gal-�-1,4-Glc; Glili penta; Gal-�-1,3-Gal-�-1,4-GlcNAc-�-1,3-Gal-�-1,4-Glc; �GalLac, Gal-�-1,3-Gal-�-1,4-Glc; �Gal2Lac, Gal-�-1,3-Gal-�-1,3-Gal-�-1,4-Glc; ABEE, p-aminobenzoic acid ethyl ester; CjArb43A, arabinanase Afrom Cellvibrio japonicus; BhCBM6, family 6 CBM from a Bacillus haloduranslaminarinase.

Key words : glycoside hydrolase family 43, exo-�-1,3-galactanase, arabinogalactan-protein, Phanerochaete chrysosporium, carbohydrate binding module family 6.

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Table 1 Purification of recombinant Pc1,3Gal43A

Purification step

Volume

(ml)

Total

protein

(mg)

Total

activity

(units)

Specific

activity

(unis/mg)

Purification

(-fold)

Yield

(%)

Crude enzyme 50 393 1501 3.8 1 100

CM-Sepharose FF 8.3 14.7 1467 99.8 26 98

Table 2 Substrate specificity of Pc1,3Gal43A toward polysaccharides

Substrate

Activity

(μM of

galactose/min)

Relative activity

(%)

�-1,3-Galactan 18±1 100

Pectic galactan (�-1,4-galactan) 0.04±0.0 0.2

Lupin galactan (�-1,4-galactan) 0.00±0.0 0

CM-curdlan (�-1,3-glucan) 0.00±0.0 0

�-1,3-Xylan 0.00±0.0 0

Laminarin (�-1,3-�-1,6-glucan) 0.00±0.0 0

�-1,3-�-1,6-Galactan 2.3±0.2 13

Native leaf AGP from radish 0.80±0.2 4

�-L-Arabinofuranosidase-treated

root AGP from radish6.1±0.8 34

The enzyme (6.9 nM) was incubated in a mixture containing 0.5% (w/v) substrate andMcIlvaine buffer, pH 4.5, at 37°C. At regular intervals, initial hydrolysis rates weredetermined by the Somogyi-Nelson method.

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Table 3 Substrate specificity of Pc1,3Gal43A toward galactooligosaccharides

SubstrateConcentrati

on

Activity

(μM of

galactose/min)

Relative activity

(%)

�-1,3-Galactan 5 mg/ml 18±1 100

�-1,3-Galactobiose 5 mM 4.2±0.3 9

�-1,3-Galactotriose 5 mM 11±1 24

�-1,4-Galactobiose 5 mM 0.00±0.0 0

�-1,4-Galactotriose 5 mM 0.00±0.0 0

�-1,4-Galactotetraose 5 mM 0.00±0.0 0

�-1,6-Galactobiose 5 mM 0.00±0.0 0

�-1,6-Galactotriose 5 mM 0.00±0.0 0

�-1,6-Galactotetraose 5 mM 0.00±0.0 0

Gal-�-1,3-GalNAc 5 mM 0.00±0.0 0

Gal-�-1,3-GlcNAc 5 mM 0.00±0.0 0

Gal-�-1,3-Ara 5 mM 0.00±0.0 0

The enzyme (6.9 nM) was incubated in McIlvaine buffer, pH 4.5, containing 5 mMsubstrate at 37°C. The initial hydrolysis rate was determined periodically by theSomogyi-Nelson method.Abbreviations: Gal-�-1,3-GalNAc, �-1,3-galactosyl galactosaminide; Gal-�-1,3-GlcNAc, �-1,3-galactosyl glucosaminide; Gal-�-1,3-Ara, �-1,3-galactosyl L-arabinofuranoside.

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Table 4 Rate of �� -1,3-linked galactooligosaccharide hydrolysis by Pc1,3Gal43A

Degree of polymerization of

�-1,3-galactooligosaccharides

kcat/Km

min-1·mM

-1Log(kcat/Km)

2 1500±152 3.17±0.04

3 2749±166 3.44±0.03

4 3609±22.0 3.56±0.01

5 3039±36.0 3.48±0.01

The activity of Pc1,3Gal43A towards �-1,3-galactooligosaccharides was determined by

analyzing the hydrolysis rates of substrates by HPAEC-PAD. The enzyme (1.6-1.8 nM) was

incubated with 10 μM substrate in McIlvaine buffer, pH 4.5, at 37°C. The oligosaccharideswere quantified periodically by HPAEC-PAD. Progress curves of oligosaccharides cleavage

were used to determine kcat/Km.

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17

Figure legends

Figure 1 The nucleotide sequence of the Pc1,3Gal43A gene and the deducedprimary structureA: The putative N-terminal cleavable signal peptide is boxed. The amino acidsequences corresponding to Il1,3Gal are underlined. Possible N-glycosylation sites aredouble underlined. B: Sequence alignment of catalytic amino acid betweenPc1,3Gal43A and CjArb43A. The alignment was performed with ClustalW. Identicalamino acid residues are enclosed in black boxes. Conserved and semi-conservedamino acids residues are in gray boxes and underlined, respectively. Putative catalyticamino acid is asterisked. C: Sequence alignment of selected CBM6s. The alignmentwas performed with ClustalW. The open circles above the sequences mark the threeresidues involved in ligand binding. The black circles above the sequences mark thepositions of two aromatic residues situated in cleft B. Abbreviations: Pc1,3Gal43Aand PcCBM6, exo-�-1,3-galactanase from P. chrysosporium (DDBJ/GenBankaccession number AB200390); CjArb43A, endo-�-L-1,5-arabinanase from C.japonicus (Swiss-Prot accession number Y10458); BhCBM6, ORF BH0236 fromBacillus halodurans C-125 (Swiss-Prot accession number Q9KG76); CtCBM6,xylanase U from Clostridium thermocellum (Swiss-Prot accession number O07653);CsCBM6-1 and CsCBM6-3, ORF from Clostridium stercorarium NCIB11745 (Swiss-Prot accession number Q93AQ5); CmCBM6, endo-�-1,4-glucanase B from Cellvibriomixtus (Swiss-Prot accession number O07653).

Figure 2 SDS-PAGE of recombinant Pc1,3Gal43A and PcCBM6A: lane 1, molecular-mass markers (1 μg each); lane 2, purified recombinantPc1,3Gal43A (1 μg). B: lane 1, molecular-mass markers (1 μg each); lane 2, purifiedrecombinant PcCBM6 (1.5 μg); lane 3, endo-glycosidase H-treated recombinantPcCBM6 (1.5 μg).

Figure 3 HPAEC-PAD analysis of hydrolysis products of �� -1,3-galactooligosaccharides.Pc1,3Gal43A (6.9 nM) was incubated with 5 mM �-1,3-galactotoriose or 5 mM methyl�-1,3-galactopentaoside in McIlvaine buffer, pH4.5 at 37°C. Reaction products wereanalyzed by HPEAC-PAD as described in the Experimental section. A, standards; B,�-1,3-galactotriose hydrolysis; C, methyl �-1,3-galactopentaoside hydrolysis.

Figure 4 HPAEC-PAD analysis of hydrolysis products of gum arabic afterseveral Smith degradation treatments and radish AGPs generated by Pc1,3G43APc1,3Gal43A (6.9 nM) was incubated with 0.5% (w/v) substrate in McIlvaine buffer,pH 4.5 at 37°C. Reaction products were analyzed by means of HPAEC-PAD asdescribed in the Experimental section. A, standards; B, �-1,3-galactan (gum arabicafter 3-times-repeated Smith degradation); C, gum arabic after twice-repeated Smithdegradation; D, �-L-arabinofuranosidase-treated radish root AGP.

Figure 5 Affinity gel electrophoresis of PcCBM6A: no substrate, B: gum arabic, C: gum arabic after twice-repeated Smith degradation,D: �-1,3-galactan (gum arabic after 3-times-repeated Smith degradation). Lanes 1 and3: BSA (5 μg), lane 2: Pc1,3Gal43A (5 μg), lane 4: PcCBM6 (5 μg).

Figure 6 Frontal affinity chromatography analysis of PcCBM6

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18

A: The Lineweaver-Burk type plots of the FAC data used to determine the affinity ofPcCBM6 for �1,3Gal3-ABEE. The Bt value was obtained with Equation 1 described inthe Experimental section. From the Bt value, the Kd value was calculated by applyingEquation 2. B: Structure of oligosaccharides used for FAC. C: Elution profiles of PA-oligosaccharides after application to immobilized PcCBM6. The structures of PA-oligosaccharide are shown above the elution patterns. The numbers indicated in theelution patterns are detected retardation volume (Vf – V0 in μl) and the dissociationconstant (Kd in μM) for each PA-oligosaccharides. Abbreviations: �1,3Gal3-ABEE,Gal-�-1,3-Gal-�-1,3-Gal-p-aminobenzoic acid ethyl ester; Lac, Gal-�-1,4-Glc;�GalLac, Gal-�-1,3-Gal-�-1,4-Glc; Gb3, Gal-�-1,4-Gal-�-1,4-Glc; LNT, Gal-�-1,3-GlcNAc-�-1,3-Gal-�-1,4-Glc; LNnT, Gal-�-1,4-GlcNAc-�-1,3-Gal-�-1,4-Glc; GA1,Gal-�-1,3-GalNAc-�-1,4-Gal-�-1,4-Glc; �Gal2Lac, Gal-�-1,3-Gal-�-1,3-Gal-�-1,4-Glc; Galili penta, Gal-�-1,3-Gal-�-1,4-GlcNAc-�-1,3-Gal-�-1,4-Glc.

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Figure 1

A

CGTTTGCCCGAGGGTCAGCCATGCAGATCTTTGCACACTTGCTGCTACCCGCGCTCTCACTG 62

M Q I F A H L L L P A L S L

CTACTGCCCGCTTACGCACAAAATCAGATCGTCTCTGGTGCGGCGTGGACAGACACTGCCGGC 125

L L P A Y A Q N Q I V S G A A W T D T A G

AACACGATACAGGCGCACGGTGCTGGTATCCTCCAGGTCGGCAGCACTTTCTACTGGTTCGGC 188

N T I Q A H G A G I L Q V G S T F Y W F G

GAAGATAAGTCACACAATAGTGCCTTGTTCAAAGCCGTATCGTGCTACACCTCGTCCGACTTG 251

E D K S H N S A L F K A V S C Y T S S D L

GTTAACTGGTCTCGCCAAAATGACGCGCTCTCGCCAATTGCTGGAACTATGATCTCGACATCG 314

V N W S R Q N D A L S P I A G T M I S T S

AATGTTGTCGAACGGCCCAAGGTCATCTTCAACCAGAAGAACTCTGAGTATGTCATGTGGTTC 377

N V V E R P K V I F N Q K N S E Y V M W F

CATTCTGACAGCTCCAACTATGGCGCTGCGATGGTCGGAGTAGCGACGGCCAAAACGCCATGC 440

H S D S S N Y G A A M V G V A T A K T P C

GGCCCGTACACCTACAAGGGGAGCTTCAAGCCGCTTGGGGCTGACTCCCGGGACGAAAGCATC 503

G P Y T Y K G S F K P L G A D S R D E S I

TTCCAGGATGATGACTCCGCCCAGACGGCGTACCTGCTGTACGCGTCCGATAACAACCAGAAC 566

F Q D D D S A Q T A Y L L Y A S D N N Q N

TTCAAGATCTCGCGCCTCGACGCCAACTACTACAACGTGACGGCGCAGGTCAGCGTGATGAAC 629

F K I S R L D A N Y Y N V T A Q V S V M N

GGCGCGACCCTGGAGGCACCCGGTATCGTGAAGCACAACGGGGAATACTTCCTGATCGCGTCC 692

G A T L E A P G I V K H N G E Y F L I A S

CACACCAGCGGCTGGGCGCCGAACCCGAACAAGTGGTTCTCCGCGTCGTCGCTCGCGGGCCCG 755

H T S G W A P N P N K W F S A S S L A G P

TGGTCGGCGCAGCAGGACATCGCGCCGAGCGCGACGCGGACGTGGTACTCGCAGAACGCGTTC 818

W S A Q Q D I A P S A T R T W Y S Q N A F

GACCTGCCGCTCGGCAGCAACGCGATCTACATGGGCGACCGCTGGCGCCCGAGCCTCCTGGGC 881

D L P L G S N A I Y M G D R W R P S L L G

AGCAGCCGGTACATCTGGTACCCGCTCGACTTCTCGAGCGGCGCGCCGCAGATCGTGCACGCG 944

S S R Y I W Y P L D F S S G A P Q I V H A

GACGTGTGGAGTGTGAACGTCCAGGCAGGGACGTACTCGGTCGCCAGTGGGACCAGCTACGAG 1007

D V W S V N V Q A G T Y S V A S G T S Y E

GCTGAGAACGGGCAGCGCGGCGGATCCTCGACAATTCTGTCGGGCTCGGGATTCTCTGGAGGA 1070

A E N G Q R G G S S T I L S G S G F S G G

AAGGCTGTTGGCTATCTCGGCCACGGTGGAACGGTCACCATAAACAACGTCCAGAGCAACGGC 1133

K A V G Y L G H G G T V T I N N V Q S N G

GGTTCTCACTGGGTCGCGTTGTACTTTGCGAACGGCGATTCCACGTACAGAAATGTCACTGTC 1196

G S H W V A L Y F A N G D S T Y R N V T V

AGTGTGAACGGCGGGCCATCTGTGCTCGTCGATCAGCCAGACAGCGGGGGCGGCAATGTGGTC 1259

S V N G G P S V L V D Q P D S G G G N V V

ATCAGTGTACCTGTGAAGCTCAATTTGAATAGCGGCGAAAATTCGATAACGTTCGGATCCGGG 1322

I S V P V K L N L N S G E N S I T F G S G

CAGAGCAACTACGCCGCCGATCTTGACAAGATCATTGTGTACTGAGACTGCTGCTGCGTCTCC 1385

Q S N Y A A D L D K I I V Y *

GTGGCTCTCAGCCGAAGGTCGCAGAAGAAGAGTGTAGGACAGCATGTGGACAGTGTCTTTCCA 1448

GGACGTTCTCCCGCAGCCGCAGCGAGGCGTACCTTGTTGTGTACATAGGGTTCCCGAAAGCAC 1511

TGTATCTGGCATGGATTGCGCCATGGTCGCGCGCCGGGCTTCAAAAAAAAAAAAAAAAAA 1571

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Fig

ure

1

B Pc1,3Gal43A 21 QNQIVSGAAWTDTAGNTIQAHGAGILQVGS 50

CjArb43A 35 DVHDPVMTREGDTWYLFSTGPGITIYSSKD 64

Pc1,3Gal43A

51 TFYWFGEDKSHNSALFKAVSCYTSSDLVNW 80

CjArb43A

65 RVNWRYSDRAFGTEPTWAKRVSPSFDGHLW 94

Pc1,3Gal43A

81SRQNDALSPIAGTMISTSNVVERPKVIFNQ 110

CjArb43A

95APDIYQHKGLFYLYYSVSAFGKNTSAIGVT 124

Pc1,3Gal43A111 KNSEYVMWFHSDSSNYGAAMVGVATAKTPC 140

CjArb43A

125 VN----KTLNPASPDYRWEDKGIVIESVPQ 150

Pc1,3Gal43A141 GPYTYKGSFKPLGADSRDESIFQDDDSAQT 170

CjArb43A

151 RDLWNAIDPAIIADDHGQVWMSFGSFWGGL 180

Pc1,3Gal43A171 AYLLYASDNNQNFKIS---RLDANYYNVTA 197

CjArb43A

181 KLFKLNDDLTRPAEPQEWHSIAKLERSVLM 210

Pc1,3Gal43A198QVSVMNGATLEAPGIVKHNGEYFLIASHTS 227

CjArb43A

211DDSQAGSAQIEAPFILRKGDYYYLFAS--- 237

Pc1,3Gal43A228 GWAPNPNKWFSASSLAGPWSAQQDIAPSAT 257

CjArb43A

238 -WGLCCRKGDSTYHLVVGRSKQ------VT 260

Pc1,3Gal43A258 RTWYSQNAFDLPLGSNAIYMGDRWRPSLLG 287

CjArb43A

261 GPYLDKTGRDMNQGGGSLLIKGNKRWVGLG 290

Pc1,3Gal43A288 S-SRYIWYPLDFSSG-APQIVHADVWSVNV 315

CjArb43A

291 HNSAYTWDGKDYLVLHAYEAADNYLQKLKI 320

Pc1,3Gal43A316 QAGTYSVASGTSY 328

CjArb43A

321 LNLHWDGEGWPQV 333

C�

PcCBM6 329 EAENGQRGGSSTILSGSGFSGGKAVGYLGH 358

BhCBM6 799 QAEAYDAMSGIQTEGTDDDGGGDNIGWIND 828

CtCBM6 255 ESEEYNSLKSSTIQTIGTSDGGSGIGYIES 284

CsCBM6-1 19 EAEEYNSTNSSTLQVIGTPNNGRGIGYIEN 48

CsCBM6-3 296 QAEDYDSSYGPNLQIFSLPGGGSAIGYIEN 325

CmCBM6 498 QAEDHSQQSGTQQETTTDTGGGKNVGYIDA 527

�

PcCBM6

359GGTVTINNVQSNGGSHW---VALYFAN--- 382

BhCBM6

829GDWVKYERVHFERDASS---IEVRVAS-DT 854

CtCBM6

285GDYLVFNKINFGNGANS---FKARVASGAD 311

CsCBM6-1

49 GNTVTYSNIDFGSGATG---FSATVAT--E 73

CsCBM6-3

326GYSTTYKNIDFGDGATS---VTARVAT--Q 350

CmCBM6

528GDWLSYAGTPVNIPSSGSYLIEYRVAS--Q 555

�

PcCBM6

383 GDSTYRNVTVSVNGGPSVLVDQPDSGGGNV 412

BhCBM6

855 PGGRIEIRTGSPTGTLLGDVQVPNTGGWQQ 884

CtCBM6

312 TPTNIQLRLGSPTGTLIGTLTVASTGGWNN 341

CsCBM6-1

74 VNTSIQIRSDSPTGTLLGTLYVSSTGSWNT 103

CsCBM6-3

351 NATTIQVRLGSPSGTLLGTIYVGSTGSFDT 380

CmCBM6

556 NGGGSLTFEEAGGAPVHGTIAIPATGGWQT 585

�

PcCBM6

413 VISVPVKLNLNSGENS--ITFGSGQSN--Y 438

BhCBM6

885 WQTVTGNVQIQPGTYDVYLVFKGSPEYDLM 914

CtCBM6

342 YEEKSCSITNTTGQHDLYLVFSGP-----V 366

CsCBM6-1

104 YQTVSTNISKITGVHDIVLVFSGP-----V 128

CsCBM6-3

381 YRDVSATISNTAGVKDIVLVFSGP-----V 405

CmCBM6

586 WTTIQHTVNLSAGSHQFGIKANAGG----W 611

�

PcCBM6

439 AADLDKIIV 447

BhCBM6

915 NVNWFVFRA 923

CtCBM6

367 NIDYFIFDS 375

CsCBM6-1

129 NVDNFIFSR 137

CsCBM6-3

406 NVDWFVFSK 414

CmCBM6

612 NLNWIRINK 620

*

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1 2 3

31 kDa

21 kDa

14 kDa

97 kDa

66 kDa

45 kDa

97 kDa

31 kDa

21 kDa

1 2

66 kDa

45 kDa

Figure 2

A B

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Figure 3

AStandards

-0.02

0

0.02

0.04

0.06

0.08

0 5 10 15

Gal Gal2 Gal3

Gradient shock

MeGal4 MeGal5

PA

D r

espo

nse

(μC

)P

AD

res

pons

e (μ

C)

Retention time (min)

Retention time (min)

-0.02

0

0.02

0.04

0.06

0.08

0.1

0 5 10 15 20

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Figure 3

B�-1,3-Galactotriose

-0.005

0.005

0.015

0.025

0.035

0.045

0.055

-0.005

0.005

0.015

0.025

0.035

0.045

-0.005

0.005

0.015

0.025

0.035

0.045

0 5 10 15

Gradientshock

GalGal2

10 min

20 min

60 min

Gal3

PA

D r

espo

nse

(μC

)P

AD

res

pons

e (μ

C)

PA

D r

espo

nse

(μC

)

Retention time (min)

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Figure 3

CMethyl �-1,3-galactopentaoside

5 min

10 min

20 min

60 min

-0.02

0

0.02

0.04

0.06

0.08

0.1

0.12

-0.02

0

0.02

0.04

0.06

0.08

0.1

-0.02

0

0.02

0.04

0.06

0.08

0.1

-0.02

0

0.02

0.04

0.06

0.08

0.1

0 5 10 15 20

PA

D r

espo

nse

(μC

)

Retention time (min)

PA

D r

espo

nse

(μC

)P

AD

res

pons

e (μ

C)

PA

D r

espo

nse

(μC

)

MeGal4 MeGal5

Gal

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Figure 4

AStandards

PA

D r

espo

nse

(μC

)

-0.01

0

0.01

0.02

0.03

0.04

PA

D r

espo

nse

(μC

)

4-MeGlcA-�-1,6-Gal2

Retention time (min)

-0.01

0

0.01

0.02

0.03

0.04

0 5 10 15 20 25

PA

D r

espo

nse

(μC

)

4-MeGlcA-�-1,6-Gal

-0.01

0

0.01

0.02

0.03

0.04

0.05

PA

D r

espo

nse

(μC

) Gal�-1,6-Gal2

�-1,6-Gal3

-0.01

0

0.01

0.02

0.03

0.04 L-Ara-�-1,3-Gal-�-1,6-Gal

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Figure 4

B�-1,3-Galactan

-0.02

0

0.02

0.04

0.06

0.08

0.1

-0.02

0

0.02

0.04

0.06

0.08

-0.02

0

0.02

0.04

0.06

0.08

-0.02

0

0.02

0.04

0.06

0.08

0 5 10 15 20 25

Retention time (min)

0 min

10 min

30 min

60 minPA

D r

espo

nse

(μC

)

Gal

PA

D r

espo

nse

(μC

)P

AD

res

pons

e (μ

C)

PA

D r

espo

nse

(μC

)

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Figure 4

CGum arabic after twice-repeated Smith degradation

-0.05

0

0.05

0.1

0.15

0.2

PA

D r

espo

nse

(μC

)P

AD

res

pons

e (μ

C)

PA

D r

espo

nse

(μC

)

Retention time (min)

0h

1h

3h

-0.05

0

0.05

0.1

0.15

-0.05

0

0.05

0.1

0.15

0 5 10 15 20 25

Gal

�-1,6-Gal2

L-Ara-�-1,3-Gal-�-1,6-Gal

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Figure 4

D�-L-Arabinofuranosidase-treated AGP from radish root

-0.05

0

0.05

0.1

0.15

0.2

0.25

-0.05

0

0.05

0.1

0.15

0.2

-0.05

0

0.05

0.1

0.15

0.2

0 5 10 15 20 25

0h

1h

3h

Retention time (min)

PA

D r

espo

nse

(μC

)P

AD

res

pons

e (μ

C)

PA

D r

espo

nse

(μC

)

Gal

�-1,6-Gal2

�-1,6-Gal3

4-MeGlcA-�-1,6-Gal2

4-MeGlcA-�-1,6-Gal

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�

� � � �

� � �

� � � � � � � �� � � �

�����

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0.05

0.10

0.15

0.20

0.25

0.30

0 100 200 300

[�1,3Gal3-ABEE] (μM)

Figure 6

A

Bt = 2.3 nmolKd = 265 μM

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0 1 2 0 1 2

Retention time (min)

0204060

80

100

0 1 2

Figure 6

C

�GalLac �Gal2Lac Others

22.8 μl(82 μM)

10.6 μl(217 μM)

<1.0 μl( - )

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KanekoYoichi Tsumuraya, Masahiro Samejima, Jun Hirabayashi, Hideyuki Kobayashi and Satoshi

Hitomi Ichinose, Makoto Yoshida, Toshihisa Kotake, Atsushi Kuno, Kiyohiko Igarashi,phanerochaete chrysosporium

An exo-A-1,3-galactanase having a novel A-1,3-galactan binding module from

published online May 2, 2005J. Biol. Chem. 

  10.1074/jbc.M501024200Access the most updated version of this article at doi:

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