Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences,Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan
Received 29 October 2001/Accepted 19 December 2001
Aspergillus nidulans possessed an �-glucosidase with strong transglycosylation activity. The enzyme, desig-nated �-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising74- and 55-kDa subunits and catalyzed hydrolysis of maltose along with formation of isomaltose and panose.Approximately 50% of maltose was converted to isomaltose, panose, and other minor transglycosylationproducts by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The genecomprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. Thededuced amino acid sequence contained the chemically determined N-terminal and internal amino acidsequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptideprecursor. AgdB showed low but overall sequence homology to �-glucosidases of glycosyl hydrolase family 31.However, AgdB was phylogenetically distinct from any other �-glucosidases. We propose here that AgdB is anovel �-glucosidase with unusually strong transglycosylation activity.
�-Glucosidases (EC 3.2.1.20) catalyze liberation of glucosefrom nonreducing ends of �-glucosides, �-linked oligosaccha-rides, and �-glucans. They show diverse substrate specificities;some prefer �-linked di-, oligo-, and/or polyglucans, while oth-ers preferentially hydrolyze heterogeneous substrates such asaryl glucosides and sucrose (1, 5). Theoretically �-glucosidaseis capable of catalyzing transglycosylation, since it is a retainingglycosyl hydrolase (GH) (2), and some �-glucosidases indeedexhibit clear transglycosylation activity. For example, Aspergil-lus niger �-glucosidase catalyzes formation of �-1,6 glucosidiclinkages in addition to hydrolysis, resulting in productionof isomaltose (6-O-�-D-glucopyranosyl-D-glucopyranose) andpanose (6-O-�-glucopyranosyl-maltose) from maltose (3, 15,21). Buckwheat �-glucosidase produces kojibiose (2-O-�-glu-cosyl-glucose), nigerose (3-O-�-glucosyl-glucose), maltose, andisomaltose from soluble starch (1), and �-glucosidases fromBacillus stearothermophilus and brewer’s yeast produce oligo-saccharides consisting of �-1,3, �-1,4, and �-1,6 linkages (13).Transglycosylation activity of the �-glucosidases has been ap-plied in industries to produce isomaltooligosaccharides andalso to conjugate sugars to biologically useful materials, aimingto improve their chemical properties and physiological func-tions (18, 33).
The main physiological role of most exo-type glycosidasessuch as �-glucosidase is to produce monosaccharides that areutilized as carbon and energy sources. However, transglycosy-lation activities of exo-type glycosidases sometimes play phys-iologically important roles in gene regulation involved in car-bohydrate utilization. A well-known example is induction ofthe lac operon in Escherichia coli. The physiological inducer of
the operon, allolactose (6-O-�-D-galactopyranosyl-D-glucose),is synthesized from lactose by transglycosylation activity of�-galactosidase encoded by lacZ (16). In the filamentousfungus Trichoderma reesei, the strongest cellulase inducer, so-phorose (2-O-�-D-glucopyranosyl-D-glucose) (14, 27), can beformed from cellooligosaccharides by transglycosylation activ-ity of �-glucosidase or endoglucanase (11, 31).
�-Amylase synthesis in Aspergillus oryzae is induced by var-ious �-linked oligosaccharides, including kojibiose, maltose,isomaltose, and panose. Isomaltose has the strongest inducingactivity among them, and A. oryzae produces intracellularlytransglycosylation activity that transforms maltose to isomalt-ose (30). Isomaltose was also shown to be the strongest amy-lase inducer in Aspergillus nidulans (N. Kato et al., unpublisheddata), suggesting that the similar triggering process of amylasesynthesis including transformation of the �-linked oligosac-charides to isomaltose, would be shared in A. nidulans and A.oryzae. In this work, a novel �-glucosidase, designated �-glu-cosidase B (AgdB), was purified from A. nidulans from a view-point of formation of isomaltose. AgdB possessed strong trans-glycosylation activity with preferential formation of �-1,6linkages. Sequence analysis of the agdB gene revealed thatAgdB was a member of the GH family 31 but phylogeneticallydistant from any other �-glucosidases of the family.
MATERIALS AND METHODS
Purification of �-glucosidase B (AgdB) from A. nidulans. A. nidulans ABPU1(pyrG89 biA1 wA3 argB2 pyroA4) (17, 28) was kindly supplied by H. Horiuchi andM. Takagi, University of Tokyo, Japan. A. nidulans ABPU1 was cultivatedaerobically at 37°C for 24 h with rotary shaking at 180 rpm in 2-liter Erlenmeyerflasks with baffles, each containing 500 ml of the standard minimal medium withappropriate supplements (24), except that the carbon source was replaced with2% starch. The culture filtrate and mycelia were separated by filtration with filterpaper no. 1 (285 mm; Toyo Roshi, Tokyo, Japan). The mycelia (30 g [wetweight]) were ground to fine powder under liquid nitrogen and suspended at0.2 g of mycelial powder/ml in 0.2 M 2-(N-morpholino)ethanesulfonic acid(MES)-KOH buffer (pH 5.5) containing 0.5% Triton X-100, 1 mM EDTA, and
* Corresponding author. Mailing address: Department of BiologicalMechanisms and Functions, Graduate School of Bioagricultural Sci-ences, Nagoya University, Chikusa-ku, Nagoya 464-8601, Japan. Phone:81-52-7894086. Fax: 81-52-7894087. E-mail: [email protected].
2 mM phenylmethylsulfonyl fluoride. The suspension was homogenized withPolytron (Kinematica, Littau-Lucerne, Switzerland) and centrifuged at 16,000 �g for 30 min at 4°C. The resultant supernatant was used as the cell extract.
All the following steps were carried out at 4°C. The cell extract was dialyzedagainst 20 mM MES-KOH buffer (pH 5.5) containing 1 mM EDTA and 0.5 mMphenylmethylsulfonyl fluoride and applied to a DEAE-Toyopearl 650 M column(2.5 by 10 cm; Tosoh, Tokyo, Japan) equilibrated with 20 mM MES-KOH buffer(pH 5.5). After the column was washed with 100 ml (2 column volumes) of theMES buffer, bound proteins were eluted with a 200-ml (4 column volumes) lineargradient of 0 to 0.5 M NaCl in the MES buffer at a flow rate of 1.5 ml/min (0.3cm/min). The active fractions, eluted at about 0.1 M NaCl, were combined anddialyzed against the MES buffer containing 1.5 M ammonium sulfate. The dia-lysate was loaded onto a Phenyl Sepharose CL-4B column (1.0 by 12 cm; Am-ersham Pharmacia Biotech, Uppsala, Sweden) equilibrated with the same buffer.Bound proteins were eluted at a flow rate of 0.5 ml/min (0.6 cm/min) with a 40-ml(4-column-volume) linear gradient of 1.5 to 0 M ammonium sulfate in the MESbuffer. The active fractions, eluted at nearly 0 M ammonium sulfate, werecollected, dialyzed against 20 mM HEPES-KOH buffer (pH 7.4), and concen-trated to about 1 ml by Centriprep YM-10 (Millipore, Bedford, Mass.). Theconcentrated sample was applied to a RESOURCE Q column (1.6 by 3.0 cm;Amersham Pharmacia Biotech), which was under the control of an ÄKTAexplorer 10S system (Amersham Pharmacia Biotech), equilibrated with 20 mMHEPES buffer (pH 7.4). After the column was washed with 12 ml (2 columnvolumes) of the HEPES buffer, bound proteins were eluted with a 60-ml (10column volumes) linear gradient of 0 to 1 M NaCl in the HEPES buffer at a flowrate of 6 ml/min (3 cm/min). The active fractions, eluted at about 0.3 M NaCl,were collected, dialyzed against the HEPES buffer, and used as a purified en-zyme.
Enzyme assay. The AgdB activity was measured by incubating the enzyme with0.5% (wt/vol) maltose in 40 mM acetate buffer (pH 5.5) for 30 min at 45°C. Thereaction was terminated by boiling for 3 min, and liberated glucose was measuredby the glucose oxidase-peroxidase method with a Glucose B Test (Wako PureChemical Ind. Ltd., Osaka, Japan). One unit of the AgdB activity was defined asthe amount of enzyme that catalyzes hydrolysis of 1 �mol of maltose per min.Note that this unit definition is apparent, since the transglycosylation activity ofAgdB is not taken into account. Kinetic parameters for hydrolysis of variousoligosaccharides were calculated from a Hanes-Woolf plot. Chemical structuresof the oligosaccharides used, except for maltooligosaccharides, are shown in Fig.1. The pH optimum of enzyme catalysis was determined at 45°C in Britton-Robinson buffer of various pH values (pH 2 to 10; 40 mM acetic acid–40 mMphosphoric acid–40 mM boric acid adjusted with NaOH to the desired pHvalues). Thermal and pH stabilities were determined by measuring residualactivity after incubation of the enzyme solutions of 60 �g/ml at various temper-atures of 30 to 70°C for 1 h and at various pH values of 2.0 to 12.0 for 1 h at 45°C.
TLC. Thin-layer chromatography (TLC) was carried out using Silica Gel 60plates (Merck, Darmstadt, Germany) with three ascents of 2-propanol–1-buta-nol–water (12:3:4, vol/vol/vol) as the solvent (19). The solvent path length was 8.5cm for each ascent. The products were visualized by spraying 1% p-anisaldehydeand 2% sulfuric acid in acetic acid (vol/vol) followed by heating at 110°C for 10min. The quantification of oligosaccharides separated by TLC was performed bydensitometry scanning of the TLC plates with NIH Image software (version 1.61;NIH Research Services Branch [http://rsb.info.nih.gov/nih-image/]).
HPAEC-PAD analysis. For high-performance anion-exchange chromatogra-phy with pulsed amperometric detection (HPAEC-PAD) analysis, the DX-500chromatography system (Dionex, Sunnyvale, Calif.) was used with a GP-50 gra-
dient pump, an ED-40 electrochemical detector, and CarboPac PA1 column(4 by 25 mm; Dionex). The reaction products were separated with a lineargradient of 0 to 300 mM sodium acetate for 15 min in 100 mM NaOH at a flowrate of 1 ml/min.
Determination of the N-terminal and internal amino acid sequences. Thepurified enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE), transferred electrophoretically to a Sequi-Blotpolyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.), and stainedwith Coomassie brilliant blue R-250. The stained bands, corresponding to the 74-and 55-kDa subunits, were excised and analyzed for N-terminal amino acidsequences by an Applied Biosystems (Foster City, Calif.) model 473A gas phasesequencer. For determination of the internal amino acid sequences, 74- and55-kDa subunits of AgdB were separated by SDS–10% PAGE and eluted elec-trophoretically from the acrylamide gel. Each subunit was digested with Lysylendopeptidase (Wako). The peptides generated were resolved by SDS–15%PAGE, and major peptides were subjected to amino acid sequencing.
FIG. 1. Structures of five �-linked glucooligosaccharides describedin this study.
FIG. 2. Isomaltose-producing activity in the culture filtrate and cellextract of A. nidulans. The 35-fold-concentrated culture filtrate (CF)and the cell extract (CE) were incubated with 0.5% maltose in 40 mMacetate buffer (pH 5.5) at 45°C for 2 h, and the reaction products wereanalyzed by TLC. The volumes of CF and CE used for the reactionswere 48 and 10 �l, corresponding to 2 ml and 200 �l of the originalculture suspension, respectively. The culture filtrate was concentratedby Ultrafree MC (Millipore). Glucose (G), maltose (M), maltotriose(G3), maltotetraose (G4), maltopentaose (G5), isomaltose (I), panose(P), and isomaltotriose (IG3) were used as standards (Std).
Molecular cloning of the agdB gene. A part of the agdB gene was amplified bynested PCR using two pairs of degenerate primers designed based on the N-terminal and internal amino acid sequences of the 74-kDa subunit. The primerpairs N1-N2 and I1-I2 correspond to the N-terminal and internal amino acidsequences, respectively (Table 1). The first PCR was carried out with A. nidulanstotal DNA as a template using the primer pair N1-I1, and a part of the reactionmixture was subjected to the second PCR using the primer pair N2-I2. A 440-bpfragment was specifically amplified and used as a probe for Southern and colonyhybridizations.
Total DNA of A. nidulans was digested with HindIII and size fractionated bypreparative agarose gel electrophoresis. DNA fragments of approximately 5.0 to7.0 kb hybridized to the PCR amplified probe were ligated to pBluescript IIKS(�) and introduced into E. coli JM109. Transformants were screened bycolony hybridization. A plasmid carrying the agdB gene on a 5.6-kb HindIIIfragment was obtained and designated pGBH6.
Parts of cDNA were obtained by reverse transcriptase PCR with A. nidulanstotal RNA as a template. First-strand cDNA was synthesized using an oligo(dT)primer and subjected to PCR using the primer sets S x-A x (where x is 1 to 4[Table 1]). The PCR products were cloned into pGEM T-vector (Promega,Madison, Wis.) and sequenced.
Phylogenetic analysis. Multiple alignment of GH family 31 �-glucosidases wasperformed by using the CLUSTAL W program (version 1.80; DDBJ [http://www.ddbj.nig.ac.jp]) (29). The phylogenetic relationship of the �-glucosidaseswas calculated by using the neighbor-joining method (25). Positions with gaps inthe alignment were excluded from the calculation. The unrooted phylogenetictree was produced using the TREE VIEW program (version 1.6.5; Taxonomyand Systematics, University of Glasgow [http://taxonomy.zoology.gla.ac.uk/]).Accession numbers of the following amino acid sequences used in the phy-logenetic analysis are as indicated: Schwanniomyces occidentalis glucoamylase,GenBank AAA33923; Candida albicans glucoamylase, AAC31968; A. niger �-glucosidase, BAA23616; A. oryzae �-glucosidase, BAA95702; A. nidulans �-glu-cosidase AgdA, AAF17102; Schizosaccharomyces pombe �-glucosidase, BAB43946;S. pombe ORF SPAC922.02c, CAB63549; S. pombe ORF SPAC30D11.01c,SwissProt Q09901; Mucor javanicus �-glucosidase, GenBank BAA11053; Can-dida tsukubaensis �-glucosidase, CAA39501; spinach �-glucosidase, BAA19924;
sugar beet �-glucosidase, BAA20343; Arabidopsis thaliana �-glucosidase I,AAB82656; barley high-pI �-glucosidase, AAF76254; potato �-glucosidase,CAB96077; mouse lysosomal �-glucosidase, AAB06943; human lysosomal �-glu-cosidase, AAA52506; rabbit sucrase-isomaltase, AAA31459; human sucrase-iso-maltase, CAA45140; human maltase-glucoamylase, AAC39568. The sequencesof Neurospora crassa putative �-glucosidase genes were obtained from an N. crassagenome database (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/).The amino acid sequences of putative �-glucosidases 1 and 2, which showed thehighest sequence homology to A. nidulans AgdA and AgdB, respectively, werededuced based on alignment with sequences of AgdA and AgdB.
Other methods. Standard DNA manipulations were performed according tothe method of Sambrook et al. (26). Total DNA from A. nidulans was isolated byusing the cetyltrimethylammonium bromide method (4). Labeling of the probesand detection of hybridization signals in Southern and colony hybridizationswere carried out using an enhanced chemiluminescence nucleotide labeling anddetection system (Amersham Pharmacia Biotech). Nucleotide sequences were
FIG. 3. SDS-PAGE of the purified AgdB. The purified enzyme(lane 2) was analyzed on an SDS–10% polyacrylamide gel. Phosphor-ylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa),carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa),and �-lactalbumin (14.4 kDa) were used as molecular mass markers(lane 1).
FIG. 4. (A) Time course of the AgdB reaction with maltose as asubstrate. The enzyme (0.6 �g) was incubated with 0.5% maltose in100 �l of 40 mM acetate buffer (pH 5.5) at 45°C. Aliquots werewithdrawn at the indicated times and analyzed by TLC. (B) TLCanalysis of the transglycosylation products from various �-glucobioses.The purified AgdB (0.6 �g) was incubated with 0.5% (wt/vol) maltose(M), isomaltose (I), nigerose (N), and kojibiose (K) as substrates(Subs.) in 100 �l of 40 mM acetate buffer (pH 5.5) for 2 h at 45°C ([�]or without [�] enzyme [Enz.]), and the reaction products were ana-lyzed by TLC. Glucose (G), maltose (M), isomaltose (I), panose (P),and isomaltotriose (IG3) were used as standards (Std).
TABLE 2. Purification of �-glucosidase B from A. nidulans
determined by the dideoxy chain termination method with a DNA sequencer(LI-COR model 4000). Protein was determined by a Bio-Rad protein assay kitwith bovine immunoglobulin G as a standard. SDS-PAGE was carried out ac-cording to the method of Laemmli (12), and proteins were visualized by Coo-massie brilliant blue staining. An LMW marker kit (Amersham Pharmacia Bio-tech) was used for standard protein markers. The molecular mass of the nativeenzyme was estimated by gel filtration on a Superdex 200 column (1.0 by 30 cm;Amersham Pharmacia Biotech) calibrated with gel filtration low- and high-molecular-weight calibration kits (Amersham Pharmacia Biotech). The columnwas equilibrated and run with the MES buffer containing 0.15 M NaCl at a flowrate of 0.5 ml/min.
Nucleotide sequence accession number. The nucleotide sequence of agdB hasbeen deposited in the GenBank/EMBL/DDBJ databases under the accessionnumber AB057788.
RESULTS AND DISCUSSION
Purification of AgdB from A. nidulans. A. nidulans culturefiltrates and cell extracts were determined for isomaltose-pro-ducing activity with maltose as a substrate (Fig. 2). The cellextracts were found to convert maltose to glucose and a seriesof transglycosylation products, including two dominant specieswhich, by the TLC analysis, showed Rf values nearly identicalto those of maltotriose or isomaltose and maltotetraose orpanose, respectively. Little conversion of maltose was observedwith the culture filtrates.
The purification of AgdB, which catalyzed conversion ofmaltose to isomaltose, from the cell extract is summarized inTable 2. The enzyme was purified 52-fold with a specific activ-ity of 9.6 U/mg, and the overall yield was 22%. The purifiedenzyme gave two polypeptide bands of 74 and 55 kDa onSDS-PAGE (Fig. 3), while the approximate molecular mass ofthe native AgdB determined by an analytical gel filtration onSuperdex 200 was 130 kDa. Both subunits of AgdB werestained with periodic acid oxidation-silver staining (32), indi-cating that AgdB was a glycoprotein (data not shown). AgdBhad a pH optimum of 5.5, and less than 10% of the activity waslost over a wide pH range of 5.0 to 8.5 and at temperatures upto 45°C.
Transglycosylation activity and substrate specificity of AgdB.The time course of the AgdB reaction was examined by TLCwith 0.5% maltose as a substrate (Fig. 4A). Within the first 2 hof the reaction, more than 90% of maltose was consumed, andglucose and a series of transglycosylation products wereformed as the reaction products. Two dominant transglycosy-lation products gave Rf values by the TLC analysis identical tothose of isomaltose and panose, respectively. However, as de-scribed earlier for Fig. 2, isomaltose and panose were notseparated from maltotriose and maltotetraose, respectively,under the TLC conditions used here. In order to identify fur-ther the dominant transglycosylation products, HPEAC-PADanalysis of the reaction products was carried out. As shown inFig. 5A and B, glucose, maltose, maltotriose, maltotetraose,and panose were clearly assigned by HPEAC-PAD, althoughisomaltose and isomaltotriose were not separated from eachother. In the initial stage of the reaction, the products con-tained isomaltose and/or isomaltotriose, panose, and glucoseas major species (Fig. 5B), and later isomaltose and/or isomal-totriose and glucose dominated other reaction products (Fig.5C). However, only trace amounts of maltotriose and maltote-traose were detected. Taken together with the results of theTLC analysis, the dominant transglycosylation products were
FIG. 5. HPAEC elution profiles of the reaction products of AgdBfrom maltose. The enzyme (2.5 �g) was incubated with 0.5% maltosein 100 �l of 40 mM acetate buffer (pH 5.5) at 45°C for 5 (B) and 60(C) min. The reaction products were subjected to HPAEC-PAD anal-ysis. (A) Elution profile of the standard sugars. Glucose (G), maltose(M), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), iso-maltose (I), panose (P), and isomaltotriose (IG3) were used as stan-dards.
TABLE 3. Kinetic parameters for hydrolysis of variousoligosaccharides by AgdB
FIG. 6. Comparison of the amino acid sequence of A. nidulans AgdB with those of GH family 31 �-glucosidases. (A) Multiple alignment of theregions corresponding to two GH family 31 signature sequences. Conserved amino acids are shaded, and the derived consensus sequences are givenbelow the alignment. Uppercase letters indicate conserved residues, and the asterisk indicates a catalytic residue. (B) Phylogenetic tree of�-glucosidases belonging to GH family 31. Bootstrap values (based on 1,000 bootstrap trials) are shown at each node. The scale bar correspondsto a genetic distance of 0.1 substitution per position. Abbreviations: ORF, open reading frame; SI, sucrase-isomaltase; MG, maltase-glucoamylase.
1254 ISOMALTOSE-PRODUCING �-GLUCOSIDASE OF A. NIDULANS KATO ET AL.
isomaltose and panose. As shown in Fig. 4, panose graduallydecreased as the reaction proceeded, while glucose and iso-maltose increased steadily. At 6 h of the reaction, approxi-mately 50% of maltose was converted to transglycosylationproducts, 60% of which was found to be isomaltose.
Formation of isomaltose and panose from maltose by theaction of �-glucosidase has been demonstrated in A. niger andA. oryzae (3, 15, 20–22). The A. niger enzyme possesses strongtransglycosylation activity, however, a maltose concentration of30% is used for A. niger �-glucosidase to achieve an isomaltosecontent of 30% in the final reaction products (3, 21). At asubstrate concentration of 0.5%, AgdB clearly showed highertransglycosylation activity than that of the A. niger enzyme (15).The other �-glucosidases so far reported also require highsubstrate concentrations to exhibit sufficient transglycosylationactivities (13). Therefore, AgdB may be the �-glucosidase withthe strongest transglycosylation activity among �-glucosidasesknown to date.
Kinetic parameters for hydrolysis of various substrates areshown in Table 3. AgdB exhibited high hydrolytic activity to-ward maltooligosaccharides, and the most favored substratewas maltotriose, with a k0/Km ratio of 120, which was 2.9-foldhigher than that of maltose. The k0/Km values for maltote-traose and maltopentaose decreased in this order, indicatingthat the longer maltooligosaccharides are less favored. Theenzyme also efficiently hydrolyzed kojibiose, nigerose, and iso-maltose and showed weak activity toward trehalose and p-nitrophenyl �-glucoside. Sucrose and soluble starch were theleast-favored substrates examined (data not shown).
The enzyme produced a series of transglycosylation productsfrom kojibiose, nigerose, maltose, and isomaltose (Fig. 4B).Isomaltose was also formed from nigerose and kojibiose, whileisomaltotriose was the main transglycosylation product fromisomaltose. These facts imply that the enzyme preferentiallyforms an �-1,6 linkage.
Cloning and sequence analysis of the agdB gene. The N-terminal amino acid sequences of the 74- and 55-kDa subunitwere chemically determined to be SQAGVDPLDRPGNDLYVKD and QSHRQLGAGRWRSAVRH, respectively. Lysylendopeptidase digestion of the 74- and 55-kDa subunits gen-erated major peptides of 30 and 15 kDa, respectively, and theN-terminal amino acid sequences for 30- and 15-kDa peptideswere determined to be THLPQNPHLYGLGE and DVSHWLGDNISDWLSYRLSI, respectively. Based on these pieces ofinformation, genomic and cDNA clones including the agdBgene were obtained as described in Materials and Methods.Nucleotide sequences of the cloned DNA fragments revealedthat the agdB gene comprised 3,055 bp, interrupted by threeshort introns of 57 to 72 bp, and encoded a polypeptide of 955amino acids. The N-terminal amino acid sequences of the 74-and 55-kDa subunits were identified at residues 21 to 39 and515 to 531 of the derived amino acid sequence, respectively,and the internal amino acid sequences were also found atresidues 167 to 180 and 637 to 656. This indicates that theenzyme is synthesized as a single polypeptide precursor andthen the precursor is processed to form the mature het-erodimeric protein. The first 20 amino acids at the N terminusof AgdB showed a typical feature of signal peptides: a basicresidue Arg at position 2 followed by 12 hydrophobic residues.Taken together with the cellular distribution and glycosylation
of the enzyme, AgdB appears to be an extracellular enzymepresent in the cell wall.
Comparison with other �-glucosidases. �-Glucosidases havebeen classified into two families, GH families 13 and 31, basedon hydrophobic cluster analysis (http://afmb.cnrs-mrs.fr/�pedro/CAZY/ghf.html) (6). AgdB showed low but overall sequenceidentity to the GH family 31 enzymes such as A. nidulans�-glucosidase AgdA (32%), A. niger �-glucosidase (31%), S. oc-cidentalis glucoamylase (31%), C. albicans glucoamylase (31%),and A. oryzae �-glucosidase (30%). The GH family 31 enzymesshare two conserved sequences, designated as the GH family31 signature sequence 1 and 2 (5, 8), which corresponded toresidues 434 to 448 and residues 671 to 704 in AgdB. Multiplealignment of the two signature sequences of eukaryotic GHfamily 31 �-glucosidases is shown in Fig. 6A. Six conservedresidues in the signature sequence 1, including the catalyticresidue Asp (7, 9, 10, 23), were conserved in AgdB except forAla 436. Among 14 conserved residues in sequence 2, AgdBhad a single substitution, Thr to Pro at position 698. In addi-tion, Asp 643, which functions possibly as a proton donor (19),was also conserved. These facts indicate that AgdB is a mem-ber of GH family 31.
Phylogenetic analysis of the GH family 31 �-glucosidasesrevealed three clusters comprised of the enzymes from mam-mals, plants, and fungi (Fig. 6B). A. niger �-glucosidase, whichhas been shown to exhibit strong transglycosylation activity (3,15, 21), was located in the fungal cluster with close geneticdistances to �-glucosidases (AgdAs) from A. oryzae and A. nid-ulans. However, AgdB was phylogenetically distant from thoseAspergillus �-glucosidases, and surprisingly, it was located out-side of the fungal cluster. Furthermore, AgdB was obviouslydistant from mammalian and plant �-glucosidases. The phylo-genetic location of AgdB, being independent from those ofother �-glucosidases, indicates that AgdB is a novel �-gluco-sidase that appears to have diverged from the other �-gluco-sidases at an early stage of fungal evolution. Homologues ofagdB were found in an N. crassa genome database (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/) and in theA. oryzae EST database (http://www.aist.go.jp/RIODB/ffdb/index.html). Enzymes similar to AgdB may be widely distrib-uted in filamentous fungi.
ACKNOWLEDGMENTS
We thank Kikkoman Co. Ltd. for the generous gifts of kojibiose andnigerose. We also thank Shinji Go for performing the HPAEC-PADanalysis.
This work was partially supported by a grant to T. Kobayashi fromNoda Institute for Scientific Research and by a grant-in-aid for scien-tific research (C) to T. Kobayashi from the Ministry of Education,Science, Sports, and Culture.
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