ABSTRACT
Aspergillus nidulans possessed an α-glucosidase with strong transglycosylation activity. The enzyme, designated α-glucosidase B (AgdB), was purified and characterized. AgdB was a heterodimeric protein comprising 74- 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 transglycosylation products by AgdB, even at low maltose concentrations. The agdB gene was cloned and sequenced. The gene comprised 3,055 bp, interrupted by three short introns, and encoded a polypeptide of 955 amino acids. The deduced amino acid sequence contained the chemically determined N-terminal and internal amino acid sequences of the 74- and 55-kDa subunits. This implies that AgdB is synthesized as a single polypeptide precursor. AgdB showed low but overall sequence homology to α-glucosidases of glycosyl hydrolase family 31. However, AgdB was phylogenetically distinct from any other α-glucosidases. We propose here that AgdB is a novel α-glucosidase with unusually strong transglycosylation activity.
α-Glucosidases (EC 3.2.1.20) catalyze liberation of glucose from nonreducing ends of α-glucosides, α-linked oligosaccharides, and α-glucans. They show diverse substrate specificities; some prefer α-linked di-, oligo-, and/or polyglucans, while others preferentially hydrolyze heterogeneous substrates such as aryl glucosides and sucrose (1, 5). Theoretically α-glucosidase is capable of catalyzing transglycosylation, since it is a retaining glycosyl hydrolase (GH) (2), and some α-glucosidases indeed exhibit clear transglycosylation activity. For example, Aspergillus niger α-glucosidase catalyzes formation of α-1,6 glucosidic linkages in addition to hydrolysis, resulting in production of isomaltose (6-O-α-d-glucopyranosyl-d-glucopyranose) and panose (6-O-α-glucopyranosyl-maltose) from maltose (3, 15, 21). Buckwheat α-glucosidase produces kojibiose (2-O-α-glucosyl-glucose), nigerose (3-O-α-glucosyl-glucose), maltose, and isomaltose from soluble starch (1), and α-glucosidases from Bacillus stearothermophilus and brewer's yeast produce oligosaccharides consisting of α-1,3, α-1,4, and α-1,6 linkages (13). Transglycosylation activity of the α-glucosidases has been applied in industries to produce isomaltooligosaccharides and also to conjugate sugars to biologically useful materials, aiming to improve their chemical properties and physiological functions (18, 33).
The main physiological role of most exo-type glycosidases such as α-glucosidase is to produce monosaccharides that are utilized as carbon and energy sources. However, transglycosylation activities of exo-type glycosidases sometimes play physiologically important roles in gene regulation involved in carbohydrate utilization. A well-known example is induction of the lac operon in Escherichia coli. The physiological inducer of the operon, allolactose (6-O-β-d-galactopyranosyl-d-glucose), is synthesized from lactose by transglycosylation activity of β-galactosidase encoded by lacZ (16). In the filamentous fungus Trichoderma reesei, the strongest cellulase inducer, sophorose (2-O-β-d-glucopyranosyl-d-glucose) (14, 27), can be formed from cellooligosaccharides by transglycosylation activity of β-glucosidase or endoglucanase (11, 31).
α-Amylase synthesis in Aspergillus oryzae is induced by various α-linked oligosaccharides, including kojibiose, maltose, isomaltose, and panose. Isomaltose has the strongest inducing activity among them, and A. oryzae produces intracellularly transglycosylation activity that transforms maltose to isomaltose (30). Isomaltose was also shown to be the strongest amylase inducer in Aspergillus nidulans (N. Kato et al., unpublished data), suggesting that the similar triggering process of amylase synthesis including transformation of the α-linked oligosaccharides to isomaltose, would be shared in A. nidulans and A. oryzae. In this work, a novel α-glucosidase, designated α-glucosidase B (AgdB), was purified from A. nidulans from a viewpoint of formation of isomaltose. AgdB possessed strong transglycosylation activity with preferential formation of α-1,6 linkages. Sequence analysis of the agdB gene revealed that AgdB was a member of the GH family 31 but phylogenetically distant from any other α-glucosidases of the family.
MATERIALS AND METHODS
Purification of α-glucosidase B (AgdB) from A. nidulans. A. nidulans ABPU1 (pyrG89 biA1 wA3 argB2 pyroA4) (17, 28) was kindly supplied by H. Horiuchi and M. Takagi, University of Tokyo, Japan. A. nidulans ABPU1 was cultivated aerobically at 37°C for 24 h with rotary shaking at 180 rpm in 2-liter Erlenmeyer flasks with baffles, each containing 500 ml of the standard minimal medium with appropriate supplements (24), except that the carbon source was replaced with 2% starch. The culture filtrate and mycelia were separated by filtration with filter paper no. 1 (285 mm; Toyo Roshi, Tokyo, Japan). The mycelia (30 g [wet weight]) were ground to fine powder under liquid nitrogen and suspended at 0.2 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 2 mM phenylmethylsulfonyl fluoride. The suspension was homogenized with Polytron (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 dialyzed against 20 mM MES-KOH buffer (pH 5.5) containing 1 mM EDTA and 0.5 mM phenylmethylsulfonyl 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 the MES buffer, bound proteins were eluted with a 200-ml (4 column volumes) linear gradient of 0 to 0.5 M NaCl in the MES buffer at a flow rate of 1.5 ml/min (0.3 cm/min). The active fractions, eluted at about 0.1 M NaCl, were combined and dialyzed against the MES buffer containing 1.5 M ammonium sulfate. The dialysate was loaded onto a Phenyl Sepharose CL-4B column (1.0 by 12 cm; Amersham 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 MES buffer. The active fractions, eluted at nearly 0 M ammonium sulfate, were collected, dialyzed against 20 mM HEPES-KOH buffer (pH 7.4), and concentrated to about 1 ml by Centriprep YM-10 (Millipore, Bedford, Mass.). The concentrated sample was applied to a RESOURCE Q column (1.6 by 3.0 cm; Amersham Pharmacia Biotech), which was under the control of an ÄKTA explorer 10S system (Amersham Pharmacia Biotech), equilibrated with 20 mM HEPES buffer (pH 7.4). After the column was washed with 12 ml (2 column volumes) of the HEPES buffer, bound proteins were eluted with a 60-ml (10 column volumes) linear gradient of 0 to 1 M NaCl in the HEPES buffer at a flow rate 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 enzyme.
Enzyme assay.The AgdB activity was measured by incubating the enzyme with 0.5% (wt/vol) maltose in 40 mM acetate buffer (pH 5.5) for 30 min at 45°C. The reaction was terminated by boiling for 3 min, and liberated glucose was measured by the glucose oxidase-peroxidase method with a Glucose B Test (Wako Pure Chemical Ind. Ltd., Osaka, Japan). One unit of the AgdB activity was defined as the amount of enzyme that catalyzes hydrolysis of 1 μmol of maltose per min. Note that this unit definition is apparent, since the transglycosylation activity of AgdB is not taken into account. Kinetic parameters for hydrolysis of various oligosaccharides were calculated from a Hanes-Woolf plot. Chemical structures of 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 mM phosphoric acid-40 mM boric acid adjusted with NaOH to the desired pH values). Thermal and pH stabilities were determined by measuring residual activity after incubation of the enzyme solutions of 60 μg/ml at various temperatures 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.
Structures of five α-linked glucooligosaccharides described in this study.
TLC.Thin-layer chromatography (TLC) was carried out using Silica Gel 60 plates (Merck, Darmstadt, Germany) with three ascents of 2-propanol-1-butanol-water (12:3:4, vol/vol/vol) as the solvent (19). The solvent path length was 8.5 cm for each ascent. The products were visualized by spraying 1% p-anisaldehyde and 2% sulfuric acid in acetic acid (vol/vol) followed by heating at 110°C for 10 min. The quantification of oligosaccharides separated by TLC was performed by densitometry 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 chromatography with pulsed amperometric detection (HPAEC-PAD) analysis, the DX-500 chromatography system (Dionex, Sunnyvale, Calif.) was used with a GP-50 gradient pump, an ED-40 electrochemical detector, and CarboPac PA1 column (4 by 25 mm; Dionex). The reaction products were separated with a linear gradient of 0 to 300 mM sodium acetate for 15 min in 100 mM NaOH at a flow rate of 1 ml/min.
Determination of the N-terminal and internal amino acid sequences.The purified enzyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred electrophoretically to a Sequi-Blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, Calif.), and stained with Coomassie brilliant blue R-250. The stained bands, corresponding to the 74- and 55-kDa subunits, were excised and analyzed for N-terminal amino acid sequences by an Applied Biosystems (Foster City, Calif.) model 473A gas phase sequencer. For determination of the internal amino acid sequences, 74- and 55-kDa subunits of AgdB were separated by SDS-10% PAGE and eluted electrophoretically from the acrylamide gel. Each subunit was digested with Lysyl endopeptidase (Wako). The peptides generated were resolved by SDS-15% PAGE, and major peptides were subjected to amino acid sequencing.
Molecular cloning of the agdB gene.A part of the agdB gene was amplified by nested PCR using two pairs of degenerate primers designed based on the N-terminal and internal amino acid sequences of the 74-kDa subunit. The primer pairs N1-N2 and I1-I2 correspond to the N-terminal and internal amino acid sequences, respectively (Table 1). The first PCR was carried out with A. nidulans total DNA as a template using the primer pair N1-I1, and a part of the reaction mixture was subjected to the second PCR using the primer pair N2-I2. A 440-bp fragment was specifically amplified and used as a probe for Southern and colony hybridizations.
Primers used in this study
Total DNA of A. nidulans was digested with HindIII and size fractionated by preparative agarose gel electrophoresis. DNA fragments of approximately 5.0 to 7.0 kb hybridized to the PCR amplified probe were ligated to pBluescript II KS(+) and introduced into E. coli JM109. Transformants were screened by colony hybridization. A plasmid carrying the agdB gene on a 5.6-kb HindIII fragment was obtained and designated pGBH6.
Parts of cDNA were obtained by reverse transcriptase PCR with A. nidulans total 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 α-glucosidases was performed by using the CLUSTAL W program (version 1.80; DDBJ [http://www.ddbj.nig.ac.jp ]) (29). The phylogenetic relationship of the α-glucosidases was calculated by using the neighbor-joining method (25). Positions with gaps in the alignment were excluded from the calculation. The unrooted phylogenetic tree was produced using the TREE VIEW program (version 1.6.5; Taxonomy and Systematics, University of Glasgow [http://taxonomy.zoology.gla.ac.uk/ ]). Accession numbers of the following amino acid sequences used in the phylogenetic analysis are as indicated: Schwanniomyces occidentalis glucoamylase, GenBank AAA33923 ; Candida albicans glucoamylase, AAC31968; A. niger α-glucosidase, BAA23616; A. oryzae α-glucosidase, BAA95702; A. nidulans α-glucosidase AgdA, AAF17102; Schizosaccharomyces pombe α-glucosidase, BAB43946; S. pombe ORF SPAC922.02c, CAB63549; S. pombe ORF SPAC30D11.01c, SwissProt Q09901; Mucor javanicus α-glucosidase, GenBank BAA11053 ; Candida tsukubaensis α-glucosidase, CAA39501; spinach α-glucosidase, BAA19924; sugar beet α-glucosidase, BAA20343; Arabidopsis thaliana α-glucosidase I, AAB82656; barley high-pI α-glucosidase, AAF76254; potato α-glucosidase, CAB96077; mouse lysosomal α-glucosidase, AAB06943; human lysosomal α-glucosidase, AAA52506; rabbit sucrase-isomaltase, AAA31459; human sucrase-isomaltase, CAA45140; human maltase-glucoamylase, AAC39568. The sequences of Neurospora crassa putative α-glucosidase genes were obtained from an N. crassagenome database (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/ ).The amino acid sequences of putative α-glucosidases 1 and 2, which showed the highest sequence homology to A. nidulans AgdA and AgdB, respectively, were deduced based on alignment with sequences of AgdA and AgdB.
Other methods.Standard DNA manipulations were performed according to the method of Sambrook et al. (26). Total DNA from A. nidulans was isolated by using the cetyltrimethylammonium bromide method (4). Labeling of the probes and detection of hybridization signals in Southern and colony hybridizations were carried out using an enhanced chemiluminescence nucleotide labeling and detection system (Amersham Pharmacia Biotech). Nucleotide sequences were determined by the dideoxy chain termination method with a DNA sequencer (LI-COR model 4000). Protein was determined by a Bio-Rad protein assay kit with bovine immunoglobulin G as a standard. SDS-PAGE was carried out according to the method of Laemmli (12), and proteins were visualized by Coomassie brilliant blue staining. An LMW marker kit (Amersham Pharmacia Biotech) was used for standard protein markers. The molecular mass of the native enzyme 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 column was equilibrated and run with the MES buffer containing 0.15 M NaCl at a flow rate of 0.5 ml/min.
Nucleotide sequence accession number.The nucleotide sequence of agdB has been deposited in the GenBank/EMBL/DDBJ databases under the accession number AB057788 .
RESULTS AND DISCUSSION
Purification of AgdB from A. nidulans. A. nidulans culture filtrates and cell extracts were determined for isomaltose-producing activity with maltose as a substrate (Fig. 2). The cell extracts were found to convert maltose to glucose and a series of transglycosylation products, including two dominant species which, by the TLC analysis, showed Rf values nearly identical to those of maltotriose or isomaltose and maltotetraose or panose, respectively. Little conversion of maltose was observed with the culture filtrates.
Isomaltose-producing activity in the culture filtrate and cell extract of A. nidulans. The 35-fold-concentrated culture filtrate (CF) and the cell extract (CE) were incubated with 0.5% maltose in 40 mM acetate buffer (pH 5.5) at 45°C for 2 h, and the reaction products were analyzed by TLC. The volumes of CF and CE used for the reactions were 48 and 10 μl, corresponding to 2 ml and 200 μl of the original culture suspension, respectively. The culture filtrate was concentrated by 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).
The purification of AgdB, which catalyzed conversion of maltose to isomaltose, from the cell extract is summarized in Table 2. The enzyme was purified 52-fold with a specific activity of 9.6 U/mg, and the overall yield was 22%. The purified enzyme gave two polypeptide bands of 74 and 55 kDa on SDS-PAGE (Fig. 3), while the approximate molecular mass of the native AgdB determined by an analytical gel filtration on Superdex 200 was 130 kDa. Both subunits of AgdB were stained with periodic acid oxidation-silver staining (32), indicating that AgdB was a glycoprotein (data not shown). AgdB had a pH optimum of 5.5, and less than 10% of the activity was lost over a wide pH range of 5.0 to 8.5 and at temperatures up to 45°C.
SDS-PAGE of the purified AgdB. The purified enzyme (lane 2) was analyzed on an SDS-10% polyacrylamide gel. Phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and α-lactalbumin (14.4 kDa) were used as molecular mass markers (lane 1).
Purification of α-glucosidase B from A. nidulans
Transglycosylation activity and substrate specificity of AgdB.The time course of the AgdB reaction was examined by TLC with 0.5% maltose as a substrate (Fig. 4A). Within the first 2 h of the reaction, more than 90% of maltose was consumed, and glucose and a series of transglycosylation products were formed as the reaction products. Two dominant transglycosylation products gave Rf values by the TLC analysis identical to those of isomaltose and panose, respectively. However, as described earlier for Fig. 2, isomaltose and panose were not separated from maltotriose and maltotetraose, respectively, under the TLC conditions used here. In order to identify further the dominant transglycosylation products, HPEAC-PAD analysis of the reaction products was carried out. As shown in Fig. 5A and B, glucose, maltose, maltotriose, maltotetraose, and panose were clearly assigned by HPEAC-PAD, although isomaltose and isomaltotriose were not separated from each other. In the initial stage of the reaction, the products contained isomaltose and/or isomaltotriose, panose, and glucose as major species (Fig. 5B), and later isomaltose and/or isomaltotriose and glucose dominated other reaction products (Fig. 5C). However, only trace amounts of maltotriose and maltotetraose were detected. Taken together with the results of the TLC analysis, the dominant transglycosylation products were isomaltose and panose. As shown in Fig. 4, panose gradually decreased as the reaction proceeded, while glucose and isomaltose increased steadily. At 6 h of the reaction, approximately 50% of maltose was converted to transglycosylation products, 60% of which was found to be isomaltose.
(A) Time course of the AgdB reaction with maltose as a substrate. The enzyme (0.6 μg) was incubated with 0.5% maltose in 100 μl of 40 mM acetate buffer (pH 5.5) at 45°C. Aliquots were withdrawn at the indicated times and analyzed by TLC. (B) TLC analysis of the transglycosylation products from various α-glucobioses. The purified AgdB (0.6 μg) was incubated with 0.5% (wt/vol) maltose (M), isomaltose (I), nigerose (N), and kojibiose (K) as substrates (Subs.) in 100 μl of 40 mM acetate buffer (pH 5.5) for 2 h at 45°C ([+] or without [−] enzyme [Enz.]), and the reaction products were analyzed by TLC. Glucose (G), maltose (M), isomaltose (I), panose (P), and isomaltotriose (IG3) were used as standards (Std).
HPAEC elution profiles of the reaction products of AgdB from maltose. The enzyme (2.5 μg) was incubated with 0.5% maltose in 100 μl 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 analysis. (A) Elution profile of the standard sugars. Glucose (G), maltose (M), maltotriose (G3), maltotetraose (G4), maltopentaose (G5), isomaltose (I), panose (P), and isomaltotriose (IG3) were used as standards.
Formation of isomaltose and panose from maltose by the action of α-glucosidase has been demonstrated in A. niger and A. oryzae (3, 15, 20-22). The A. niger enzyme possesses strong transglycosylation activity, however, a maltose concentration of 30% is used for A. niger α-glucosidase to achieve an isomaltose content of 30% in the final reaction products (3, 21). At a substrate concentration of 0.5%, AgdB clearly showed higher transglycosylation activity than that of the A. niger enzyme (15). The other α-glucosidases so far reported also require high substrate concentrations to exhibit sufficient transglycosylation activities (13). Therefore, AgdB may be the α-glucosidase with the strongest transglycosylation activity among α-glucosidases known to date.
Kinetic parameters for hydrolysis of various substrates are shown in Table 3. AgdB exhibited high hydrolytic activity toward maltooligosaccharides, and the most favored substrate was maltotriose, with a k0/Km ratio of 120, which was 2.9-fold higher than that of maltose. The k0/Km values for maltotetraose and maltopentaose decreased in this order, indicating that the longer maltooligosaccharides are less favored. The enzyme also efficiently hydrolyzed kojibiose, nigerose, and isomaltose and showed weak activity toward trehalose and p-nitrophenyl α-glucoside. Sucrose and soluble starch were the least-favored substrates examined (data not shown).
Kinetic parameters for hydrolysis of various oligosaccharides by AgdB
The enzyme produced a series of transglycosylation products from kojibiose, nigerose, maltose, and isomaltose (Fig. 4B). Isomaltose was also formed from nigerose and kojibiose, while isomaltotriose was the main transglycosylation product from isomaltose. These facts imply that the enzyme preferentially forms an α-1,6 linkage.
Cloning and sequence analysis of the agdB gene.The N-terminal amino acid sequences of the 74- and 55-kDa subunit were chemically determined to be SQAGVDPLDRPGNDLYVKD and QSHRQLGAGRWRSAVRH, respectively. Lysyl endopeptidase digestion of the 74- and 55-kDa subunits generated major peptides of 30 and 15 kDa, respectively, and the N-terminal amino acid sequences for 30- and 15-kDa peptides were determined to be THLPQNPHLYGLGE and DVSHWLGDNISDWLSYRLSI, respectively. Based on these pieces of information, genomic and cDNA clones including the agdB gene were obtained as described in Materials and Methods. Nucleotide sequences of the cloned DNA fragments revealed that the agdB gene comprised 3,055 bp, interrupted by three short introns of 57 to 72 bp, and encoded a polypeptide of 955 amino acids. The N-terminal amino acid sequences of the 74- and 55-kDa subunits were identified at residues 21 to 39 and 515 to 531 of the derived amino acid sequence, respectively, and the internal amino acid sequences were also found at residues 167 to 180 and 637 to 656. This indicates that the enzyme is synthesized as a single polypeptide precursor and then the precursor is processed to form the mature heterodimeric protein. The first 20 amino acids at the N terminus of AgdB showed a typical feature of signal peptides: a basic residue 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 enzyme present in the cell wall.
Comparison with other α-glucosidases.α-Glucosidases have been classified into two families, GH families 13 and 31, based on hydrophobic cluster analysis (http://afmb.cnrs-mrs.fr/∼pedro/CAZY/ghf.html ) (6). AgdB showed low but overall sequence identity to the GH family 31 enzymes such as A. nidulans α-glucosidase AgdA (32%), A. niger α-glucosidase (31%), S. occidentalis glucoamylase (31%), C. albicans glucoamylase (31%), and A. oryzae α-glucosidase (30%). The GH family 31 enzymes share two conserved sequences, designated as the GH family 31 signature sequence 1 and 2 (5, 8), which corresponded to residues 434 to 448 and residues 671 to 704 in AgdB. Multiple alignment of the two signature sequences of eukaryotic GH family 31 α-glucosidases is shown in Fig. 6A. Six conserved residues in the signature sequence 1, including the catalytic residue Asp (7, 9, 10, 23), were conserved in AgdB except for Ala 436. Among 14 conserved residues in sequence 2, AgdB had a single substitution, Thr to Pro at position 698. In addition, Asp 643, which functions possibly as a proton donor (19), was also conserved. These facts indicate that AgdB is a member of GH family 31.
Comparison of the amino acid sequence of A. nidulans AgdB with those of GH family 31 α-glucosidases. (A) Multiple alignment of the regions corresponding to two GH family 31 signature sequences. Conserved amino acids are shaded, and the derived consensus sequences are given below the alignment. Uppercase letters indicate conserved residues, and the asterisk indicates a catalytic residue. (B) Phylogenetic tree of α-glucosidases belonging to GH family 31. Bootstrap values (based on 1,000 bootstrap trials) are shown at each node. The scale bar corresponds to a genetic distance of 0.1 substitution per position. Abbreviations: ORF, open reading frame; SI, sucrase-isomaltase; MG, maltase-glucoamylase.
Phylogenetic analysis of the GH family 31 α-glucosidases revealed three clusters comprised of the enzymes from mammals, plants, and fungi (Fig. 6B). A. niger α-glucosidase, which has been shown to exhibit strong transglycosylation activity (3, 15, 21), was located in the fungal cluster with close genetic distances to α-glucosidases (AgdAs) from A. oryzae and A. nidulans. However, AgdB was phylogenetically distant from those Aspergillus α-glucosidases, and surprisingly, it was located outside of the fungal cluster. Furthermore, AgdB was obviously distant from mammalian and plant α-glucosidases. The phylogenetic location of AgdB, being independent from those of other α-glucosidases, indicates that AgdB is a novel α-glucosidase that appears to have diverged from the other α-glucosidases at an early stage of fungal evolution. Homologues of agdB were found in an N. crassa genome database (http://www-genome.wi.mit.edu/annotation/fungi/neurospora/ ) and in the A. oryzae EST database (http://www.aist.go.jp/RIODB/ffdb/index.html ). Enzymes similar to AgdB may be widely distributed in filamentous fungi.
ACKNOWLEDGMENTS
We thank Kikkoman Co. Ltd. for the generous gifts of kojibiose and nigerose. We also thank Shinji Go for performing the HPAEC-PAD analysis.
This work was partially supported by a grant to T. Kobayashi from Noda Institute for Scientific Research and by a grant-in-aid for scientific research (C) to T. Kobayashi from the Ministry of Education, Science, Sports, and Culture.
FOOTNOTES
- Received 29 October 2001.
- Accepted 19 December 2001.
- Copyright © 2002 American Society for Microbiology
