Insights into the reaction mechanism of glycosyl hydrolase family 49 Site-directed mutagenesis and substrate preference of isopullulanase Hiromi Akeboshi 1 , Takashi Tonozuka 1 , Takaaki Furukawa 1 , Kazuhiro Ichikawa 1 , Hiroyoshi Aoki 1,2 , Akiko Shimonishi 1 , Atsushi Nishikawa 1 and Yoshiyuki Sakano 1 1 Department of Applied Biological Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; 2 Fuence Co., Shibuya, Tokyo, Japan Aspergillus n iger isopullulanase (IPU) is the only pullulan- hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse d extran at all, while all o ther GH family 49 enzymes a re dextran-hydrolysing enzymes. T o i nvestigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three A sp and two Glu). Homology modelling of I PU was also carried out based o n the structure of Penicillium minioluteum dextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pu llulan and panose, were p redicted to b e located in the negatively numbered subsites. Three Asp- mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these r esidues are candidates f or the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and the K m value was sixfold higher and the k 0 value was 5 00-fold lower than t hose for the wild- type enzyme, suggesting that Trp402 is a residue participa- ting in subsite )1. Trp31 and Glu273, whose substitutions caused a d ecrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and b-helical domains. T he substrate p reference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar c atalytic mechanism i n their negatively numbered s ubsites in spite of the d ifference of their substrate s pecificities. Keywords: d extranase; GH family 49; isopullulanase; pullu- lan-hydrolase; site-directed mutagenesis. Isopullulanase (IPU, EC 3.2.1.57; pullulan 4 -glucanohydro- lase) f rom Aspergillus niger ATCC9642 hydrolyses pullula n to produce isopanose (Glc-a-(1fi4)-Glc-a-(1fi6)-Glc) and also hydrolyses substrates containing the panose (Glc-a- (1fi6)-Glc-a-(1fi4)-Glc) s tructure, and cleaves the a-1,4- glucosidic linkage in the panose motif [1,2]. Enzymes that hydrolyse specific sites of pullulan can be classified into the following three types (schematic action patterns of these enzymes have been illustrated previously [2]). (a) Pullulanase (EC 3 .2.1.41), which hyd rolyses a-1,6-glucosidic linkages t o produce maltotriose [3]; (b) Thermoactinomyces vulgaris R-47 a-amylase (TVA, EC 3.2.1.1) [4] and neopullulanase (EC 3.2.1.135) [5], which hydrolyse a-1,4-glucosidic linkages to produce panose; and (c) IPU, which h ydrolyses the other a-1,4-glucosidic linkages to produce isopanose. Except for IPU, these enzymes are c lassified into glycosyl h ydrolase (GH) family 13, known as the a-amylase family (reviewed in [6–8]). In contrast, IPU is the sole enzyme classified into GH family 49 [2,9,10] among these pullulan-hydrolases, and no homology between IPU a nd a-amylase family e nzymes has been found (http://afmb.cnrs-mrs.fr/cazy/CAZY/ index.html). Interestingly, IPU does not hydrolyse dextran at all, while all o ther GH family 49 enzymes are dextran-hydrolysing enzymes, such as endo-dextranase (EC 3.2.1.11) [11–14] and isomaltotrio-dextranase (EC 3.2.1.95) [15]. We have repor- ted the molecular cloning of IPU, and indicated that seven highly co nserved regions are found among the p rimary structures of these dextran-hydrolases and IPU [2]. The expression systems of IPU have been constructed with eukaryotic hosts Aspergillus oryzae and Pichia pastoris [2,16]. Recently, a three-dimensional structure of GH family 49 dextranase (D ex49A), wh ich shows a 38% s equence identity with IPU, has b een reported, and the catalytic domain folds into a right-handed parallel b-helix [17]. Crystal structures o f polygalacturonases a nd rhamnogalac- turonases, which belong to GH family 28, have been solved by many researchers (for e xample [18–20]). Although the substrate specificities between GH family 49 and 28 are completely different, the GH family 28 polygalacturonases and rhamnogalacturonases consist of the similar b-helical structures, and GH family 49 and 28 form clan GH-N Correspondence to Y. Sakano, Department of Applied Biological Science, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo, 183-8509 Japan. Fax: +81 42 367 5705, E - mail: s akano@cc.tuat.ac.jp Abbreviations: IPU, isopullulanase; GH, glycosyl hydrolase; BMM, buffered minimum methanol medium; YPGY, yeast peptone glycerol medium. Enzyme: isopullulanase (EC 3.2.1.57); pullulanase (EC 3.2.1.41); neo- pullulanase ( EC 3 .2.1.135); R-47 a-amylase (TVA, EC 3.2.1.1); endo- dextranase (EC 3.2.1.11); isomaltotrio-dextranase (EC 3 .2.1.95); glucoamylase (anomer-inverting enzyme; EC 3.2.1.3); a-glucosidase (anomer-retaining en zyme; EC 3 .2.1.20). (Received 2 3 June 2004, revised 1 6 August 2004, accepted 27 September 200 4) Eur. J. Biochem. 271, 4420–4427 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04378.x [10,17]. D espite these a dvances, little is known a bout the unique substrate preference and the catalytic mechanism of IPU. To clarify the catalytic mechanism of IPU, site-directed mutagenesis was carried out. Acidic amino acid residues, Asp a nd/or Glu, are commonly r eported a s the catalytic residues of GHs [6,21–26], and it is probable that IPU has Asp a nd/or Glu as the catalytic residues. In addition, in the chemical modification experiment IPU was inactivated by N-bromosuccinimide, i ndicating that a Trp residue is required f or IPU activity [27]. Therefore, several Asp and/ or Glu, and Trp residues located in the conserved regions are predicted to be indispensable for IPU a nd the other GH family 49 enzymes. Here we determined the residues that are essential for the catalytic activity of IPU, and also investi- gated some detailed properties of this enzyme. The results indicated that the functionally important residues o f GH family 49 enzymes are conserved in the negatively numbered subsites, and the substrate preference of the negatively numbered subsites of IPU also resembles that of GH family 49 dextranases. Materials and methods Host strains and media Escherichia coli JM109 was used for the plasmid con- structions. P. pastoris GS115 (Invitrogen) was used for the heterologous expression of IPU. The Luria–Bertani medium for E. coli, and the yeast peptone dextrose medium and buffered minimum methanol medium (BMM) for P. pastoris were prepared according t o t he manufacturer’s r ecommendations. Y east peptone glycerol medium (YPGY: 1% yeast extract, 2% peptone, and 1% glycerol) was prepared for the propagat ion of recombinant P. pastoris strain for t he expression of enzymes. All cultivation was done at 37 °CforE. coli and 30 °Cfor P. pastoris. Mutant constructions All c loning procedures were carried out by applying standard molecular b iological t echniques [28]. Transforma- tion of P. pastoris was done according to the manufacturer’s instructions for the Pichia Expression Kit (Invitrogen), which has been described elsewhere [16]. Plasmids coding for the W31F, W32F, W240F, and W402F enzymes were constructed by PCR using p IPA118(–), a plasmid DNA coding for mature I PU. T he complementary m utagenic primers encoding the desired mutations (Table 1) were paired with universal primers, M13 forward and M13 reverse, respectively, and two partial DNA fragments were amplified. Subsequently, PCR was performed with these two amplified fragments as the tem plates and pri mers, M13 forward a nd M13 r everse. After sequence confirmation, the EcoRI–BamHI fragment w as inserted into the e xpression vector for P. pastoris, pHIL-S1 (Invitrogen). Plasmids coding for E273Q, D353N, E3 56Q, D372N, and D373N were constructed b y the method of Kunkel [ 29]. The s ites at which the mutations were introduced are located in the StyI–XbaI fragment. To construct reliable mutants, mutated St yI–XbaI fragments were verified b y DNA sequencing, and the origin al StyI–XbaI fragment of pIPA118(–) was replaced with the mutated StyI–XbaI fragment. The EcoRI–BamHI fragments o f these plasmids were further subcloned into pHIL-S1. Expression of wild-type and mutated IPU enzymes The wild-type IPU was expressed in P. pastoris harbouring pSig-PHO as d escribed [16] with slight modifications. Briefly, P. pastoris harbouring pSig-PHO w as cultured in 250 mL YPGY for 2 days, and the propagated cells collected by centrifugation (5000 g for 1 0 min) were resus- pended a nd cultivated for 6 days in 100 mL B MM. The clear supernatant of cultured BMM (crude IPU) was then obtained by c entrifugation ( 5000 g for 1 5 min). During the cultivation in BMM, 500 lL of methanol was added every 24 h for the maintenance of 0.5% (v/v) methanol as the carbon source and i nducer. The expressi on of IPU mutants was performed using the same procedure as for the wild- type IPU. Substrates Pullulan w as obtained from H ayashibara, Japan. Panose [30], IMTG (4 1 -a-isomaltotriosylglucose, also called 6 2 -a-isomaltosylmaltose; Glc-a-(1fi6)-Glc-a-(1fi6)-Glc-a- (1fi4)-Glc) [ 31], and MM (6 2 -a-malto sylmaltose; Glc-a- (1fi4)-Glc-a-(1fi6)-Glc-a-(1fi4)-Glc) [32] were prepared as described. Dextran T-2000 was from Amersham. Concentrations of the substrates dissolved in 50 m M acetate buffer (pH 3.5) were measured by a modified phenol/sulfuric acid method [33], using glucose as the standard. Table 1. Primers used for the mutant constructions. For PCR muta- genesis (W21F, W32F, W240F and W402F), the same sequences on the opposite strand were also used as described in Materials and methods. Lower c ase letters indicate the nucleotide m utations. To facilitat e the selection of mutant clones, silent mutations were made to introduce restriction enzyme reco gnition sites ( u nderlined). Pimers Nucleotide sequence (5¢fi3¢) W31F CTGACCTtcTGGCATAAC ACCGGtGAAATC AgeI W32F CCTGGTtcCATAAC ACCGGtGAAATC AgeI W240F GGTGCT gAGCTCAAGTGTGACTTtcGTCTAC SacI W402F CCGGTG GTcGAcTTTGGTTtcACGCCC SalI E273Q ACGTACTGCTgTCCGGAA AGtACtCCATGGCCGC ScaI D353N TCCAATCCGTtAGTC TGgCCaTAGAACGCGC MscI E356Q GGAGAAT GGTgCCAGGGTACATTTcCAATCCGTCA KpnI D372N AATACAT CTTaAGGCCGTCGTtGTCGGTGTGG AflII D373N AATACAT CTTaAGGCCGTtGCGTCGGTG AflII Ó FEBS 2004 Reaction mechanism of isopullulanase (Eur. J. Biochem. 271) 4421 Assays of IPU activity and protein concentration The p ullulan-hydrolysing activity of IPU was evaluated as described previously [34]. The activities for panose, IMTG and MM were measured as follows. A re action mixture consisting of a desired substrate (32 m M )andIPUin 40 m M acetate buffer (pH 3.5) was incubated at 40 °Cfor 30 min, and the reaction was terminated by t he addition of an equal v olume o f 0.1 M Na 2 CO 3 . T he amount of glucose p roduced by IPU was assayed with G lucose CII (Wako Pure Chemical Co., Osaka, Japan) [35,36] using a Bio-Rad550 Microplate reader. To determine the kinetic parameters of wild-type and W402F IPU for p anose, mixtures consisting of 4 lgÆmL )1 enzyme and from 43 to 216 m M substrate, and 20 lgÆmL )1 enzyme and 64 to 480 m M substrate, respectively, were used. The protein concentration was measured by the method of Lowry et al. with B SA as standard [37]. TLC TLC was p erformed to analyse t he hydrolysates of the W402F enzyme. The reaction mixture consisting of pullu- lan ( 5%) o r p anose ( 100 m M ) a nd the W402F e nzyme (0.1 mg ÆmL )1 )in50m M acetate buffer (pH 3.5) was incubated at 30 °C for 3 days, and the hydrolysates were developed by TLC with 1-butanol/ethanol/H 2 O ¼ 2/2/1 (v/v/v). The spots were detected by charring with H 2 SO 4 . Homology modelling The primary structure of IPU is homologous to that of Penicillium minioluteum dextranase (38% identity), whose three-dimensional structures of unliganded form and com- plex form with a product, isomaltose, have been reported (PDB IDs, 1OGM and 1OGO, respectively) [17]. The primary structure of mature IPU (residues 20– 564) was submitted for automatic modelling on t he Swiss-Model server (http://swissmodel.exp asy.org/) [ 38] u sing the first approach mode, a nd a m odel consisting of residues 2 5–540, which is based on the structure of 1OGM, was obtained. To determine the potential catalytic site, this mo del was superimposed on 1OGO using the program DEEPVIEW [38], and a glucosyl units in subsites +1 and +2 w ere placed i n the m odel. The figure was generated using the programs RASMOL [39], RASTOP (http://www.geneinfinity.org/rastop/), MOLSCRIPT [40] and RASTER 3 D [41]. Polarimetric assays Polarimetric measurements of IPU, glucoamylase from Rhizopus niveus (Seikagaku K ogyo, Japan; a nomer-invert- ing enzyme; EC 3.2.1.3), and a-glucosidase from Bacillus sp. (Toyobo, Japan; an omer-retaining enzyme; EC 3 .2.1.20) were compared. An enzyme solution (equivalent to 1.2 UÆmL )1 ), and 1.6–1.8% of panose (for IPU) or malto- tetraose ( for R. niveus glucoamylase and Bacillus a-glucosi- dase) were d issolved in 50 m M acetate buffer (pH 4.5), and the optical r otations were measured at 1-min intervals at 589 nm using a JASCO DIP-360 polarim eter. After 1 0 min (IPU) o r 2 0 min ( R. niveus glucoamylase and Bacillus a-glucosidase), 20 lLof15 M ammonium hydroxide w as added to the reaction mixture t o raise mutarotation and the anomeric form of the product was determined [42,43]. Results and Discussion Purification of wild-type and mutated IPU Purification of I PU using HiTrap Con A Sepharose HP column (Amersham Biosciences) has been described [ 16]. However, because the recovery of IPU by this method was low (13%), several other purification methods were tested. When a hydrophobic column, TOYOPEARL Hexyl-650C (Tosoh, Japan) was used, the recovery increased to 50%. Ammonium sulfate was added directly to the dialysed crude enzyme to adjust it to 70% s aturation and the supernatant was loaded on to the column. The specific activity of purified IPU for pullulan using this method was 40 UÆmg )1 , while the previous method gave only 25 UÆmg )1 .This specific activity was also h igher t han t hose o f IPU from original A. niger and heterologously expressed I PU fr om A. oryzae (27 a nd 38ÆUmg )1 , r espectively) [7,27]. The mutatedIPUswerepurifiedwiththesameprocedureas wild-type IPU. Properties of Trp-mutated enzymes Previous experiments indicated that some T rp residues are essential f or IPU a ctivity [27]. Four Trp residues, conserved in the seven regions of GH family 49 (Fig. 1), are replaced by Phe (W31F, W32F, W240F and W402F). The relative activity of mutated enzymes towards pullulan and panose are shown in Table 2. The W 402F enzyme lost the activity for pullulan ( 0.4% of the wild-type IPU) and the activity for p anose w as almost undetectable ( 0.1%) under the given conditions. The W31F enzyme had only 38% activity for pullulan, but the a ctivity for panose w as 1.4-fold higher than that of wild-type enzymes. The activities of W32F and W 240F were similar (90–160%) to that o f wild-type enzyme. As t he a ctivity o f W402F drastically decr eased, it s action pattern was investigated using TLC. T he W402F enzyme liberated isopanose from pullulan, and isomaltose and glucose from panose, and the action patterns of the wild- type and the W402F enzymes were almost identical (Fig. 2). The kinetic study for W402F towards panose showed that the K m value was sixfold higher and the k 0 value was 500- fold lower than those for the wild-type enzyme (Table 3). Properties of Asp- and Glu-mutated enzymes Three Asp residues (353, 372 and 373) and three Glu residues (157, 273 and 356) are found in the seven conserved regions of all the GH family 49 enzymes (Fig. 1). To determine the catalytic r esidues of I PU, these Asp a nd Glu residues are replaced by Asn and Gln, respectively. Five of these enzymes (E273Q, D353N, E356Q, D372N and D373N) were obtained in soluble form, and were purified. Their activities for pullulan and panose were compared with those of wild-type e nzyme (Table 2). All three of the Asp-mutated enzymes, D353N, D372N and D373N, virtually lost their activities. The activities of mutated enzymes, E273Q and E356Q, were also decreased but less 4422 H. Akeboshi et al. (Eur. J. Biochem. 271) Ó FEBS 2004 significantly. The mutant E356Q had 38% and 50% of the activities for pullulan and panose, respectively. In contrast, the activity of E273Q for p ullulan was 45%, w hile that for panose remained at 74%. The sixth mutant E157Q is not obtained in the P. pastoris expression system, but h as been expressed by u sing A. nidulans as a host and shown to have detectable activity to both pullulan and panose (data not shown). Prediction of the positions of amino acid residues whose substitutions resulted in the reduction of the IPU activity While the study of site-directed mutagenesis described above was carried out, a crystal structure of Penicillium minioluteum dextranase, Dex49A, complexed with a prod- uct, isomaltose, has been reported (PDB ID, 1OGO) [17]. The identity between the primary structures of Dex49A a nd IPU is 38%, and a t hree-dimensional structure of IPU was modelled based on the structure of Dex49A using the Swiss- Model server. IPU consists of a signal sequence (residues 1–19) and a mature part (residues 20–564), and the model is composed of residues 25–540. The overall structure of the model of IPU and the mutated residues in this study are shown in Fig. 3. To elucidate the mechanism of the substrate recognition of IPU, two glucosyl units (Glc +1 and +2, respectively) were forced to be placed based on the position o f isomaltose bound in the subsites +1 and + 2 of Dex49A, although IPU does not produce isomaltose. IPU was predicted to consist of two domains, N-terminal domain (residues 25–189) and b-h elical domain ( residues 190–540). Asp353, Glu356, Asp372, Asp373, and Trp402, whose substitutions resulted in the reduction of the activity for both pullulan an d panose, were predicted to be l ocated in potential subsites )1and)2 ( a d etailed description is given i n the next section). T rp31 and Glu273, whose Fig. 1. Conserved regions of GH family 49 e nzymes. Identical amino acid residues are shown in white on black, and conserved Trp, Asp and Glu residues are indicated b y asterisks. PMDEX, Penicillium minioluteum dextranase [12]; DEX49A, Penicillium minioluteum dextranase isoform [13]; PFDEXA, Penicillium funiculosum dextranase (DDBJ/EMBL/GenBank No. AJ272066); AGTDEX1 and 2, Arthrobacter globiformis T-3044 endodextranase 1 an d 2 [1 4]; AGCDEX, Arthrobacter sp. CB-8 d extranase [11]; I MTD, Brevibacterium fuscum var. dextranlyticum isomaltotrio- dextranase [15]. Table 2. Relative activities of wild-type and mutant IPUs for pullulan and panose. Activities for 0.4% (w/v) pullulan and 32 m M panose were measured. ND, Not d etected. Pullulan Panose Wild-type 100 100 W31F 38 140 W32F 90 120 W240F 130 160 W402F 0.4 0.1 E273Q 45 74 D353N ND ND E356Q 38 50 D372N ND ND D373N ND ND AB Fig. 2. Patterns of hydrolysis for pullulan (A) and panose (B) by W402F IPU. Reaction mixtures of 5% (w/v) pullulan or 100 m M panose with 0.1 m gÆmL )1 of wild-type or W402F IPU were incubated at 30 °Cfor 1 day (wild-type) or 3 days (W402F). The hydrolysates were analysed by TLC using the conditions described in Materials and methods. M, Maltooligosaccharide marker; G1–G7, maltooligosaccharides glucose to maltoheptaose; Pu, pullulan; Pa, panose; W, wild-type IPU added; W402F, W402F IPU added. The numbers indicate the time of reaction in days; 0 , no enzyme added. I P, isopanose; IM, isomaltose. Table 3. Kinetic parameters of wi ld-type and W402F IPUs for panose. K m (m M ) k 0 (s )1 ) k 0 /K m (m M )1 Æs )1 ) Wild-type 160 ± 3.8 180 ± 2.2 1.13 ± 0.04 W402F 920 ± 140 0.36 ± 0.036 (3.9 ± 1.0) · 10 )4 Ó FEBS 2004 Reaction mechanism of isopullulanase (Eur. J. Biochem. 271) 4423 substitutions caused a decrease in the activity for pullulan (38 and 45%, r espectively) but not significant for panose (140 and 74%, respectively), are located r elatively far from the potential catalytic site, and the side chains were predicted t o o rient t o the interface b etween N-terminal and b-helical domains. A structural homology search for the N-terminal domain (residues 25–189) was also carried out using the Dali server [44]. N umerous proteins containing an immunoglobulin-like fold were listed, and among glycosyl hydrolases, domain N of a pullulan-hydrolysing enzyme from Thermoactinomyces vulgaris, TVA II (PDB ID, 1BVZ; Z score of 3.2) [45] was a solution in the Dali result. It is likely that the inte rface between N-terminal and b-he lical domains participate in binding of the polysaccharide, pullulan. Comparison of the active sites of the model of IPU and Dex49A The active site structures of t he model of IPU (Fig. 4A) and Dex49A (Fig. 4B) were compared. The three Asp residues, Asp353, Asp372 and Asp373, mutation of which causes nearly complete loss of the enzymatic activity, were positioned c lose to the O4 hydroxyl group of Glc +1 residue (Fig. 4A). Larsson et al. reported that, in Dex49A, the corresponding aspartyl residues , Asp376, 395 and Asp396, are conserved within GH family 49, and Asp376 and 396 are positioned in a potential )1 subsite [17] (Fig. 4B). These findings show that Asp353, 372, and 373 are the potential catalytic r esidues of I PU. Also, Trp425 of Dex49A, which is the corresponding residue of Trp402 of IPU, is reportedly located in the vicinity of the active site and could form a binding site for a glucosyl unit i n subsite )1 (Fig. 4B). Together with the observations from the site-directed mutagenesis, Trp402 of IPU appears to be a residue participating in subsite )1. The r esidues that form potential subsites )1and)2 are reportedly more conserved in GH family 49 than the residues in s ubsites +1 and +2 [17]. Comparison of the active sites o f t he model of IPU and D ex49A c learly shows that residues located in the negatively numbered subsites are highly conserved between IPU and Dex49A (Fig. 4). In addition t o the r esidues Asp353(IPU)-376(Dex49A), Asp373–396, Glu356–379, and Trp402–425, numerous aromatic and charged residues Arg297–322, Asn323–348, Asp326–351, Tyr358–381, Lys376–399, Tyr378–401, Tyr379–402, and Tyr440–463, are conserved in the negat- ively numbered subsites. On the other hand, residues located in the positively numbered s ubsites are relatively not conserved. The report o f Dex49A shows an illustration where seven amino acid residues, Asp86, Tyr303, Lys315, Asp395, Asn417, Lys447, and Glu449, interact with Glc +1 and +2 [17] (Fig. 4B). Only two of these residues, Tyr278(IPU)-303(Dex49A), and Asp372–395, both o f which interact with Glc +1, are conserved between IPU and Dex49A. The position equivalent to Lys315 o f Dex49A is identified as Gly290 of IPU, which may enable IPU to incorporate the a-(1fi4)-linked glucose un its. In addtion, in Dex49A, Phe373 protrudes to the active cleft, which appears to restrict the c onformation of the s ubstrate and accom- modate only the a-(1fi6)-linked glucose units. In IPU, the position e quivalent to Phe373 of Dex49A is identified as Gly350, which allows IPU to have a relatively wide cleft, thus it seems to b e possible that both a-(1fi4)-linked and a- (1fi6)-linked glucose units enter the active cleft of I PU. However, residues corresponding to Asn417 and Glu449 o f Dex49A are v irtually lack ing in I PU because positions equivalent to Asn417 and Glu449 of Dex49A are ident ified as Val39 4 and Gly426 of IPU, respectively. Therefore, even if the a-( 1fi6)-linked glucose units enter to the active cleft of IPU as shown in Fig. 4 A, it would be impossible for the substrate to be retained in the cleft. In the model of IPU, several aromatic and charged residues, Trp277, Tyr349, and Asp371, are p resent in t he v icinity of G ly350, and c ould b e favourable fo r binding of Glc +2 of the a-(1fi4)-linked glucose units of pullulan (Fig. 4A). IPU is an anomer-inverting enzyme In GH family 49 enzymes, Dex49A has been identified as an anomer-inverting enzyme by using N MR spectroscopy [17]. To compar e the mechanism of h ydrolysis of IPU and o ther GH family 49 dextranases, the a nomer configuration of the hydrolysate of IPU was determined. A polarimetric assay was carried out using panose as the substrate. The anomeric forms of t he hydrolysate are equilibrated immediately by the addition of ammonium hydroxide, and the change in the optical rotation was compared with those of bacterial a-glucosidase (retaining enzyme) and fungal glucoamylase Fig. 3. Overall structure of the model of IPU. N-terminal and b-helical domains are shown in light pink and grey, respectively.Theaminoacid residues who se s ubstitu tions re sulted in the reduction of the activity for both pullulan and panose (red), and substitutions that caused a decrease in the activity for pullulan (blue), are shown. Other mutated residues are indicated in light brown. G lucosyl units in subsites +1 an d +2 (Glc +1 an d +2, respect ively), based on the position of isomaltose in the Dex49A structu re, are shown i n yellow. The figure was generated using RASTOP . 4424 H. Akeboshi et al. (Eur. J. Biochem. 271) Ó FEBS 2004 (inverting enzyme), respectively [43]. The optical rotation decreased with t he addition of ammonium hyd roxide for a-glucosidase, while it increased for glucoamylase and also IPU. The results indicated that IPU is an anomer-inverting enzyme (Fig. 5A). In the case of inverting enzymes, a single displacement mechanism has been proposed [6,17,19–21]. In this mech- anism, two catalytic residues function as a general acid (donating a p roton) and a general base ( activating the nucleophilic water m olecule) in the first step of the reaction. A carbonium ion intermediate subsequently forms, and is further attacked by the water molecule. The study of site-directed mutagenesis, however, indicated that the three Asp residues, A sp353, Asp372, and Asp373, are the potential catalytic r esidues of IPU. T he report of the crystal structure of Dex49A also mentioned that either Asp376 or Asp396, the residues corresponding to Asp353 and Asp373 of IPU, respectively, appears to be properly positioned to act as a base in the hydrolytic reaction [17]. Three Asp residues are also strictly conserved in t he catalytic centre of GH family 28 polygalacturonases and rhamnogalacturon- ases [18–20], another family of inverting enzymes forming the clan GH-N w ith GH family 49 enzymes. van San ten et al. [19] and Shimizu et al. [20] reported that a n Asp Fig. 4. Comparison of the active site structures of the model of IPU (A) and Dex49A (B). Conserved residues between IPU and De x49A are shown in red (mutated res idues i n this study) o r orange. Residu es that are u niqu ely found in IPU and may interact with the substrate (see Results and Discussion), are shown in cyan. Residues that are uniquely found in Dex49A and interact with Glc +1 and +2 are shown in green. Other colour representations a re as in Fig. 3. The figures w ere generated using MOLSCRIPT [40] and RASTER 3 D [41]. Fig. 5. Enzymatic properties of I PU. (A) Optical ro tation during the h ydrolysis of s ubstrates by a-glucosidase (top), g lu coamylase (middle), and IPU (bott om) was observed. Reaction mixtu r es consist of 1.6% maltotetraose in 50 m M acetate buffer (pH 4.5) with a-glucosidase or glucoamylase, and 1.8% panose with IPU in 50 m M acetate buffer (pH 3.5). The mutarotation was achieved by adding 20 lLof15 M ammonium hydroxide at the point indicated by arrows. (B) Hydrolysis of IPU for panose, IMTG and MM were compared. Symbols: Circle, glucose residue; Circle with line, glucose residue of the reducing end; ¾ c , a-1,6-glucosidic linkage; – ; a-1,4-glucosidic linkage; triangle, cleaving site. G rey circles indicate the residue exterior to t he panose motif. A ctivity is defined a s the amount o f product (mmol) r ele ased by 1 mg IPUÆmin )1 . Ó FEBS 2004 Reaction mechanism of isopullulanase (Eur. J. Biochem. 271) 4425 residue of the polygalacturonases (position equivalent to Asp372 of IPU) has been proposed to act as t he acid (proton donor), while there are two candidates f or a general base, t wo Asp r esidues of the polygalacturonases ( positions equivalent to Asp353 and Asp373 of IPU). Although the overall structure is completely different, glucoamylase is known as an inverting enzyme and hydrolyses a-(1fi4)-glucosidic linkages s uch as that in IPU. Glucoamylase and IPU are c lassified into d ifferent GH families o f 1 5 a nd 49, respectively, but three conserved acidic residues are found in each of the GH families, and two of them are consecutively numbered residues. In the glucoamylase from Aspergillus awamori, the corresponding acidic residues are Glu179, Glu180, and Glu400, and Glu179 and Glu400 have been reported t o function as a general a cid a nd g eneral b ase, res pectively [4 6]. S ierks et al. suggested that Glu179 is the general acid catalyst of pK a 5.9, and that the adjacent Glu180 is negatively charged, raising the p K a of the general acid catalyst [23]. I t is likely t hat catalytic residues of IPU adopt a similar catalytic mechan- ism to those of glucoamylase. Enzymatic properties of wild-type IPU The modelling s tudy of the IPU structure i ndicated that the conserved and functionally important residues of both Dex49A and I PU are found in the negatively numbered subsites. The anomeric configuration of products of both Dex49A and IPU are identical, as well. Does the substrate preference of the negatively numbered s ubsites of IPU also resemble that of GH family 49 dextranases even though IPU does not hydrolyse dextran at all? IPU not only h ydrolyses panose and a polymer of panose, pullulan [1,30], but also the oligosaccharides containing the panose structure such as IMTG (Glc-a-(1fi6)-Glc-a-(1fi6)-Glc-a-(1fi4)-Glc) [16], MM (Glc -a-(1fi4)-Glc-a-(1fi6) -Glc-a-(1fi4)-Glc) [1,16], 4 2 -a-isomaltosylisomaltose (Glc-a-(1fi6)-Glc-a-( 1fi4)- Glc-a-(1fi6)-Glc) [16], and 6 3 -a-glucosylmaltotriose (Glc-a-(1fi6)-Glc-a-(1fi4)-Glc-a-(1fi4)-Glc) [1,16]. We measured the activities of IPU for panose, IMTG and MM, because IPU releases only glucose from the reducing end side o f t hese substrates (Fig. 5B). The activities of IPU for 32 m M of IMTG, panose a nd MM were assayed, and 1 mg of the enzyme liberated 18.5, 14.0 and 7.37 lmolÆ min )1 of glucose, respectively. Although IPU w as originally reported as a pullulan-hydrolase [1], MM, part of the structure of pullulan, was the poorest substrate among these three oligosaccharides, while IMTG, whose portion bound to the negatively numbered subsites is composed of the structure of d extran (Glc-a-(1fi6)-Glc-a-(1 fi6)-Glc), was the best substrate. These findings suggest that both IPU and the GH family 49 dextranase have a s imilar catalytic mechanism i n their negatively numbered s ubsites in spite of the difference o f their substrate s pecificities. IPU h as been originally reported as the pullulan-hydro- lase [1], but the principal substrate is still not clear because of the low affinities f or pullulan a nd panose (K m ¼ 5 .7% [4 7] and 160 m M , respectively). What might be the physiological role of this enzyme in A. niger? In several Aspergillus species, A. oryzae [48] and A. nidulans [49], isomaltose and panose are known as effective inducers for amylase synthesis. In the case of A. nidulans, a mylase synthes is is induced at an extremely low concentration ( 3 l M )of isomaltose. Kato et al. reported that t wo a-glucosidases from A. nidulans, AgdA and AgdB, showed strong trans- glycosylation activity to produce isomaltose from maltose, and they are suggested to participate in the maltose- dependent induction of amylase s ynthesis along with other undetected isomaltose-forming enzymes [49,50]. 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Insights into the reaction mechanism of glycosyl hydrolase family 49 Site-directed mutagenesis and substrate preference of isopullulanase Hiromi. polysaccharide, pullulan. Comparison of the active sites of the model of IPU and Dex49A The active site structures of t he model of IPU (Fig. 4A) and Dex49A (Fig. 4B) were compared. The