Báo cáo Y học: Barley a-amylase Met53 situated at the high-affinity subsite )2 belongs to a substrate binding motif in the bfia loop 2 of the catalytic (b/a)8-barrel and is critical for activity and substrate specificity pot

Eur J Biochem 269, 5377–5390 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03185.x Barley a-amylase Met53 situated at the high-affinity subsite )2 belongs to a substrate binding motif in the bfia loop of the catalytic (b/a)8-barrel and is critical for activity and substrate specificity Haruhide Mori*, Kristian Sass Bak-Jensen and Birte Svensson Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, Copenhagen Valby, Denmark Met53 in barley a-amylase (AMY1) is situated at the highaffinity subsite )2 While Met53 is unique to plant a-amylases, the adjacent Tyr52 stacks onto substrate at subsite )1 and is essentially invariant in glycoside hydrolase family 13 These residues belong to a short sequence motif in bfia loop of the catalytic (b/a)8-barrel and site-directed mutagenesis was used to introduce a representative variety of structural changes, Met53Glu/Ala/Ser/Gly/Asp/Tyr/Trp, to investigate the role of Met53 Compared to wild-type, Met53Glu/ Asp AMY1 displayed 117/90% activity towards insoluble Blue Starch, and Met53Ala/Ser/Gly 76/58/38%, but Met53Tyr/Trp only 0.9/0.1%, even though both Asp and Trp occur frequently at this position in family 13 Towards amylose DP17 (degree of polymerization ¼ 17) and 2-chloro-4-nitrophenyl b-D-maltoheptaoside the activity (kcat/Km) of all mutants was reduced to 5.5–0.01 and 1.7– 0.02% of wild-type, respectively Km increased up to 20-fold for these soluble substrates and the attack on glucosidic linkages in 4-nitrophenyl a-D-maltohexaoside (PNPG6) and PNPG5 was determined by action pattern analysis to shift to be closer to the nonreducing end This indicated that side chain replacement at subsite )2 weakened substrate glycon moiety contacts Thus whereas all mutants produced mainly PNPG2 from PNPG6 and similar amounts of PNPG2 and PNPG3 accounting for 85% of the products from PNPG5, wild-type released 4-nitrophenol from PNPG6 and PNPG and PNPG2 in equal amounts from PNPG5 Met53Trp affected the action pattern on PNPG7, which was highly unusual for AMY1 subsite mutants It was also the sole mutant to catalyze substantial transglycosylation – promoted probably by slow substrate hydrolysis – to produce up to maltoundecaose from PNPG6 a-Amylases (a-1,4-D-glucan glucanohydrolase, EC 3.2.1.1) catalyze hydrolysis of internal a-1,4-glucosidic linkages in starch and related oligosaccharides and polysaccharides [1] and belong to glycoside hydrolase family 13 (GH13) [2–5] Family 13 and the closely related families 70 and 77 constitute glycoside hydrolase clan H (GH-H) [5] that currently comprises 28 different enzyme specificities [2–5], e.g a-glucosidase (EC 3.2.1.20), maltotetraose-forming exoamylase (EC 3.2.1.60), cyclomaltodextrinase (EC 3.2.1.54), isoamylase (EC 3.2.1.68), pullulanase (EC 3.2.1.41), oligo- 1,6-glucosidase (EC 3.2.1.10), cyclodextrin glucosyltransferase (EC 2.4.1.19), amylomaltase (EC 2.4.1.25), branching enzyme (EC 2.4.1.18), and amylosucrase (EC 2.4.1.5) Although the a-amylases possess very low sequence similarity, and only four residues are invariant in GH-H, conserved short motifs exist which are critical in substrate binding and catalysis [2–6] These motifs extend from the C-termini of certain b-strands in the catalytic (b/a)8-barrel as seen in numerous crystal structures of enzymes from GH13 and GH77 in the native state and in complex with inhibitors or substrates [6–30] The different enzymes bind substrate in a deep accessible cleft formed by bfia loops of the (b/a)8-fold (domain A) including a longer protrusion (named domain B) between b-strand and a-helix [16–30] In barley a-amylase the C-terminal antiparallel b-sheet domain (domain C) has five b-strands [15], while most GH13 enzymes have 8, 10, or 12 b-strands and GH77 lacks domain C [14], which has not yet been ascribed a role in activity in GH-H The active site cleft in a-amylases encompasses a varying number of consecutive subsites interacting with substrate glucosyl residues Enzymatic subsite mapping was developed in the 1970s to characterize the number of recognized substrate glucosyl residues, the binding affinity of individual subsites, and the position of the bond to be cleaved [31–34] The spatial distribution of binding forces illustrates how particular subsites of high or low affinity along the cleft Correspondence to B Svensson, Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Fax: + 45 33 27 47 08, Tel.: + 45 33 27 53 45, E-mail: bis@crc.dk Abbreviations: AMY1, barley a-amylase 1; AMY2, barley a-amylase 2; Cl-PNPG7, 2-chloro-4-nitrophenyl b-D-maltoheptaoside; DP17, degree of polymerization ¼ 17; GH13, glycoside hydrolase family 13; GH-H, clan H of glycoside hydrolases; PNPG, 4-nitrophenyl a-D-glucoside; PNPG2–PNPG12, 4-nitrophenyl a-D-maltoside through 4-nitrophenyl a-D-maltododecaoside; TAA, Taka-amylase A Enzyme: a-Amylase (a-1,4-D-glucan glucanohydrolase, EC 3.2.1.1) *Present address: Division of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan (Received 12 April 2002, revised 28 June 2002, accepted 14 August 2002) Keywords: glycoside hydrolase family 13; plant a-amylases; site-directed mutagenesis; binding subsite engineering; oligosaccharide hydrolysis Ó FEBS 2002 5378 H Mori et al (Eur J Biochem 269) control productive binding modes of oligosaccharide substrates Crystal structures of different Bacillus a-amylases recently described enzyme complexes in which the entire binding site, as defined by subsite mapping, was occupied by an inhibitory substrate analog [22,23] Even though the validity of subsite mapping is debated, these crystal structures show in detail stacking and hydrogen-bond interactions for glucosyl residues at an array of subsites [16–19,24] Barley a-amylase is by far the best known a-amylase from higher plants with respect to structure [15,16] and mutational analysis of structure/function relationships [35–38] Two isozyme families, AMY1 and AMY2, distinguished as the low and high pI isozymes, are de novo synthesized during germination [39–41] They possess 80% sequence identity and have distinctly different enzymatic and stability properties [39–43] The crystal structure of AMY2 – the predominant isozyme in malt – was solved in the native state and in complex with the inhibitory pseudotetrasaccharide acarbose [15,16] Subsite mapping showed 10 substrate binding subsites in AMY1 and AMY2, )6 through )1 in the direction of the nonreducing end from the catalytic site and +1 through +4 towards the reducing end [44] Molecular modeled structures of AMY2/maltodecaose [45] and AMY2/maltododecaose [46] showed substrate glycon binding subsites )1 through )6 formed by residues from domains A and B with Tyr104 stacking onto the sugar ring at subsite )6 [44–46] and subsites interacting with the leaving or aglycon part of substrates to include sequence motifs at C-terminal extensions of b-strands and as well as residues from the long b7fia7 segment of the (b/a)8-barrel [16,36–38,45,46] Alignment of bfia loop sequences that contributed to subsites )1 and ) in a-amylases and other GH-H members, identified a motif with an invariant Tyr stacking onto inhibitor and substrate at subsite )1 [7,13,14,16–27] This Tyr is succeeded by Trp in, e.g CGTases, Takaamylase A (TAA), and maltogenic amylase [7,8,17, 20,21,23–25], and by Gln in porcine and human pancreatic and B subtilis a-amylases [14,18,19,22,24,27] Glucosyl O6 at subsite )2 in enzyme/oligosaccharide complexes was hydrogen bonded to NE1 of the indole ring of Trp [7,17,20,21,23,25] or to NE2 of the carboxamide group in Gln [18,19,22,24] Asp was also common [9,13], while Phe [10], Met [16], and Ala [47,48] were rarely seen among known structures Sequence alignment moreover identified sporadic occurrence of Leu, Gly, Tyr, and His at the position in question (see Table below) In barley a-amylase the invariant Tyr51 stacked onto the valienamine ring (a sugar mimic) of acarbose bound at subsite )1 [16] Acarbose, however, only covered subsites )1 through +2 in this complex [16], but in a modeled AMY2/ ˚ maltodecaose complex, Met52 SD was 3.4 A from O6 of the glucose ring at subsite )2 [45], reminiscent of the contact between ligand and the equivalent Trp and Gln in other a-amylase structures [7,8,14,17–25,27] It is noticeable that subsite )2 of AMY1 and AMY2 had the highest binding affinity of the 10 subsites [44] suggesting that Met52 (Met53 in AMY1), which is conserved in plant a-amylases, has a role in substrate binding and activity This local region was different in structures of nonplant a-amylases having a longer bfia loop which typically contained aromatic residues that might enhance substrate binding adjacent to subsite )2 In barley a-amylase, a shorter bfia loop is proposed to cause higher accessibility at the level of subsites )3 through )6 [15,16] This study describes a series of Met53AMY1 mutants chosen with wide side chain diversity The C-terminally truncated AMY1D9, lacking residues 406–414, was used as parent enzyme because of its high yield compared to AMY2 by heterologous expression in yeast [49,50] and the absence of C-terminally proteolytically trimmed and O-glycosylated forms obtained in the case of recombinant full length AMY1 [38,51,52] However, the specific glutathionylation of Cys95, typically in 25% of the AMY1 molecules, that reduced activity to about 2% [51,52] was unavoidable and glutathionylated forms were removed by anion-exchange chromatography prior to characterization of mutants [38] The central role in activity of Met53 from a substrate glycon binding motif at the high-affinity subsite )2 was emphasized by the present seven mutants exhibiting 1100, 500, and > 40-fold variation in activity towards insoluble Blue Starch, amylose DP17, and 2-chloro-4-nitrophenyl b-D-maltoheptaoside, respectively MATERIALS AND METHODS Materials Escherichia coli DH5a and JM109 [53] (Life Technologies, Inc., MD, USA) were used for propagation of the expression plasmid derived from pPICZA (Invitrogen, Carlsbad, CA, USA) carrying the ZeocinR marker gene for selection of E coli and Pichia pastoris transformants P pastoris GS115 [54] (Invitrogen) was used for expression of AMY1 cDNA inserted into pPICZA under the control of the AOX1 promoter [55] Standard culture media were used for E coli [56] and P pastoris [50] Construction of expression plasmids, transformation, and screening Derivatives were constructed of the expression plasmid pPICZA harboring inserts encoding AMY1 flanked by EcoRI and KpnI sites For AMY1 wild-type, cDNA was amplified using primers A; 5¢-TTT GAA TTC C ATG GGG AAG AAC GGC AGC-3¢ (pos 87–114, sense orientation), and B; 5¢-TTT GGT ACC TCA GTT CTT CTC CCA GAC GGC GTA-3¢ (pos 1395–1363, antisense orientation), to generate DNA with the EcoRI and KpnI sites (underlined) Primer B contained a new stop codon to encode AMY1D9 (referred to as AMY1 in the rest of this paper) that lacks the C-terminal nonapeptide For sitedirected mutagenesis of AMY1, the mega-primer method [57] was applied using the primers: 5¢-AAC GAA GGT TAC XXX CCT GGT CGG C-3¢ (pos 217–241, sense orientation), where ÔXXXÕ was GCT, GGT, GAT, GAA, TCT, TAC, or TGG encoding Ala, Gly, Asp, Glu, Ser, Tyr, and Trp, respectively, instead of Met53 All PCRs were performed using the high fidelity Pfu DNA polymerase (Stratagene, La Jolla, CA, USA) E coli transformants harboring the constructed plasmids were screened on LB agar plates containing 25 lgỈmL)1 Zeocin and plasmid was propagated and purified from the selected transformants grown overnight by using either the classical method of alkaline-lysis of the cells and polyethylene glycol-NaCl a Cyclodextrinase b Trehalose 6-phosphate hydrolase G4-forming amylase G6-forming amylase G5-forming amylase Branching enzyme Amylopullulanase Neopullulanase CDase a Tre 6-P hydrolase b Oligo-1,6-glucosidase a-Glucosidase Pullulanase Trehalose synthase Isoamylase CGTase c Sucrose phosphorylase Dextran sucrase d Glucosyltransferase d Alternansucrase d Amylosucrase 4-Glucanotransferase Amylomaltase e c Cyclodextrin glycosyltransferase d VS -NEGYMPGRLYDIDA VA -EQGYMPGRLYDLDA VS -TQGYMPGRLYDLDA VA -PQGYMPGRLYDLDA VS -PEGYLPGQLYNLNS VS -PEGYLPGRLYDLDA VVTN PSRPWWERYQPVSYKLCTR KEGNQGDKSMS NWYWLYQPTSYQIGNR LPQTTAYG -DAYHGYWQQDIYSLNE IPDNTAYG -YAYHGYWMKNIYKINE LDTLAGTDN TGYHGYWTRDFKQIEE HS -NHKYDTIDYMEIDP ASGG YSVGYDSYDLFDLGE LSQS -DNGYGPYDLYDLGE TSQA -DVGYGAYDLYDLGE KSE YAYHGYHTYDFYAVDG IHGWVGGGTKGDFPHYAYHGYYTQDWTNLDA FSSWTDGGKS -GGGEGYFWHDFNKNGR ASQN -DVGYGAYDLYDLGE EHNWVSSGDGAP YPWWMRYQPVSYSLDRS EHPFD -RSWGYQGIGYYSATS QSPS -NHRYDTTDYTKIDE RSPS -NHKYDTADYFEVDP LSHS -THKYDTTDYYTIDP LSPQV DNGYDVANYTAIDP ESPND DNGYDISDYCKIMN DSPQQ DMGYDISNYEKVWP LSGS -VHGYDTYDYYTVDP MASPG -SNHGYDVIDHSRIND QNDANDVVPNS-DANQNYWGYMTENYFSPDR FATINYSGVTN TAYHGYWARDFKKTNP GDRGF -APADYTRVDAAFGDW ASSDK -SFLDAIVQNGYAFTDRYDIGY VSSEDG SFLDSIIQNGYAFEDRYDLAM SSGDTNYGGMSFLDSFLNNGYAFTDRYDLGF KCPEG KSDGGYAVSSYRDVNP SSIS -FHGYDVVDFYSFKA PTG YGDSPYQSFSAFAGNP DTG SCSSPYNSISSIALNP PTG FGNSPYLCYSALAINP PPGKR GNEDGSPYSGQDANCGNT PP DEGGSPYAGQDANCGNT Sequence Enzymes from GH70 e Enzymes from GH77 47–63 46–62 68–83 74–89 71–86 68–83 50–72 90–113 69–92 96–119 111–135 202–217 48–66 78–95 78–95 812–830 255–285 84–109 84–101 68–96 195–214 477–494 200–217 196–213 57–75 55–73 63–81 236–253 41–60 257–286 118–144 48–67 711–736 905–931 1104–1134 145–165 47–64 51–69 74–92 51–69 124–146 128–146 Plant Plant Plant Plant Plant Plant Mammal Bacterium Mold Yeast Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Yeast Bacterium Archaea Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Bacterium Plant Plant a-Amylase Barley (AMY1) Barley (AMY2) Rice (2A) Maize Wheat (AMY3) Black gram Hog Bacillus subtilis Aspergillus oryzae Saccharomycopsis fibuligera Bacillus stearothermophilus Bacillus acidopullulyticus Escherichia coli Bacillus amyloliquefaciens Bacillus licheniformis Paenibacillus polymyxa Escherichia coli Pseudomonas saccharophila Bacillus sp (strain 707) Pseudomonas sp Bacillus stearothermophilus Thermoanaerobacter ethanolicus Bacillus stearothermophilus Thermoanaerobacter ethanolicus Escherichia coli Bacillus cereus S cerevisiae (MAL3S) Desulfurococcus mucosus Sulfolobus acidocaldarius Pseudomonas amyloderamosa Bacillus circulans Leuconostoc mesenteroides Leuconostoc mesenteroides Streptococcus mutans Leuconostoc mesenteroides Neisseria polysaccharea Thermotoga maritima Thermus aquaticus Chlamydia trachomatis Synechocystis sp Potato Arabidopsis thaliana Position Source Enzyme P00693 P04063 P27935 Q41770 P08117 P17859 P00690 P00691 P10529 P21567 P19531 P32818 P26612 P00692 P06278 P21543 P25718 P22963 P19571 Q52516 P30538 P38939 P38940 P29964 P28904 P21332 P38158 Q9HHB0 Q53688 P10342 P30920 Q59495 Q48756 P49331 Q9RE05 Q9ZEU2 Q60035 O87172 O84089 P72785 Q06801 Q9LV91 Accession Table Multiple alignment of the partial sequence including the bfia loop2 motif in various members of glycoside hydrolase clan H Amino acid residues corresponding to Met53AMY1 are shown in bold Ó FEBS 2002 Met53 mutants at subsite )2 in barley a-amylase (Eur J Biochem 269) 5379 Ó FEBS 2002 5380 H Mori et al (Eur J Biochem 269) precipitation [56] or a GFX plasmid purification column (Pharmacia, Sweden) The entire sequence was subsequently confirmed (Applied Biosystems 377 DNA Sequencer and Taq DyeDeoxy Terminator Cycle Sequencing kit, PerkinElmer) and the plasmid was used for P pastoris transformation by electroporation [50] upon linearization at the BstXI site Screening was performed for Zeocin transformants on YPDS plates (1% yeast extract, 2% peptone, 2% glucose, M sorbitol, and 2% agar) containing 100 lgỈmL)1 Zeocin followed by transfer to MMH-starch plates containing 1.34% yeast nitrogen base, 0.4 lgỈmL)1 biotin, 0.5% methanol, and 1% soluble starch to visualize secreted active a-amylase by exposure to I2 vapor [51] Production and purification of enzyme variants P pastoris transformants were grown in L BMGY [1% yeast extract, 2% peptone, 0.67% yeast nitrogen base (Difco), 100 mM potassium phosphate buffer, pH 6.0, 1% glycerol, and 0.4 lg · mL)1 biotin] at 30 °C for d in L flasks to reach D600 > 15 The medium was replaced by L BMMY induction medium (as BMGY except for 0.5% methanol replacing glycerol) by pelleting (1500 g, min, room temperature) and resuspension of the cells for continued incubation for 29–40 h during vigorous shaking After centrifugation AMY1 variants were purified from culture supernatants by affinity chromatography on bcyclodextrin-Sepharose [49,50], followed by anion exchange chromatography using a ResourceQ column (6 mL) and the ă AKTAexplorer automated chromatograph (Pharmacia) [38] The sample was applied to the column, equilibrated in 10 mM sodium acetate buffer, mM CaCl2, pH 7.0, and eluted, at a flow rate of mLỈmin)1, by a gradient (0–50%/ 48 mL, 50–100%/24 mL) made from equilibration buffer and 10 mM sodium acetate buffer, mM CaCl2, mM NaCl, pH 5.3 The first eluted protein peak was collected, dialyzed against 10 mM Mes, 25 mM CaCl2, pH 6.8, concentrated (Centriprep YM10 or YM30, Millipore, Bedford, MA, USA), and added 0.02% (w/v) sodium azide All purification steps were carried out at °C Isoelectric focusing (IEF) was performed (PhastGel, pI 4–6.5; Phast-System, Pharmacia, Sweden) and silver-stained for protein according to the manufacturer’s recommendation, or soaked in starch solution followed by I–/I2 to develop a zymogram [51] SDS/PAGE (PhastGel, 10–15%) was performed as described [38] Enzyme concentrations were calculated from amino acid contents of protein (25 lg) hydrolysates (6 M HCl, 24 h, 110 °C) determined using an Alpha Plus amino acid analyzer equipped with OPAdetection system (Pharmacia, Sweden) Electrospray ionization mass spectrometry was done [38,52] using a VG Quattro triple quadropole mass spectrometer (Micromass Ltd, Wythenshawe, Manchester, UK) Enzyme activity assays Insoluble Blue Starch Activity was measured on insoluble Blue Starch (customer preparation, Pharmacia) suspended (6.25 mgỈmL)1) in 20 mM sodium acetate buffer, mM CaCl2, 0.5 mgỈmL)1 BSA, pH 5.5 The reaction was initiated by enzyme addition (around U) to the suspension (4 mL) and stopped after 15 at 37 °C by 0.5 M NaOH (1 mL) After centrifugation (2 min, 12 000 g) supernatants were transferred (300 lL) to a microtiter plate A620 values (Ceres UV900 HDI microplate reader, Biotek Instruments, Inc., UK) in the range 0.8–1.2 were used to calculate activity [36] One unit was defined as the amount of enzyme that during 15 reaction resulted in an increase in A620 of in the supernatant of the stopped reaction mixture Amylose Rates of hydrolysis of amylose DP17 (average degree of polymerization 17, Hayashibara Chemical Laboratories, Okayama, Japan) were determined in 20 mM sodium acetate buffer, mM CaCl2, and 0.05 mgỈmL)1 BSA, pH 5.5 at 37 °C The content of reducing sugar was measured by the copper bicinchoninate procedure on aliquots removed from the mixture during 0–10 of reaction and using maltose as standard [36,58] Samples (300 lL, in triplicates) were transferred to microtiter plates and A540 was measured as above Enzyme concentrations were 20.0–38.5 nM of AMY1 wild-type and Met53Ala/Gly/ Asp/Glu/Ser, and 220 nM of Met53Tyr and 0.82–1.03 lM of Met53Trp kcat and Km were obtained from initial rates at 5–8 substrate concentrations (0.06–9.00 mgỈmL)1) by fitting to the Michaelis–Menten equation 2-Chloro-4-nitrophenyl b-D-maltoheptaoside The initial rate of hydrolysis of Cl-PNPG7 (GranutestÒ 3, Merck, Darmstadt, Germany) at 30 °C was measured as described [36] with 20.0–103 nM wild-type and Met53Ala/Gly/Asp/ Glu/Ser/Tyr AMY1 and kcat and Km were determined as above using five to eight substrate concentrations (0.40– 8.0 mM) Bond cleavage frequencies of 4-nitrophenyl a-D-maltooligosaccharides Individual bond cleavage frequencies were analysed for PNPG7 (Boehringer Mannheim, Germany), PNPG6, and PNPG5 (both Calbiochem, Bad Soden, Germany) in 20 mM sodium acetate buffer pH 5.5, mM CaCl2, at 37 °C Hydrolysis was initiated by addition of enzyme (4.0–5 · 103 nM final concentration) to mM PNPG5-7 and aliquots (18 lL) were added at time intervals to 10% acetic acid (3 lL) to stop the reaction Products and remaining substrate were separated on a Hypersil APS2 column (4 · 250 mm, ThermoQuest, Cheshire, UK) at 30 °C, using a Waters HPLC Model 510 pump for isocratic elution by 75 : 25 (v/v) CH3CN/H2O or elution by a linear gradient from 93 : to 70 : 30 (v/v) CH3CN/H2O in 20 at a flow rate of 1.0 mLỈmin)1 PNPG1-7 and 4-nitrophenol were detected at 313 nm (Shimadzu SPD-10AU UV-VIS detector) and quantified by using standard mixtures The bond cleavage frequencies were calculated for products obtained at 4–17% substrate consumption Transglycosylation In transglycosylation experiments the same conditions as for hydrolysis were applied using 10 mM of PNPG6 Molecular graphics The structures of AMY2/acarbose and TAA/acarbose were obtained from the protein data bank [59], entry codes 1BG9 and 7TAA, respectively The figures were made using the software program INSIGHT II (98.0) (Molecular Simulations Inc., San Diego, CA) Ó FEBS 2002 Met53 mutants at subsite )2 in barley a-amylase (Eur J Biochem 269) 5381 RESULTS Choice of mutants a-Amylases and other GH-H enzymes have a short substrate glycon binding motif in the middle of the typically 35 residues bfia loop in the (b/a)8-barrel (Table 1) In barley AMY1 (for which the structure is not available) and AMY2 this loop (Pro41-Gly65, AMY1 numbering) was unusually short, or only 25 residues [15] Tyr51AMY2 (Tyr52AMY1) was invariant in GH13 (Table 1) and stacked at subsite )1 onto the valienamine ring in AMY2/acarbose (Fig 1A) Superpositioning of AMY2 and TAA guided by the catalytic acids was excellent for Tyr51AMY2 and Tyr82TAA (Fig 1A) and mutation in Saccharomycopsis fibuligera a-amylase (closely related to TAA) confirmed the corresponding Tyr83 to be involved in activity [60] Trp83TAA NE1 formed a hydrogen bond to O6 of glucosyl at subsite )2 [17] and in contrast to Tyr51AMY2 and Tyr82TAA, the geometry differed of the Met52AMY2 (Met53AMY1) and Trp83TAA (Fig 1A) Also the larger TAA loop in TAA appeared to hinder binding of the substrate beyond subsite )3/)4 as illustrated by global views of AMY2 and TAA complexes (Fig 1B) The enzymatically determined subsite maps agreed with different length of the glycon binding region in AMY2 and TAA [44,61] Comparison of structures of AMY2 and other a-amylases (not shown) also gave the impression that AMY2 might accommodate larger parts of the substrate Thus porcine pancreatic a-amylase like TAA had a larger loop segment with the location of Gln63 resembling that of Trp83TAA [18] This holds true also for Trp101 in CGTase [25] Although almost one thousand GH-H sequences were reported [5] Met53 occurred only in plant a-amylases (Table 1), with the exception of a bacterial isoamylase, which had a structural unit formed by bfia segments and [11] but lacked domain B which together with bfia loop created the glycon binding site [3] Subsite )2 had highest affinity of the 10 subsites in AMY1 and AMY2 [44] Subsites )6 and +1 were almost as strong, while )5, )4, +2, and +4 contributed intermediate, and )3 and + very weak binding energy The catalytic subsite )1 had a large negative affinity [44] due to energy spent to distort of the bound glucose residue in catalysis SD of Met52AMY2 (Met53AMY1) in a computed AMY2/malto˚ decaose complex was 3.4 A from O6 of glucose at subsite )2 [45] reminiscent of the enzyme/substrate interaction for the corresponding Trp and Gln (Table 1, Fig 1A) from certain GH13 enzymes [7,8,17–24,27] Met53AMY1 was investigated by exchange with Trp and Tyr because Trp was common [7,8,17,20,21,23,25,] (Table 1), and, although Tyr is only rarely found, a reinforced aromatic character, of the binding crevice was expected to influence recognition and stabilization of enzyme/substrate complexes Asp occurred widely in GH-H members of varying specificity, e.g in bacterial a-amylase, pullulanase, amylopullulanase, neopullulanase, cyclodextrinase, trehalose 6-phosphate hydrolase, different a-glucosidases (including oligo-1,6-glucosidase), trehalose synthase, and 4-glucosyltransferase (Table 1) The Glu exchange combined the acidic character of the Asp with the length and hydrogen bond forming potential of Gln present in mammalian [9,18,24,27] and certain bacterial enzymes [22] Finally, some smaller and less common residues in GH-H were chosen; Ala from amylosucrase [47] and GH70 glucansucrases [48] having no subsite )2; Ser from GH77; and Gly from certain Bacillus a-amylases (Table 1) Gly was included as it increased the polypeptide chain flexibility; biased random mutagenesis of the F286VDAMY1 motif in bfia segment gave functional FVG and FGG variants which, although no reported natural sequences contained Gly, were unusual [36–38, 61,62] These mutants moreover were highly unusual among the already described AMY1 subsite mutants [34–36,59,60] by having improved activity towards Cl-PNPG7 and less than 10% activity for insoluble starch [37] Inspection of Met53AMY1 replacements in AMY2/ acarbose [16] indicated Asp, Glu, Ser, Asn, and Ala, as readily accommodated, whereas Trp53AMY1 and perhaps Tyr53AMY1 might obstruct the binding cleft Production and purification of AMY1 mutants The P pastoris transformants secreted the mutants at 14 (Met53Glu), 22 (Met53Ala), 16 (Met53Ser), 3.9 (Met53Gly), 1.9 (Met53Asp), 6.0 (Met53Tyr), and 20 (Met53Trp) mgỈL)1 as calculated from the activity in the culture supernatants towards insoluble Blue Starch and the specific activity of the purified enzymes All mutants gave as a single band in SDS/PAGE after purification on b-cyclodextrin-Sepharose, but resolved into two components of pI 4.8 and 4.7 in IEF (Fig 2) The form of pH 4.8 eluting first in anion exchange chromatography (see Materials and methods) was used for enzymatic characterization, and the exceptionally abundant Cys95-glutathionylated form constituting 26% (Met53Tyr) to 55% (Met53Trp) was almost inactive [38,52] and therefore discarded Mass spectrometry confirmed the two forms were distiguished by Cys95AMY1 glutathionylation in the lower pI form [38,50–52] The aromatic replacements Met53Trp/Tyr had no visible activity in the zymogram and exchange by Asp (Fig 2) and Glu (not shown) did not significantly decrease the pI Enzyme kinetic properties of AMY1 mutants The activity of Met53 mutants and wild-type AMY1 was compared using three different substrates; insoluble Blue Starch; amylose DP17 that spans the 10 subsites; and ClPNPG7, a maltoheptaoside (Table 2) binding at subsites )6 through +1/+2 in accordance with the subsite map [44] The mutants appeared in three groups based on activity: (a) Met53Glu/Asp were highly active towards insoluble starch and had modest activity for soluble substrates including the three 4-nitrophenyl-malto-oligosaccharides (see below); (b) Met53Ser/Gly/Ala showed intermediate activity for the insoluble and somewhat further reduced activity than mutants in the first category for the soluble substrates; and (c) Met53Trp/Tyr had very low activity on all substrates (Table 2) In the light of Trp being common at this position in microbial a-amylases and cyclodextrin glucosyltransferases, Met53Trp and Met53Tyr compared to wild-type AMY1 were surprisingly poor catalysts showing 0.1 and 0.9% activity, respectively, towards insoluble Blue Starch Moreover, the catalytic efficiency (kcat/Km) of these mutants was reduced 103- to 104-fold for both amylose DP17 and Cl-PNPG7 (Table 2) This was chiefly due a low kcat for 5382 H Mori et al (Eur J Biochem 269) Ó FEBS 2002 4.30 25.0 10.9 41.6 24.2 22.8 16.2 ND 26 3270 1150 4190 3090 3170 1000 ND 11.7 16.3 42.4 14.8 11.6 27.8 16.7 9.5 111 1.04 1.91 0.406 0.356 0.820 0.025 ND 1.1 ± 0.1 ND ND ND 11.1 ± 3.8 17.8 ± 4.2 ND ND 122 ± ND ND ND 3.95 ± 1.36 14.6 ± 2.8 ND ND 477 26.0 20.8 16.9 8.64 18.7 0.405 0.050 0.01 1.8 0.2 0.7 0.4 1.0 2.0 2.6 ± ± ± ± ± ± ± ± Substrate concentration 6.25 mgỈmL)1 Wild type M53E M53A M53S M53G M53D M53Y M53W a 2900 3400 2200 1700 1100 2600 25 Enzyme 248 208 51.9 115 95.0 93.6 1.50 0.316 ± ± ± ± ± ± ± ± 16 34 3.5 7.4 22 0.60 0.21 0.52 8.0 2.5 6.8 11 5.0 3.7 6.3 BS/Cl-PNPG7 Activ./(kcat/Km) (mg)1ỈsỈmM)1) BS/Amyl Activ./kcat (mg)1Ỉs) Km (s)1ỈmM)1) Km (mM) kcat (s)1) kcat/Km (s)1ỈmLỈmg)1) Cl-PNPG7 Km (mgỈmL)1) kcat (s)1) Fig Comparison of the structure of complexes of inhibitory substrate analogues derived from acarbose and barley a-amylase (AMY2 [16]); and Taka-amylase A (TAA [17]) (A) Stereo view of interactions involving segments of bfia loops and (i.e domain B) from AMY2 (in green) and TAA (in black) The superimpositioning was guided by the catalytic acids (D179AMY2, E204AMY2, and D289AMY2 and D206TAA, E230TAA, and D297TAA) The invariant Y51AMY2 and Y82TAA are at subsite )1 as are H92AMY2 and H122TAA; M52AMY2 (M53AMY1) and W83TAA are at subsite )2; T94AMY2 (C95AMY1) at subsite-5 [38,45]; and Y104AMY2 at subsite-6 (B) Stereo view of the global structure of the AMY2 (top) and TAA (bottom) complexes [16,17] The inhibitors are in green and the arrow indicates the nitrogen (in dark blue) that corresponds to the oxygen of the scissile glycosidic bond Loop (AMY2 residues 40–65 and TAA residues 63–97; indicated by arrow) is in dark blue The catalytic acids (see A above) are colored in yellow M52AMY2 (M53AMY1) and W83TAA are in red (indicated by arrow) Y51AMY2 and Y82TAA are in orange Other binding residues (W9AMY2, H92AMY2, T94AMY2, A95AMY2, Y130AMY2, A145AMY2, F180AMY2, K182AMY2, W206AMY2, S208AMY2, Y211AMY2, H288AMY2, Q294AMY2, M296AMY2 and Q35TAA, H122TAA, R204TAA, K209TAA, H210TAA, G234TAA, D340TAA, R344TAA) are in purple Amylose DP17 amylose DP17 of only 0.1 and 0.6% of the wild-type value for Met53Trp and Met53Tyr, respectively, while the Km increased about 10-fold as for other Met53 mutants For Blue starch activity a (mg)1) Fig Isoelectric focusing in the pH range 4.0–6.5 of AMY1 wild-type and mutants (60 ng each) produced in P pastoris and purified on b-cyclodextrin-Sepharose (A) Protein silver staining (B) Activity staining Lane 1: pI marker proteins; lane 2: wild-type AMY1; lanes 3–8: Met53Trp, Met53Tyr, Met53Asp, Met53Gly, Met53Ala, and Met53Ser AMY1 Amyl/Cl-PNPG7 (kcat/Km)/(kcat/Km) (mg)1ỈmLỈmM) Met53 mutants at subsite )2 in barley a-amylase (Eur J Biochem 269) 5383 Table Activity and kinetic parameters of Met53 AMY1 mutants and wild-type towards insoluble Blue Starch, amylose DP17, and Cl-PNPG7 BS, Blue Starch; Amyl, amylose DP17 ND, not determined, (Km too high) Ó FEBS 2002 5384 H Mori et al (Eur J Biochem 269) Cl-PNPG7, however, kcat and Km could not be determined due to low affinity and while the second order rate constant (kcat/Km) of Met53Tyr was 0.025% of wild-type, it could not be estimated for Met53Trp AMY1 as it had low activity (Table 2) The two other groups of Met53 mutants had considerably reduced catalytic efficiency on amylose DP17 and Cl-PNPG7, kcat/Km corresponding to 1.8–5.5% and 0.3–1.7%, respectively, of the wild-type values On amylose DP17 Met53Glu was the most active mutant with a kcat of 84%, while Km increased 15 times compared to wild-type Met53Asp/Ser/Gly had a kcat of 38–45% and 10–20 times increased Km For Met53Ala on amylose DP17 kcat was 21%, while Km increased only five times Due to the limited solubility of Cl-PNPG7 and poor affinity of mutants for this substrate, kinetic parameters were only obtained for Met53Gly/Asp (Table 2) The values suggested that exchange of Met53 highly reduced affinity and activity each by at least an order of magnitude Met53Gly had the highest Km for amylose DP17, and this mutant probably also had highly increased Km for Cl-PNPG7 as suggested by the high Km determined for Met53Asp and the failure to determine kcat and Km for Met53Glu/Ala of kcat/Km superior to Met53Asp AMY1 (Table 2) kcat of Met53Glu/Ala AMY1 was thus assessed to be ‡ 30 s)1 Remarkably, the Met53 mutants, except for Met53Trp/ Tyr, showed good activity towards insoluble Blue Starch of 38–117% compared to wild-type The five most active mutants also gave similar ratios of activity towards insoluble Blue Starch over kcat/Km for amylose DP17 in the range of 100–140, while the ratios were around 60 for Met53Trp/Tyr and for wild-type The noted expansion to fourfold variation of the ratio of the activity towards starch over kcat for amylose (10–43; Table 2) suggested that in certain mutants reduced affinity for insoluble Blue Starch accompanied the low affinity for amylose DP17 This property, however, was not further investigated Compared to values calculated for wild-type AMY1, for all mutants the relative activities insoluble Blue Starch/ Cl-PNPG7 and amylose DP17/Cl-PNPG7 were 40- to 160and 2.5- to 10-fold in favor of starch and amylose DP17 hydrolysis, respectively Clearly Met53 situated at subsite )2 had an extraordinary role in substrate specificity as the various Met53 mutants showed particularly suppressed action on oligosaccharides For the individual mutant enzymes, however, the relative specificity values vary within less than a factor of four and thus indicate that Met53 substitution moderately modulated relative substrate preferences For the five most active mutants, the amylose DP17/Cl-PNPG7 specificity ratio (Table 2) reflected, however, that enzyme–substrate interaction along the entire binding cleft counteracted favorably the severe losses in activity encountered with Cl-PNPG7 that cannot cover the full length of the binding site Malto-oligosaccharide bond cleavage frequencies of AMY1 mutants As Met53 is situated at subsite )2 with the highest subsite affinity in AMY1 [42] its mutation was expected to affect the cleavage propensity of individual bonds in oligosaccharides, as confirmed by quantitative analysis of Ó FEBS 2002 hydrolysis products from PNPG7, PNPG6, and PNPG5 (Table 3) Six Met53 mutants and wild-type AMY1 primarily hydrolyzed the second glucosidic bond in PNPG7 to release PNPG and G6, but Met53Trp also released PNPG2 and G5 to constitute 30% of the products, PNPG and G6 being formed in 50%, and PNPG5 and G2 in 17% of its cleavages (Table 3) Thus even subsites +4/+5 may be occupied in productive Met53Trp–PNPG7 complexes The action patterns of wild-type and the majority of the mutants, however, reflected the importance of the high-affinity subsite )6 [44], where Tyr104AMY1 (Tyr105AMY1) was stacking onto glucosyl at the nonreducing end of PNPG7 and PNPG6 [45] Introduction of Trp at subsite )2 (Fig 1A) partially suppressed productive binding beyond subsite )2 This mutant thus acquired exo-amylase character, which on the other hand was accompanied by severe activity loss Met53Asp/Gly produced small amounts of PNPG2 and Met53Tyr released more 4-nitrophenol (10%) than wildtype or any other M53 mutant (Table 3) Thus while PNPG7 productive complexes covering subsite )6 were highly populated for most mutants, a certain deviation was found especially for Met53Trp This most likely results from adverse effects on both kcat and Km for PNPG7 as indicated by the effect on Cl-PNPG7 (Table 2) for which kinetic parameters were not determined due to the high Km and/or low kcat or for both reasons The rate of product release was 8% of that of wild-type for the most active mutants, Met53Glu/Ala, and 2% for the second most active group, Met53Ser/Gly/Asp, whereas very low values of 0.1% and 0.006% for Met53Tyr and Met53Trp, respectively, presumably stemmed from a dominating loss of rate of catalysis as suggested by the kinetics properties of these mutants on amylose DP17 and Cl-PNPG7 (Table 3) Most remarkably binding of PNPG6 at subsites )6 through +1 to release 4-nitrophenol was favored only by wild-type AMY1 This mode reflected the high affinity in AMY1 at subsite )6 (7.68 kJỈmol)1) compared to +2 (4.94 kJỈmol)1) [44] Thus although the activity of Met53 mutants towards PNPG6 was distributed in the same three categories as for PNPG7 (Table 3), PNPG6 occupied preferably subsites )4 through +3 in the mutants and applied the predominant wild-type binding mode to low (1–19%) extent Moreover PNPG6 showed greater degree of multiple binding for mutants than wild-type leading to products ranging from 4-nitrophenol to PNPG5 Subsite )6 thus seemed unimportant in PNPG6 binding by AMY1 mutated at subsite )2 AMY1 was reported to have four aglycon binding subsites +1 to +4 [44], and as PNPG4 and PNPG5 constituted 11–28% of the products from mutants, PNPG6 interactions also involved areas corresponding to subsites +5 and +6, i.e exterior to the kinetically determined wildtype binding cleft Wild-type AMY1 in contrast released 1% PNPG4 and no PNPG5 (Table 3) Met53Trp differed by releasing as little as 1% 4-nitrophenol from PNPG6, compared to 9–19% formed by the other mutants The two structurally similar mutants Met53Ala and Met53Gly AMY1 showed closely related action patterns, which also resembled that of Met53Asp, while Met53Glu/Ser/Tyr shared a different trend in their action pattern (Table 3) Remarkably, the Met53 mutants hydrolysed PNPG5 and PNPG6 at essentially the same rate, whereas wild-type Met53 mutants at subsite )2 in barley a-amylase (Eur J Biochem 269) 5385 Ó FEBS 2002 Table Action pattern for hydrolysis of PNPG7, PNPG6, and PNPG5 by Met53 AMY1 mutant and wild-type [PNPG5-7] ¼ 1.0 mM AMY1 Cleavage frequency (%) [E] (nM) PNPG7 Wild-type M53E M53A M53S M53G M53D M53Y M53W G – G – G – G – G – G – G – PNP 96 1 93 96 95 94 90 90 10 17 0 31 51 6.7 4.0 67 6.7 67 67 333 5000 PNPG6 Wild-type M53E M53A M53S M53G M53D M53Y M53W G – G – G – G – G – G – PNP 1 12 24 62 13 15 37 19 12 14 12 42 20 12 21 33 18 12 15 42 21 11 19 11 43 17 11 12 34 24 19 18 16 48 17 67 167 167 667 667 667 833 5000 PNPG5 Wild-type M53E M53A M53S M53G M53D M53Y M53Wa G – G – G – G – G – PNP 14 44 41 32 53 15 40 44 16 48 35 16 40 44 16 39 46 15 32 54 11 43 42 11 167 167 167 833 167 167 833 5000 Time (min) 1.5 30 60 10 10 15 45 15 20 10 30 30 120 90 3.0 7.0 16 10 60 60 90 17 Degree of cleavage (%) Relative activityb (%) 8.3 8.4 14.0 8.8 13.0 11.9 3.9 11.0 100 (0.83) 8.4 8.4 2.6 2.3 2.1 0.09 0.006 12.7 12.3 11.4 11.6 16.5 15.1 5.9 10.5 100 (0.095) 5.2 3.6 1.8 0.87 0.79 0.062 0.025 9.6 7.3 11.0 15.3 7.4 8.6 7.4 12.9 100 (0.019) 32.9 21.7 9.7 3.9 4.5 0.52 0.80 a Transglycosylation was apparent by the formation of PNP malto-oligosaccharides longer than PNPG5; b the activity relative to wild-type AMY1 as estimated from the substrate consumption, reaction time, and enzyme concentration The wild-type AMY1 values given in parenthesis are calculated as [product]/[enzyme] per minute corresponding to the entire period of incubation AMY1 hydrolysed PNPG6 about five times faster than PNPG5 The mutant activity, however, still grouped for PNPG5 as for PNPG6 and PNPG7 in three categories, which for the most active mutants Met53Glu/Ala AMY1 in opposition to their behaviour on PNPG6 and PNPG7 approached that of wild-type (Table 3) The Met53 mutants produced PNPG2 and PNPG3 from PNPG5 in roughly equal amounts representing 83–86%, with PNPG constituting 11–16% of the aglycon products, as opposed to only 14% PNPG3, 44% PNPG2, and 41% PNPG produced by wild-type AMY1 As found for PNPG6, Met53Ala/Gly/Asp had essentially the same action pattern Met53Glu/Tyr also resembled each other, whereas Met53Ser was peculiar by the amount of PNPG3 surpassing that of PNPG2 With PNPG5 and PNPG6, all Met53 mutants thus apparently disfavoured substrate glycon binding interactions compared to wild-type AMY1 while this in PNPG7 hydrolysis appeared only for Met53Trp AMY1 Transglycosylation by Met53Ala/Tyr/Trp Retaining glycoside hydrolases are able to catalyze transglycosylation [3] as depicted in the schematics of the reaction mechanism (Fig 3) Under the present assay Fig Schematics of the double displacement mechanism of retaining glycoside hydrolases [3,66] In transglycosylation the covalent intermediate is attacked at C1 by another sugar molecular, HO-R2, which in hydrolysis would be replaced by water R and R3 signify other substrate chain parts Ó FEBS 2002 5386 H Mori et al (Eur J Biochem 269) Table Product distribution from 10 mM PNPG6 by action of selected AMY1 mutants Distribution (%) Enzyme PNPG>6a PNPG5 PNPG4 PNPG3 PNPG2 PNPG PNP [E] (nM) Time (min) Substrate consumption (%) M53A M53Y M53W 15 16 13 17 12 14 12 34 32 35 26 21 13 167 833 5000 17 90 90 10.2 4.4 9.2 a The sum of oligosaccharides longer than PNPG6 Fig Time course of the formation of products from 10 mM PNPG6 catalyzed by AMY1 Met53Ala (A), Met53Trp (B), and Met53Tyr (C) mutants *, include PNPG7-11; j, PNPG5; h, PNPG4; n, PNPG3; m, PNPG2; s, PNPG; d, PNP Enzyme concentrations are given in Table conditions, Met53Trp AMY1 formed 1.8 lM PNPG7 (not included in Table 4) from mM PNPG5 This significant transglycosylation corresponded to 1.5% of the enzyme catalyzed events After another 25 of reaction 2.2 lM PNPG7 had accumulated at a substrate consumption of 14.7%, still corresponding to a frequency of 1.5% No other transglycosylation products were detected in this or any of the reaction mixtures from mM PNPG6 and PNPG7 While only Met53Trp of the seven AMY1 mutants catalyzed transglycosylation with PNPG5, Met53Trp/Tyr/ Ala AMY1 formed longer oligosaccharides from 10 mM PNPG6 (Figs and 5; Table 4) In the case of Met53Trp this amounted to 15% of the total products, compared to £ 3% and £ 1% for Met53Tyr and Met53Ala AMY1, respectively It is noted that action patterns at mM and 10 mM PNPG6 were not completely identical (Tables and 4; Fig 4), for example 5–9% PNPG5 was formed from 10 mM PNPG6 (Table 4) but lacking in mM PNPG6 reaction mixtures (Table 3) The degree of polymerization of the various transglycosylation products from Met53Trp could not be confirmed as proper reference compounds are not available However, from the number of peaks in the HPLC chromatogram (Fig 5B), Met53Trp presumably gave PNPG7-11, while Met53Ala/Tyr gave PNPG8-10 PNPG8 was always the predominant product (note, PNPG7 is a contaminant in the substrate) Thus Met53Tyr/Ala similarly to Met53Trp AMY1 catalyzed transglycosylation, but since Met53Tyr and Met53Ala hydrolysed PNPG6 (1 mM) with roughly 3- and 140-fold higher rates than Met53Trp AMY1, and PNPG7 with 16- and 1400-fold higher rates (Table 3), transglycosylation products would be hydrolysed relatively fast This may in fact be reflected in the rate of accumulation of the different products, Met53Tyr and Met53Ala AMY1 thus both formed higher amounts of PNPG3 and PNPG4 than of 4-nitrophenol (Fig 4), in contrast to the ratio of these products in the action pattern analysis, where transglycosylation was kept at a minimum Fig HPLC profiles of the reaction products from 10 mM PNPG6 catalyzed by AMY1 Met53Ala (A), Met53Trp (B), and Met53Tyr (C) mutants, and substrate before the reaction (D) Enzyme concentrations and reaction times are given in Table The arrows indicate presumed PNPG8-11 (Table 3) The longest transglycosylation product from a single catalytic event was PNPG12, which can only be present in trace amounts (Fig 5) The anticipated dominant product was PNPG10 generated by nucleophilic attack of PNPG6 as acceptor on the enzyme maltotetraoseintermediate (Fig 3), which arose by release of the major product PNPG2 (Fig 4) Although PNPG10, however, appeared in higher amounts than the products in neighbouring peaks in the chromatogram (Fig 5), the shorter PNPG8 predominated Thus significant hydrolysis of the longer products took place Although monitoring of the 4-nitrophenyl chromophor fails to detect both substrate glycon moieties after hydrolysis and – where such products acted as acceptors – the transglycosylation products, underivatized maltodextrins were assumed to arise in trace amounts only Ó FEBS 2002 Met53 mutants at subsite )2 in barley a-amylase (Eur J Biochem 269) 5387 DISCUSSION Role of the Met53 region in AMY1 and GH-H Barley a-amylase Tyr52AMY1 and Met53AMY1 from a sequence motif in bfia loop (Table 1) are involved in substrate binding at subsites )1 and )2 as illustrated in the structure of AMY2 (Fig 1A) The Met is essentially unique to plant a-amylases and has been subjected to mutational analysis, while the invariant tyrosine was not investigated here It was anticipated that local changes by exchange of Met53 would radically influence barley a-amylase activity Thus three selected mutant residues, Trp, Asp, and Ala, which are common in related enzymes caused very different changes of enzymatic properties in AMY1/Trp a drastic loss of activity towards starch and maltodextrin, and Ala and Asp both retained activity on starch but highly decreased activity for amylose DP17 and a maltoheptaoside In comparison Leu, Phe, and Tyr replacement of the corresponding Trp84 in S fibuligera a-amylase resulted in 19–38% activity of wild-type towards an oligosaccharide [64] Even the Trp and Phe mutants of the preceding essentially invariant Tyr83 in this enzyme had and 20% of the wild-type catalytic efficiency (kcat/Km), respectively [60] Interestingly Trp84Leu, but not the conservative mutants, promoted transglycosylation [65] The higher sensitivity to changes in barley compared to fungal a-amylase may stem from the shorter and perhaps less adaptable bfia loop in the plant enzyme and a requirement of structural integrity at a longer glycon binding crevice Noticeably, sequence variation is sparse in eukaryotes at the position corresponding to Met53AMY1 Thus plants have Met and occasionally Leu, animals Gln, yeast and fungi Trp, whereas bacterial a-amylases have Gln, Trp, Gly, Asp, His, or Tyr, and not include the plant type Finally, in non-a-amylase GH-H members Phe, Gly, Asp, Met, Trp, Ala, Gln, and Ser occur (Table 1) In TAA/ acarbose NE1 of Trp83 (corresponds to Met53AMY1) made a hydrogen bond with O6 of the glucose ring at subsite )2 [17], and in porcine [18] and human pancreatic a-amylases [19] NE2 of Gln63 participated in an analogous hydrogen bond, as did Trp101 NE1 in cyclodextrin glycosyltransferase from Bacillus circulans [20,21] Of the non-a-amylase members which not utilize subsite )2, trehalose-6phosphate hydrolase, oligo-1,6-glucosidase, and a-glucosidase have Asp, sucrose phosphorylase has Thr, and different glucansucrases (GH13 and GH70) have Ala Interestingly, neopullulanase that produces panose from pullulan and thus has an O6-substituted glucosyl ring at subsite )2, also has Asp aligned to Met53 (Table 1) However, because Trp, Asp, Gln, Leu, Gly, and His corresponding to Met53AMY1 exist in a-amylases and Phe in the maltotetraose-forming exo-amylase and some of the residues were present in other enzymes which are not possessing subsite )2, the sequence motif and specificity were not unequivocally correlated Enzymatic properties of AMY1 Met53 mutants Using the AMY2/acarbose structure as a starting point, AMY2/maltodecaose interactions at subsites )6 through +4 were described by molecular modeling [45] and a groove formed by domain B and bfia loop constituted subsites )1 through )6 accommodating the substrate glycon moiety In this complex SD of Met52AMY2 (Met53AMY1) formed a hydrogen bond with glucose O6 at subsite )2 [45], the subsite with highest affinity in AMY1 [44] and Tyr51AMY2 and Tyr104AMY2 were stacking onto rings at subsites )1 and )6, respectively The latter contact was proposed to contribute importantly [45] to the high subsite affinity [44] and Tyr104AMY2 is conserved in plant a-amylases Thus the subsite map of kidney bean a-amylase composed of six glycon and two aglycon binding subsites similarly had high affinity at subsite-6 [65] The action pattern changes for the Met53 mutants produced in bfia loop showed that modification at subsite )2 could importantly influence utilization of the outermost subsite-6 Such long-range interactions in the substrate-mutant enzyme complex between subsite )2 and other parts of the binding cleft emphasized the intimate contact in between bfia loop and domain B and its importance in activity [15] The activity towards insoluble Blue Starch of seven Met53AMY1 mutants representing characteristic GH-H side chains varied from being slightly superior (Met53Glu) to less than 0.1% (Met53Trp) of wild-type AMY1 Interestingly, Trp was present in many fungal and bacterial a-amylases and certain other GH13 members The mutagenized position as evident from the crystal structure was expected to play a major role in activity Even with amylose DP17, that filled the entire binding site, all Met53 mutants displayed 5- to 20-fold higher Km accompanied by large variation in kcat ranging from values similar to wild-type to 0.3% of its value Indeed some of these mutants (Met53Asp/Ala) had high activity for insoluble Blue Starch and moderate kcat towards amylose DP17 of approximately 20–40% of the wild-type value It was typical of most other AMY1 mutants [36–38] that the bond cleavage pattern of PNPG7 was identical to or very similar to wild-type with the notable present exception of Met53Trp Thus even though the rates of hydrolysis of the oligosaccharide substrates greatly decreased, the binding mode was most probably controlled by the outermost highaffinity subsite )6 and was retained by the different mutants Moreover, Met53Glu/Asp/Ala had similar activity to wild-type AMY1 for the large substrate insoluble Blue Starch, even though Glu was not found in the nearly one thousand deposited GH-H sequences [5] The increased Km for amylose DP17 together with high activity for insoluble Blue Starch may reflect that substrate binds at (an) as yet unidentified site(s) which is (are) situated far from the site of catalysis and can compensate for hampered substrate contact, caused by mutation at subsite )2, along the 10 subsites long crevice Very low activity of Met53Tyr/Trp mutants seemed mostly due to reduced kcat, as Km for amylose was rather similar for these two and the other mutants This suggested proper transition state stabilization be hampered by introduction of an aromatic side chain in the middle of the AMY1 binding cleft which apparently disturbed crucial steps in the mechanism, perhaps involving contacts between domains A and B This effect on substrate transition state stabilization suggested the presence of active site interactions which would normally control development both of substrate distortion and a negative electrostatic field at the site of catalysis The bulky side-chain in the cleft in Ó FEBS 2002 5388 H Mori et al (Eur J Biochem 269) Met53Trp AMY1 suppressed binding at subsites )3 through )6 as demonstrated in the action pattern analysis Further protein engineering, however, would be needed to convert this endo-acting into an exo-acting a-amylase such as the natural maltotetraose-forming exo-amylase [10] or B stearothermophilus maltogenic a-amylase [23] Met53 was indicated in the modelled AMY2/maltodecaose to contribute to the high affinity of subsite )2 [45,46], perhaps via van der Waals’ interactions as a few plant a-amylases had leucine at this position and a binding role of SD Met53 seemed not adopted in Met53Asp/Glu AMY1 having high Km and low kcat/Km Thus substrate hydrogen bonding, in contrast to the situation seen for the corresponding Trp and Gln in animal, fungal, and bacterial a-amylases, may not play a critical role for this residue from plant enzymes Moreover, a charged residue may be inappropriate at this position in plant a-amylases as Met53Glu in spite of an activity superior to wild-type for insoluble Blue Starch, lost activity for amylose DP17 and oligosaccharides Met53Trp AMY1 promoted transglycosylation even in mM PNPG5, i.e at subsaturating substrate concentration (Km was estimated to be > 10 mM, D Tull and B Svensson, unpublished), while hydrolytic activity of Met53Trp compared to wild-type AMY1 and most other Met53 mutants was poor Met53Trp thus produced PNPG7 from PNPG5 consistent with the major productive binding mode of this substrate-mutant combination leading to maltose and PNPG3, the maltosyl unit covalently linked to the nucleophile Asp180 then acting as donor attacked at C1 by the acceptor PNPG5 Although the six other mutants had essentially the same binding mode preference, transglycosylation from PNPG5 was not demonstrated, probably due to a different balance between transglycosylation and hydrolysis rates From 10 mM PNPG6, however, both Met53Tyr and Met53Ala AMY1 catalyzed transglycosylation to significant albeit small extent The earlier unique transglycosylation by the corresponding Trp84Leu S fibuligera a-amylase was explained by the longer retention at the active site of the substrate glycon part after cleavage [64] The Trp84Leu thus enhanced the transglycosylation/hydrolysis ratio of that enzyme This explanation may also apply to the AMY1 mutant, although the shape of the binding cleft of S fibuligera a-amylase is very similar to that of TAA [60] and thus different from AMY1 (Fig 1A,B) target for engineering transglycosylation ability For other Met53 mutants low activity towards oligosaccharides suggested a similar effect, which however, was overcome to considerable degree by longer substrates probably through numerous interactions along the binding cleft, and – for polysaccharides – presumably also at sites elsewhere on the enzyme surface One possible candidate is the so-called starch granule binding surface site that includes Trp278 and Trp279 in AMY1 [16,67] Mutational analysis of the side chain adjacent to the invariant Tyr of GH-H stacking onto substrate at the catalytic subsite )1 as shown for barley AMY1 provided an excellent instrument for modification of enzymatic properties High activity was thus maintained (e.g in Met53Glu) on starch although substrate affinity for the model amylose DP17 and oligosaccharides decreased dramatically and the rate of oligosaccharide hydrolysis was very much reduced Remarkably, side chains found in other naturally occurring and thoroughly examined enzymes seemed not suitable in the plant enzymes, as they did not appear in any of these sequences This selective adverse effect may stem from variation seen at the sequence level and hence in the structures for the second and third (domain B) bfia connecting segments that create the glycon binding region Although comprehensive sequence/specificity correlation was not demonstrated for the short motif in bfia loop 2, noticeably introduction of Asp, which is rare in a-amylases but common in other GH-H members, maintained starch hydrolysis at 90% of the wild-type AMY1 activity, whereas introduction of Gly, another rare residue in a-amylases, gave only 35% It is proposed that combination of the present mutations and mutations at other subsites can accentuate the suppression of activity for shorter substrates and further develop the enzyme specificity as done recently for dual subsite mutants in AMY1 [37,38,61,62] To this end the present mutants may also be put through a directed evolution programme ACKNOWLEDGEMENTS The authors are grateful to C Vincentsen for expert technical assistance, L H Sørensen and the late B Corneliussen for amino acid analysis, and M.-B Rask and the late J Sage for DNA sequencing This work was supported by the EU 4th Framework Programme on Biotechnology (CT98-0022) to the project AGADE REFERENCES CONCLUSION In AMY1, Met53 was required for wild-type kinetic properties especially for affinity and in action on maltooligosaccharides and maltodextrins Indeed substitution of Met53 enabled modulation of activity and kinetic parameters for maltodextrins Introduction of a bulky aromatic group misguided the substrate glycon part to loose interaction with the high-affinity subsite )6 Furthermore, inadequate binding at subsite )2 caused by a bulky residue presumably influenced the typical distortion of the substrate ring at subsite )1 in GH-H catalyzed reactions [66], resulting in very low rate of catalysis, this mutation also promoted transglycosylation This, together with transglycosylation by a corresponding S fibuligera a-amylase mutant [60,64], identifies the position as an interesting Yamamoto, T (1995) Enzyme Chemistry and Molecular Biology of Amylases and Related Enzymes CRC Press, Inc., Boca Raton, FL Svensson, B (1994) Protein engineering in the a-amylase family: catalytic mechanism, substrate specificity, and stability Plant Mol Biol 25, 141–157 MacGregor, E.A., Janecek, S & Svensson, B (2001) Relationship of sequence and structure to specificity in the a-amylase family Biochim Biophys Acta 1546, 1–20 Henrissat, B & Bairoch, A (1996) Updating the sequence-based classification of glycosyl hydrolases Biochem J 316, 695–696 Coutinho, P.M & Henrissat, B (1999) Carbohydrate-active enzymes server at http://afmb.cnrs-mrs.fr/CAZY/index.html Janecek, S (2000) Structural features and evolutionary relationships in the alpha-amylase family In Glycoenzymes (Ohnishi, M., Hayashi, T., Ishima, S & Kuriki, T eds), pp 19–54 The Japanese Scientific Societies Press, Tokyo Ó FEBS 2002 Met53 mutants at subsite )2 in barley a-amylase (Eur J Biochem 269) 5389 Klein, C., Hollender, J., Bender, H & Schulz, G.E (1992) Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis Biochemistry 31, 8740–8746 Knegtel, R.M.A., Wind, R.D., Rozebom, H.J., Kalk, K.H., Buitelaar, R.M., Dijkhuizen, L & Dijkstra, B.W (1996) Crystal ˚ structure at 2.3 A resolution and revised nucleotide sequence of the thermostable cyclodextrin glycosyltransferase from Thermonanaerobacterium thermosulfurigenes EM1 J Mol Biol 256, 611– 622 Watanabe, K., Hata, Y., Kizaki, H., Katsube, Y & Suzuki, Y (1997) The refined crystal structure of Bacillus cereus oligo-1,6˚ glucosidase at 2.0 A resolution: structural characterization of proline-substitution sites for protein thermostabilization J Mol Biol 269, 142–153 10 Morishita, Y., Hasegawa, K., Matsuura, Y., Katsube, Y., Kubota, Y & Sakai, S (1997) Crystal structure of a maltotetraose-forming exo-amylase from Pseudomonas stutzeri J Mol Biol 267, 661–672 11 Katsuya, Y., Mezaki, Y., Kubota, M & Matsuura, Y (1998) ˚ Three-dimensional structure of Pseudomonas isoamylase at 2.2 A resolution J Mol Biol 281, 885–897 12 Kamitori, S., Kondo, S., Okuyama, K., Yokota, T., Shimura, Y., Tonuzuka, T & Sakano, Y (1999) Crystal structure of Thermoactinomyces vulgaris R-47 a-amylase II (TVAII) hydrolyzing ˚ cyclodextrins and pullulan at 2.6 A resolution J Mol Biol 287, 907–921 13 Kim, J.S., Cha, S.S., Kim, H.J., Kim, T.J., Ha, N.C., Oh, S.T., Cho, H.S., Cho, M.J., Kim, M.J., Lee, H.S., Kim, J.W., Choi, K.Y., Park, K.H & Oh, B.H (1999) Crystal structure of a maltogenic amylase provides insights into a catalytic versatility J Biol Chem 274, 26279–26286 14 Przylas, I., Tomoo, K., Terada, Y., Takaha, T., Fujii, K., Saenger, W & Strater, N (2000) Crystal structure of amylomaltase from ă Thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans J Mol Biol 296, 873–886 15 Kadziola, A., Abe, J., Svensson, B & Haser, R (1994) Crystal and molecular structure of barley a-amylase J Mol Biol 239, 104– 121 16 Kadziola, A., Søgaard, M., Svensson, B & Haser, R (1998) Molecular structure of a barley a-amylase-inhibitor complex: implications for starch binding and catalysis J Mol Biol 278, 205–217 17 Brzozowski, A.M & Davies, G.J (1997) Structure of the Aspergillus oryzae a-amylase complexed with the inhibitor acarbose at ˚ 2.0 A resolution Biochemistry 36, 10837–10845 ´ 18 Qian, M., Haser, R., Buisson, G., Duee, E & Payan, F (1994) The active center of a mammalian a-amylase Structure of the complex of a pancreatic a-amylase with a carbohydrate ˚ inhibitor refined to 2.2-A resolution Biochemistry 33, 6284– 6294 19 Nahoum, V., Roux, G., Anton, V., Rouge, P., Puigserver, A., Bischoff, H., Henrissat, B & Payan, F (2000) Crystal structures of human pancreatic a-amylase in complex with carbohydrate and proteinaceous inhibitors Biochem J 346, 201–208 20 Parsiegla, G., Schmidt, A.K & Schulz, G.E (1998) Substrate binding to a cyclodextrin glycosyltransferase and mutations increasing the c-cyclodextrin production Eur J Biochem 255, 710–717 21 Schmidt, A.K., Cottaz, S., Driguez, H & Schulz, G.E (1998) Structure of cyclodextrin glycosyltransferase complexed with a derivative of its main product b-cyclodextrin Biochemistry 37, 5909–5915 22 Fujimoto, Z., Takase, K., Doui, N., Momma, M., Matsumoto, T & Mizuno, H (1998) Crystal structure of a catalytic-site mutant a-amylase from Bacillus subtilis complexed with maltopentaose J Mol Biol 277, 393–407 23 Dauter, Z., Dauter, M., Brzozowski, A.M., Christensen, S., Borchert, T.V., Beier, L., Wilson, K.S & Davies, G.J (1999) X-ray structure of Novamyl, the five domain ÔmaltogenicÕ a-amylase from Bacillus stearothermophilus: maltose and acarbose ˚ complexes at 1.7 A resolution Biochemistry 38, 8385–8392 24 Brayer, G.D., Sidhu, G., Maurus, R., Rydberg, E.H., Braun, C., Wang, Y., Nguyen, N.T., Overall, C.M & Withers, S.G (2000) Subsite mapping of the human pancreatic a-amylase active site through structural, kinetic, and mutagenesis techniques Biochemistry 39, 4778–4791 25 Uitdehaag, J.C.M., van Alebeek, G.-J.W.M., van der Veen, B.A., Dijkhuizen, L & Dijkstra, B.W (2000) Structures of maltohexaose and maltoheptaose bound at the donor sites of cyclodextrin glycosyltransferase give insight into the mechanisms of transglycosylation activity and the cyclodextrin size specificity Biochemistry 39, 7772–7780 26 Yoshioka, Y., Hasegawa, K., Matsuura, Y., Katsube, Y & Kubota, M (1997) Crystal structures of a mutant maltotetraoseforming exo-amylase cocrystallized with maltopentaose J Mol Biol 271, 619–628 ´ 27 Machius, M., Vertesy, L., Huber, R & Wiegand, G (1996) Carbohydrate and protein-based inhibitors of porcine pancreatic a-amylase: structure analysis and comparison of their binding characteristics J Mol Biol 260, 409–421 28 Ramasubbu, N., Paloth, V., Luo, Y., Brayer, G.D & Levine, M.J ˚ (1996) Structure of human salivary a-amylase at 1.6 A resolution: implications for its role in the oral cavity Acta Crystallogr D52, 435–446 29 Aghajari, N., Feller, G., Gerday, C & Haser, R (1998) Crystal structures of the psychrophilic a-amylase from Altermonas haloplanctic in its native form and complexed with an inhibitor Prot Sci 7, 564–572 30 Brzozowski, A.M., Lawson, D.M., Turkenburg, J.P., BisgaardFrantzen, H., Svendsen, A., Borchert, T.V., Dauter, Z., Wilson, K.S & Davies, G.J (2000) Structural analysis of a chimeric bacterial a-amylase High-resolution analysis of native and ligand complexes Biochemistry 39, 9099–9107 31 Robyt, J.F & French, D (1970) The action pattern of porcine pancreatic a-amylase in relationship to the substrate binding site of the enzyme J Biol Chem 245, 454–465 32 Hiromi, K (1970) Interpretation of dependency of rate parameters on the degree of polymerization of substrate in enzyme-catalyzed reactions Evaluation of subsite affinities of exo-enzyme Biochem Biophys Res Commun 40, 1–6 33 Thoma, J.A., Brothers, C & Spradlin, J (1970) Subsite mapping of enzymes Studies on Bacillus subtilis amylase Biochemistry 9, 1768–1775 34 Suganuma, T., Matsuno, R., Ohnishi, M & Hiromi, K (1978) A study of the mechanism of action of Taka-amylase A1 on linear oligosaccharides by product analysis and computer simulation J Biochem (Tokyo) 84, 293–316 35 Søgaard, M., Kadziola, A., Haser, R & Svensson, B (1993) Sitedirected mutagenesis of histidine 93, aspartic acid 180, glutamic acid 205, histidine 290, and aspartic acid 291 at the active site and tryptophan 279 at the raw starch binding site in barley a-amylase J Biol Chem 268, 22480–22484 36 Matsui, I & Svensson, B (1997) Improved activity and modulated action pattern obtained by random mutagenesis at the fourth b-a loop involved in substrate binding to the catalytic (b/a)8-barrel domain of barley a-amylase J Biol Chem 272, 22456–22463 37 Gottschalk, T.E., Tull, D., Aghajari, N., Haser, R & Svensson, B (2001) Specificity modulation of barley a-amylase through biased random mutagenesis involving a conserved tripeptide in bfia loop of the catalytic (b/a)8-barrel domain Biochemistry 40, 12844–12854 38 Mori, H., Bak-Jensen, K.S., Gottschalk, T.E., Motawia, M.S., Damager, I., Møller, B.L & Svensson, B (2001) Modulation of 5390 H Mori et al (Eur J Biochem 269) 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 activity and substrate binding modes by single and double subsites +1/+2 and )5/)6 mutation of barley a-amylase Eur J Biochem 268, 6545–6558 Svensson, B., Mundy, J., Gibson, R.M & Svendsen, I (1985) Partial amino acid sequences of a-amylase isozymes from barley malt Carlsberg Res Commun 50, 15–22 Jones, R.L & Jacobsen, J.V (1991) Regulation of synthesis and transport of secreted proteins in cereal aleurone Int Rev Cytol 126, 49–88 Bertoft, E., Andtfolk, C & Kulp, S.-E (1984) Effect of pH, temperature, and calcium ions on barley malt a-amylase isoenzymes J Inst Brew 90, 298–302 Rogers, J.C & Milliman, C (1983) Isolation and sequence analysis of a barley a-amylase cDNA clone J Biol Chem 258, 8169– 8174 Rogers, J.C (1985) Two barley a-amylase gene families are regulated differently in aleurone cells J Biol Chem 260, 3731– 3738 Ajandouz, E.H., Abe, J., Svensson, B & Marchis-Mouren, G (1992) Barley malt a-amylase Purification, action pattern and subsite mapping of isozyme and two members of the isozyme subfamily using p-nitrophenylated maltooligosaccharide substrates Biochim Biophys Acta 1159, 193–202 ´ ´ Andre, G., Buleon, A., Haser, R & Tran, V (1999) Amylose chain behavior in an interacting context III Complete occupancy of the AMY2 barley a-amylase cleft and comparison with biochemical data Biopolymers 50, 751–762 ´ Andre, G & Tran, V (1999) Molecular modelling of complexes between a-amylases and amylose fragments of high DP In: Recent Advances in Carbohydrate Bioengineering (H.J Gilbert, G.J Davies, B Henrissat, B Svensson, eds.) Royal Soc Chem pp 165–175 Skov, L.K., Mirza, O., Henriksen, A., Potocki de Montalk, G., Remaud-Simeon, M., Sarcabal, P., Willemot, R.-M., Monsan, P & ¸ Gajhede, M (2001) Amylosucrase, a glucan-synthesizing enzyme from the a-amylase family J Biol Chem 276, 25273–25278 Monchois, V., Willemot, R.-M & Monsan, P (1999) Glucansucrases: Structure-function relationships FEMS Microbiol Rev 23, 131–151 Søgaard, M & Svensson, B (1990) Expression of cDNAs encoding barley a-amylase and in yeast andcharacterization of the secreted proteins Gene 94, 173–179 Juge, N., Andersen, J.S., Tull, D., Roepstorff, P & Svensson, B (1996) Overexpression, purification, and characterization of recombinant barley a-amylase and secreted by the methylotrophic yeast Pichia pastoris Protein Expr Purif 8, 204–214 Søgaard, M., Olsen, F.L & Svensson, B (1991) C-terminal processing of barley a-amylase in malt, aleurone protoplasts, and yeast Proc Natl Acad Sci USA 88, 8140–8144 Søgaard, M., Andersen, J.S., Roepstorff, P & Svensson, B (1993) Electrospray mass spectrometry characterization of post-translation almodifications of barley a-amylase produced in yeast Bio/ Technology 11, 1162–1165 Yanisch-Perron, C., Vieira, J & Messing, J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors Gene 33, 103–119 Ó FEBS 2002 54 Cregg, J.M., Barringer, K.J., Hessler, A.Y & Madden, K.R (1985) Pichia pastoris as a host system for transformations Mol Cell Biol 5, 3376–3385 55 Ellis, S.B., Brust, P.F., Koutz, P.J., Waters, A.F., Harpold, M.M & Gingeras, T.R (1985) Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris Mol Cell Biol 5, 1111–1121 56 Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular Cloning: a Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 57 Datta, A.K (1995) Efficient amplification using ÔmegaprimerÕ by asymmetric polymerase chain reaction Nucl Acids Res 23, 4530– 4531 58 McFeeters, R.F (1980) A manual method for reducing sugar determinations with 2,2¢-bicinchoninate reagent Anal Biochem 103, 302–306 59 Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N., Weissig, H., Shindyalov, I.N & Bourne, P.E (2000) The Protein Data Bank Nucl Acids Res 28, 235–242 60 Matsui, I., Yoneda, S., Ishikawa, K., Miyairi, S., Fukui, S.B., Umeyama, H & Honda, K (1994) Roles of the aromatic residues conserved in the active center of Saccharomycopsis a-amylase for transglycosylation and hydrolysis activity Biochemistry 33, 451– 458 61 Nitta, Y., Mizushima, M., Hiromi, K & Ono, S (1971) Influence of molecular structures of substrate analogues I effect of chain length of linear substrates J Biochem (Tokyo) 69, 567–576 62 Svensson, B., Sauer, J., Mori, H., Jensen, M.T., Bak-Jensen, K.B., Kramhøft, B., Juge, N., Nøhr, J., Greffe, L., Frandsen, T.P., Palcic, M.M., Williamson, G & Driguez, H (2002) Gluco (Amylases), What Have We Learned So Far? In Proceedings of the 4th Carbohydrate Bioengineering Meeting (Teeri, T.T., Svensson, B., Gilbert, H., & Feizi, T., eds) pp 67–75 Royal Society of Chemistry, Cambridge, UK 63 Svensson, B., Jensen, M.T., Mori, H., Bak-Jensen, K.S., Bønsager, B., Nielsen, P.K., Kramhøft, B., Prætorius-Ibba, M., Nøhr, J., Juge, N., Greffe, L., Williamson, G & Driguez, H (2002) Fascinating facets of function and structure of amylolytic enzymes of glycoside hydrolase family 13 Biologia in press 64 Matsui, I., Ishikawa, K., Miyairi, S., Fukui, S & Honda, K (1991) An increase in the transglycosylation activity of Saccharomycopsis a-amylase altered by site-directed mutagenesis Biochim Biophys Acta 1077, 416–419 65 Mori, H., Tatematsu, A., Saito, A., Matsui, H., Kimura, A & Chiba, S (1995) Substrate specificitiy and subsite affinities of a-amylase form germinating cotyledons of Phaseolus vulgaris L cv Toramame Oyo Toshitsu Kagaku (J Glycosci.) 42, 387–394 66 Uitdehaag, J.C.M., Mosi, R., Kalk, K.H., van der Veen, B.A., Dijkhuizen, L., Withers, S.G & Dijkstra, B.W (1999) X-ray structures along the reaction pathway of cyclodextrin glycosyltransferase elucidate catalysis in the a-amylase family Nature Struct Biol 6, 432–436 67 Gibson, R.M & Svensson, B (1987) Identification of tryptophanyl residues involved in binding of carbohydrate ligands to barley a-amylase Carlsberg Res Commun 52, 373–379  ... D289AMY2 and D206TAA, E230TAA, and D297TAA) The invariant Y5 1AMY2 and Y8 2TAA are at subsite )1 as are H92AMY2 and H 122 TAA; M52AMY2 (M53AMY1) and W83TAA are at subsite )2; T94AMY2 (C95AMY1) at subsite- 5... in yellow M52AMY2 (M53AMY1) and W83TAA are in red (indicated by arrow) Y5 1AMY2 and Y8 2TAA are in orange Other binding residues (W9AMY2, H92AMY2, T94AMY2, A9 5AMY2, Y1 30AMY2, A1 45AMY2, F180AMY2,... K182AMY2, W206AMY2, S208AMY2, Y2 11AMY2, H288AMY2, Q294AMY2, M296AMY2 and Q35TAA, H 122 TAA, R204TAA, K209TAA, H210TAA, G234TAA, D340TAA, R344TAA) are in purple Amylose DP17 amylose DP17 of only