Rational design of cyclodextrin glycosyltransferase from Bacillus circulans strain 251 to increase alpha-cyclodextrin production

Cyclodextrin glycosyltransferases (CGTase) (EC 2.4.1.19) are extracellular bacterial enzymes that generate cyclodextrins from starch. All known CGTases produce mixtures of alpha, beta, and gamma-cyclodextrins. A maltononaose inhibitor bound to the active site of the CGTase from Bacillus circulans strain 251 revealed sugar binding subsites, distant from the catalytic residues, which have been proposed to be involved in the cyclodextrin size specificity of these enzymes. To probe the importance of these distant substrate binding subsites for the alpha, beta, and gamma-cyclodextrin product ratios of the various CGTases, we have constructed three single and one double mutant, Y89G, Y89D, S146P and Y89D/S146P, using site-directed mutagenesis. The mutations affected the cyclization, coupling; disproportionation and hydrolyzing reactions of the enzyme. The double mutant Y89D/S146P showed a twofold increase in the production of alpha-cyclodextrin from starch. This mutant protein was crystallized and its X-ray structure, in a complex with a maltohexaose inhibitor, was determined at 2.4 A resolution. The bound maltohexaose molecule displayed a binding different from the maltononaose inhibitor, allowing rationalization of the observed change in product specificity. Hydrogen bonds (S146) and hydrophobic contacts (Y89) appear to contribute strongly to the size of cyclodextrin products formed and thus to CGTase product specificity. Changes in sugar binding subsites -3 and -7 thus result in mutant proteins with changed cyclodextrin production specificity.     

Conversion of cyclodextrin glycosyltransferase into a starch hydrolase by directed evolution: the role of alanine 230 in acceptor subsite +1

Cyclodextrin glycosyltransferase (CGTase) preferably catalyzes transglycosylation reactions, whereas many other alpha-amylase family enzymes are hydrolases. Despite the availability of three-dimensional structures of several transglycosylases and hydrolases of this family, the factors that determine the hydrolysis and transglycosylation specificity are far from understood. To identify the amino acid residues that are critical for the transglycosylation reaction specificity, we carried out error-prone PCR mutagenesis and screened for Bacillus circulans strain 251 CGTase mutants with increased hydrolytic activity. After three rounds of mutagenesis the hydrolytic activity had increased 90-fold, reaching the highest hydrolytic activity ever reported for a CGTase. The single mutation with the largest effect (A230V) occurred in a residue not studied before. The structure of this A230V mutant suggests that the larger valine side chain hinders substrate binding at acceptor subsite +1, although not to the extent that catalysis is impossible. The much higher hydrolytic than transglycosylation activity of this mutant indicates that the use of sugar acceptors is hindered especially. This observation is in favor of a proposed induced-fit mechanism, in which sugar acceptor binding at acceptor subsite +1 activates the enzyme in transglycosylation [Uitdehaag et al. (2000) Biochemistry 39, 7772-7780]. As the A230V mutation introduces steric hindrance at subsite +1, this mutation is expected to negatively affect the use of sugar acceptors. Thus, the characteristics of mutant A230V strongly support the existence of the proposed induced-fit mechanism in which sugar acceptor binding activates CGTase in a transglycosylation reaction.     

Effects of essential carbohydrate/aromatic stacking interaction with Tyr100 and Phe259 on substrate binding of cyclodextrin glycosyltransferase from alkalophilic Bacillus sp. 1011

The stacking interaction between a tyrosine residue and the sugar ring at the catalytic subsite -1 is strictly conserved in the glycoside hydrolase family 13 enzymes. Replacing Tyr100 with leucine in cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. 1011 to prevent stacking significantly decreased all CGTase activities. The adjacent stacking interaction with both Phe183 and Phe259 onto the sugar ring at subsite +2 is essentially conserved among CGTases. F183L/F259L mutant CGTase affects donor substrate binding and/or acceptor binding during transglycosylation [Nakamura et al. (1994) Biochemistry 33, 9929-9936]. To elucidate the precise role of carbohydrate/aromatic stacking interaction at subsites -1 and +2 on the substrate binding of CGTases, we analyzed the X-ray structures of wild-type (2.0 A resolution), and Y100L (2.2 A resolution) and F183L/F259L mutant (1.9 A resolution) CGTases complexed with the inhibitor, acarbose. The refined structures revealed that acarbose molecules bound to the Y100L mutant moved from the active center toward the side chain of Tyr195, and the hydrogen bonding and hydrophobic interaction between acarbose and subsites significantly diminished. The position of pseudo-tetrasaccharide binding in the F183L/F259L mutant was closer to the non-reducing end, and the torsion angles of glycosidic linkages at subsites -1 to +1 on molecule 1 and subsites -2 to -1 on molecule 2 significantly changed compared with that of each molecule of wild-type-acarbose complex to adopt the structural change of subsite +2. These structural and biochemical data suggest that substrate binding in the active site of CGTase is critically affected by the carbohydrate/aromatic stacking interaction with Tyr100 at the catalytic subsite -1 and that this effect is likely a result of cooperation between Tyr100 and Phe259 through stacking interaction with substrate at subsite +2.     

Engineering cyclodextrin glycosyltransferase into a starch hydrolase with a high exo-specificity

Cyclodextrin glycosyltransferase (CGTase) enzymes from various bacteria catalyze the formation of cyclodextrins from starch. The Bacillus stearothermophilus maltogenic alpha-amylase (G2-amylase is structurally very similar to CGTases, but converts starch into maltose. Comparison of the three-dimensional structures revealed two large differences in the substrate binding clefts. (i) The loop forming acceptor subsite +3 had a different conformation, providing the G2-amylase with more space at acceptor subsite +3, and (ii) the G2-amylase contained a five-residue amino acid insertion that hampers substrate binding at the donor subsites -3/-4 (Biochemistry, 38 (1999) 8385). In an attempt to change CGTase into an enzyme with the reaction and product specificity of the G2-amylase, which is used in the bakery industry, these differences were introduced into Thermoanerobacterium thermosulfurigenes CGTase. The loop forming acceptor subsite +3 was exchanged, which strongly reduced the cyclization activity, however, the product specificity was hardly altered. The five-residue insertion at the donor subsites drastically decreased the cyclization activity of CGTase to the extent that hydrolysis had become the main activity of enzyme. Moreover, this mutant produces linear products of variable sizes with a preference for maltose and had a strongly increased exo-specificity. Thus, CGTase can be changed into a starch hydrolase with a high exo-specificity by hampering substrate binding at the remote donor substrate binding subsites.     

Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.

Cyclodextrin glycosyltransferase (CGTase) uses an alpha-retaining double displacement mechanism to catalyze three distinct transglycosylation reactions. To investigate these reactions as catalyzed by the CGTase from Thermoanaerobacterium thermosulfurigenes the enzyme was overproduced (8 mg.L(-1) culture) using Bacillus subtilis as a host. Detailed analysis revealed that the three reactions proceed via different kinetic mechanisms. The cyclization reaction (cyclodextrin formation from starch) is a one-substrate reaction, whereas the other two transglycosylation reactions are two-substrate reactions, which obey substituted enzyme mechanism kinetics (disproportionation reaction) or ternary complex mechanism kinetics (coupling reaction). Analysis of the effects of acarbose and cyclodextrins on the disproportionation reaction revealed that cyclodextrins are competitive inhibitors, whereas acarbose is a mixed type of inhibitor. Our results show that one molecule of acarbose binds either in the active site of the free enzyme, or at a secondary site of the enzyme-substrate complex. The mixed inhibition thus indicates the existence of a secondary sugar binding site near the active site of T. thermosulfurigenes CGTase.     

The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism. Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis-Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism. In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.     

环糊精葡萄糖基转移酶的结构特征与催化机理

随着环糊精在食品、医药等领域的应用越来越广,生产环糊精所必需的环糊精葡萄糖基转移酶(CGT酶)已经成为当今研究的热点。特别是近二十年来,国外对该酶进行了比较深入的研究。首先介绍了CGT酶的功能特性与结构特征。CGT酶是一种多功能型酶,能催化三种转糖基反应(歧化、环化和耦合反应)和水解反应,其中,能将淀粉转化为环糊精的环化反应是特征反应;作为α-淀粉酶家族的成员,CGT酶除了具有与α-淀粉酶相同的A、B、C结构域外,还存在D和E结构域。另外,对CGT酶的催化机理包括底物结合方式、转糖苷反应机理以及环化机理等进行了详细的讨论。     

Catalytic reaction mechanism of goose egg-white lysozyme by molecular modelling of enzyme-substrate complex

Despite the low similarity between their amino acid sequences, the core structures of the fold between chicken-type and goose-type lysozymes are conserved. However, their enzymatic activities are quite different. Both of them exhibit hydrolytic activities, but the goose-type lysozyme does not exhibit transglycosylation activity. The chicken-type lysozyme has a retaining-type reaction mechanism, while the reaction mechanism of the goose-type lysozyme has not been clarified. To clarify the latter mechanism, goose egg-white lysozyme (GEL)-N-acetyl-D-glucosamine (GlcNAc)6 complexes were modelled and compared with hen egg-white lysozyme (HEL)-(GlcNAc)6 complexes. By systematic conformational search, 48 GEL-(GlcNAc)6 complexes were modelled. The right and left side, and the amino acid residues in subsites E-G were identified in GEL. The GlcNAc residue D could bind towards the right side without distortion and there was enough room for a water molecule to attack the C1 carbon of GlcNAc residue D from alpha-side in the right side and not for acceptor molecule. The result of molecular dynamics simulation suggests that GEL would be an inverting enzyme, and Asp97 would act as a second carboxylate and that the narrow space of the binding cleft at subsites E-G in GEL may prohibit the sugar chain to bind alternative site that might be essential for transglycosylation.     

The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a gamma-cyclodextrin-CGTase complex at 1.8-A resolution

The enzyme cyclodextrin glycosyltransferase is closely related to alpha-amylases but has the unique ability to produce cyclodextrins (circular alpha(1-->4)-linked glucoses) from starch. To characterize this specificity we determined a 1.8-A structure of an E257Q/D229N mutant cyclodextrin glycosyltransferase in complex with its product gamma-cyclodextrin, which reveals for the first time how cyclodextrin is competently bound. Across subsites -2, -1, and +1, the cyclodextrin ring binds in a twisted mode similar to linear sugars, giving rise to deformation of its circular symmetry. At subsites -3 and +2, the cyclodextrin binds in a manner different from linear sugars. Sequence comparisons and site-directed mutagenesis experiments support the conclusion that subsites -3 and +2 confer the cyclization activity in addition to subsite -6 and Tyr-195. On this basis, a role of the individual residues during the cyclization reaction cycle is proposed.     

Addition of polar organic solvents can improve the product selectivity of cyclodextrin glycosyltransferase. Solvent effects on cgtase

Cyclodextrin glycosyltransferase (EC 2.4.1.19, CGTase) is an enzyme that produces cyclodextrins from starch via an intramolecular transglycosylation reaction. Addition of small amounts (10% v/v) of polar organic solvents can affect both the overall production yield and the type of cyclodextrin produced from a maltodextrin substrate under simulated industrial process conditions. Using CGTase from Thermoanaerobacter sp. all solvents produced an increase in cyclodextrin yield when compared with a control, the greatest increase being obtained with addition of ethanol (26%). In addition product selectivity was affected by the nature of the organic solvent used: beta-cyclodextrin was favoured in the absence of any solvent and on the addition of dimethylsulphoxide, t-butanol and dimethylformanide while alpha-cyclodextrin was favoured by addition of acetonitrile, ethanol and tetrahydrofuran. With CGTase from Bacillus circulans strain 251 relatively smaller increases in overall cyclodextrin production were achieved (between 5-10%). Addition of t-butanol to a B. circulans catalysed reaction however did produce the largest selectivity for beta-cyclodextrin of any solvent-enzyme combination (82%). The effect of solvent addition was shown not to be related to the product inhibition of CGTase, but may be related to reduced competition from the intermolecular transglycosylation reaction that causes degradation of cyclodextrin products. This rate of this reaction was shown to be dependent on the nature of the organic solvent used.     

Enzymatic preparation of maltohexaose, maltoheptaose, and maltooctaose by the preferential cyclomaltooligosaccharide (cyclodextrin) ring-opening reaction of Pyrococcus furiosus thermostable amylase

Specific-length maltooligosaccharides, particularly maltohexaose, maltoheptaose, and maltooctaose, were prepared from cyclomaltooligosaccharides (cyclodextrins, CDs) by the preferential cyclodextrin ring-opening reaction of an amylolytic enzyme from Pyrococcus furiosus. The enzyme primarily produces maltohexaose, maltoheptaose, and maltooctaose by hydrolyzing alpha-, beta-, and gamma-CD, respectively. This study aims to develop a high-efficiency synthesis of specific maltooligosaccharides at high-purity. [formula: see text]     

Transfer Action of Cyclodextrin Glycosyltransferase on Starch

The transglycosylation reaction of the cyclodextrin glycosyltransferase from Bacillus megaterium (No. 5 enzyme) and Bacillus macerans (BMA) were examined. No. 5 enzyme was more efficient in transglycosylation reaction than BMA in the every acceptor employed in the present study. The order of the efficient acceptors for No. 5 enzyme was maltose (G2), glucose (Gl), maltotriose (G3) and sucrose (GF). On the other hand, that found for BMA was Gl, G2, GF and G3. The transglycosylation products to glucose formed by the action of No. 5 enzyme on starch were G2, G3, maltotetraose (G4), maltopentaose (G5), maltohexaose (G6) and maltoheptaose (G7) in the order of their quantities, while, in the case of BMA, they were G2, G3, G5, G7=G4 and G6. The larger transglycosylation products to sucrose formed by the action of No. 5 enzyme on starch were maltosylfructose. On the other hand, that formed by the action of BMA was maltoheptaosylfructose.

It was suggested that cyclodextrin glycosyltransferase could transfer the glucosyl residues to an acceptor directly from starch, as well as through cyclodextrin.     

Chemical modification of lysine side chains of cyclodextrin glycosyltransferase from Thermoanaerobacter causes a shift from cyclodextrin glycosyltransferase to alpha-amylase specificity

Cyclodextrin glycosyltransferases and alpha-amylases are two groups of enzymes with related secondary structures. However, cyclodextrin glycosyltransferases display transferase activities not present in alpha-amylases, probably derived from the existence of two more domains and different amino acid sequences. The hydrolytic activity of cyclodextrin glycosyltransferases is generally quite low, except for two cyclodextrin glycosyltransferases from termophiles. In this work, we have carried out the chemical modification (with acetic anhydride) of the amino groups of cyclodextrin glycosyltransferase from Thermoanaerobacter to assess their contributions to protein function. The acetylated cyclodextrin glycosyltransferase showed a significant reduction of its cyclization, coupling and disproportionation activities. Surprisingly, the hydrolytic (saccharifying) activity was slightly enhanced. These results suggest the participation of one or more lysine side chains in the interactions contributing to the transferase activity, either in any of the S11 subsites or in the acceptor binding site.     

Characterisation of a glycosylated alkyl polyglycoside produced by a cyclodextrin glycosyltransferase by HPLC-ELSD and -MS

A transglycosylation reaction between an alkyl polyglycoside and α-cyclodextrin catalysed by cyclodextrin glycosyltransferase (CGTase) from Bacillus macerans was investigated. The reaction products were identified by comparison with standards generated by CGTase catalysed modification of pure alkyl glycosides using HPLC-ELSD and -MS analysis. The main products were alkyl glucopyranosides (substrates present in the alkyl polyglycoside) glycosylated with 6 (primary coupling products) or 12 (secondary coupling products) glucose residues. Both α and β anomers were glycosylated.     

Functional and structural effects of mutagenic replacement of Asn37 at subsite F on the lysozyme-catalyzed reaction

To investigate the functional role of subsites E and F in lysozyme catalysis, Asn37 of hen egg-white lysozyme (HEL), which is postulated to participate in sugar residue binding at the right-sided subsite F through hydrogen bonding, was replaced by Ser or Gly by site-directed mutagenesis. The mutations of Asn37 neither significantly affected the binding constant for chitotriose nor the enzymatic activity toward the substrate glycol chitin. However, kinetic analysis with the substrate N-acetylglucosamine pentamer, (GlcNAc)(5), revealed that the conversion of Asn37 to Gly decreased the binding free energies for subsites E and F, while the conversion to Ser increased the substrate affinity at subsite F. It was further found that the rate constant of transglycosylation was reduced by these mutations. These results suggest that Asn37 is involved not only in substrate binding at subsite F but also in transglycosylation activity. No remarkable change in the tertiary structure except the side chain of the 37th residue was detected on X-ray analysis of the mutant proteins, indicating that the alterations in the enzymatic function between the wild type and mutant enzymes depend on limited structural change around the substitution site. It is thus speculated that the slight conformational difference in the side chain of position 37 may affect the substrate and acceptor binding at subsites E and F, leading to lower the efficiency of the transglycosylation activities of the mutant proteins.     

Characterization of Bacillus stearothermophilus cyclodextrin glucanotransferase in ascorbic acid 2-O-alpha-glucoside formation

In this study, we characterized cyclodextrin glucanotransferase (CGTase) from Bacillus stearothermophilus in L-ascorbic acid-2-O-alpha-D-glucoside (AA-2G) formation and compared its enzymological properties with those of rat intestinal and rice seed alpha-glucosidases which had the ability to form AA-2G. CGTase formed AA-2G efficiently using alpha-cyclodextrin (alpha-CD) as a substrate and ascorbic acid (AA) as an acceptor. Several AA-2-oligoglucosides were also formed in this reaction mixture, and they could be converted to AA-2G by the additional treatment of glucoamylase. The optimum temperature for AA-2G formation was 70 degrees C and its optimum pH was around 5.0. CGTase also utilized beta- and gamma-CDs, maltooligosaccharides, dextrin, amylose, glycogen and starch as substrates, but not any disaccharides except maltose. CGTase showed the same acceptor specificity as two alpha-glucosidases, whereas its hydrolyzing activity towards AA-2G was very low compared with those of alpha-glucosidases. Cleavage profiles of AA-2-oligoglucosides by CGTase present a possible mechanism for AA-2G formation that CGTase transfers a glucose-hexamer to an acceptor at the first step and then a glucose is stepwisely removed from the non-reducing end of the product through glucoamylase-like action of this enzyme. These results indicate that CGTase is able to synthesize AA-2G more efficiently than rat and rice alpha-glucosidases and utilization of this enzyme makes the mass production of AA-2G possible.     

Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase

Cyclodextrin-glycosyltransferases (CGTases) (EC ) preferably catalyze transglycosylation reactions with glucosyl residues as acceptor, whereas the homologous alpha-amylases catalyze hydrolysis reactions using water as acceptor. This difference in reaction specificity is most likely caused by the acceptor binding site. To investigate this in detail we altered the acceptor site residues Lys-232, Phe-183, Phe-259, and Glu-264 of Bacillus circulans strain 251 CGTase using site-directed mutagenesis. Lys-232 is of general importance for catalysis, which appears to result mainly from stabilization of the conformation of the loop containing the catalytic nucleophile Asp-229 and His-233, a residue that has been implied in transition state stabilization. Glu-264 contributes to the disproportionation reaction only, where it is involved in initial binding of the (maltose) acceptor. Phe-183 and Phe-259 play important and distinct roles in the transglycosylation reactions catalyzed by CGTase. Mutation of Phe-183 affects especially the cyclization and coupling reactions, whereas Phe-259 is most important for the cyclization and disproportionation reactions. Moreover, the hydrophobisity of Phe-183 and Phe-259 limits the hydrolyzing activity of the enzyme. Hydrolysis can be enhanced by making these residues more polar, which concomitantly results in a lower transglycosylation activity. A double mutant was constructed that yielded an enzyme preferring hydrolysis over cyclization (15:1), whereas the wild type favors cyclization over hydrolysis (90:1).     

Enzymatic circularization of a malto-octaose linear chain studied by stochastic reaction path calculations on cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) is an enzyme belonging to the alpha-amylase family that forms cyclodextrins (circularly linked oligosaccharides) from starch. X-ray work has indicated that this cyclization reaction of CGTase involves a 23-A movement of the nonreducing end of a linear malto-oligosaccharide from a remote binding position into the enzyme acceptor site. We have studied the dynamics of this sugar chain circularization through reaction path calculations. We used the new method of the stochastic path, which is based on path integral theory, to compute an approximate molecular dynamics trajectory of the large (75-kDa) CGTase from Bacillus circulans strain 251 on a millisecond time scale. The result was checked for consistency with site-directed mutagenesis data. The combined data show how aromatic residues and a hydrophobic cavity at the surface of CGTase actively catalyze the sugar chain movement. Therefore, by using approximate trajectories, reaction path calculations can give a unique insight into the dynamics of complex enzyme reactions.     

Site-directed mutations in Alanine 223 and Glycine 255 in the acceptor site of gamma-Cyclodextrin glucanotransferase from Alkalophilic Bacillus clarkii 7364 affect cyclodextrin production

A cyclodextrin glucanotransferase (CGTase) from Bacillus clarkii 7364 converts starch into gamma-cyclodextrin (gamma-CD) with high specificity. Comparison of the deduced amino acid sequence of this CGTase with those of other typical CGTases revealed that several amino acids are deleted or substituted with others at several subsites. Of these amino acids, Ala223 at subsite +2 and Gly255 at subsite +3 in the acceptor site of the enzyme were replaced by several amino acids through site-directed mutagenesis. The replacement of Ala223 by lysine, arginine and histidine strongly enhanced the gamma-CD-forming activity in the neutral pH range. On the other hand, all mutants obtained on replacing Gly255 with the above amino acids showed significant decreases in the gamma-CD-forming activity. Taking into account both the kinetic parameters and pKa values of the side chains of the three basic amino acids, the protonation state of the amino groups in their side chains at subsite +2 seems to enhance the hydrogen bonding interaction between these basic amino acids and the glucose residues of linear oligosaccharides. The enhancement of the interaction may play an important role by helping the substrate reach subsite +1, hence increasing the gamma-CD-forming activity and kcat value.     

Identification of acceptor substrate binding subsites +2 and +3 in the amylomaltase from Thermus thermophilus HB8

Glycoside hydrolase family 77 (GH77) belongs to the alpha-amylase superfamily (Clan H) together with GH13 and GH70. GH77 enzymes are amylomaltases or 4-alpha-glucanotransferases, involved in maltose metabolism in microorganisms and in starch biosynthesis in plants. Here we characterized the amylomaltase from the hyperthermophilic bacterium Thermus thermophilus HB8 (Tt AMase). Site-directed mutagenesis of the active site residues (Asp293, nucleophile; Glu340, general acid/base catalyst; Asp395, transition state stabilizer) shows that GH77 Tt AMase and GH13 enzymes share the same catalytic machinery. Quantification of the enzyme's transglycosylation and hydrolytic activities revealed that Tt AMase is among the most efficient 4-alpha-glucanotransferases in the alpha-amylase superfamily. The active site contains at least seven substrate binding sites, subsites -2 and +3 favoring substrate binding and subsites -3 and +2 not, in contrast to several GH13 enzymes in which subsite +2 contributes to oligosaccharide binding. A model of a maltoheptaose (G7) substrate bound to the enzyme was used to probe the details of the interactions of the substrate with the protein at acceptor subsites +2 and +3 by site-directed mutagenesis. Substitution of the fully conserved Asp249 with a Ser in subsite +2 reduced the activity 23-fold (for G7 as a substrate) to 385-fold (for maltotriose). Similar mutations reduced the activity of alpha-amylases only up to 10-fold. Thus, the characteristics of acceptor subsite +2 represent a main difference between GH13 amylases and GH77 amylomaltases.