Crystal structure of amylomaltase from thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans

Amylomaltase is involved in the metabolism of starch, one of the most important polysaccharides in nature. A unique feature of amylomaltase is its ability to catalyze the formation of cyclic amylose. In contrast to the well studied cyclodextrin glucanotransferases (CGTases), which synthesize cycloamylose with a ring size (degree of polymerization or DP) of 6-8, the amylomaltase from Thermus aquaticus produces cycloamyloses with a DP of 22 and higher. The crystal structure of amylomaltase from Thermus aquaticus was determined to 2.0 A resolution. It is a member of the alpha-amylase superfamily of enzymes, whose core structure consists of a (beta, alpha)(8) barrel. In amylomaltase, the 8-fold symmetry of this barrel is disrupted by several insertions between the barrel strands. The largest insertions are between the third and fifth barrel strands, where two insertions form subdomain B1, as well as between the second and third barrel strands, forming the alpha-helical subdomain B2. Whereas part of subdomain B1 is also present in other enzyme structures of the alpha-amylase superfamily, subdomain B2 is unique to amylomaltase. Remarkably, the C-terminal domain C, which is present in all related enzymes of the alpha-amylase family, is missing in amylomaltase. Amylomaltase shows a similar arrangement of the catalytic side-chains (two Asp residues and one Glu residue) as in previously characterized members of the alpha-amylase superfamily, indicating similar mechanisms of the glycosyl transfer reaction. In amylomaltase, a conserved loop of around eight amino acid residues is partially shielding the active center. This loop, which is well conserved among other amylomaltases, may sterically hinder the formation of small cyclic products.     

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.     

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.     

Use of random and saturation mutageneses to improve the properties of Thermus aquaticus amylomaltase for efficient production of cycloamyloses

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of alpha-1,4 glucans to produce cyclic alpha-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.     

Use of Random and Saturation Mutageneses To Improve the Properties of Thermus aquaticus Amylomaltase for Efficient Production of Cycloamyloses

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of α-1,4 glucans to produce cyclic α-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.     

Oligosaccharide binding to barley alpha-amylase 1

Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."     

X-ray structure of Novamyl, the five-domain "maltogenic" alpha-amylase from Bacillus stearothermophilus: maltose and acarbose complexes at 1.7A resolution.

The three-dimensional structure of the Bacillus stearothermophilus "maltogenic" alpha-amylase, Novamyl, has been determined by X-ray crystallography at a resolution of 1.7 A. Unlike conventional alpha-amylases from glycoside hydrolase family 13, Novamyl exhibits the five-domain structure more usually associated with cyclodextrin glycosyltransferase. Complexes of the enzyme with both maltose and the inhibitor acarbose have been characterized. In the maltose complex, two molecules of maltose are found in the -1 to -2 and +2 to +3 subsites of the active site, with two more on the C and E domains. The C-domain maltose occupies a position identical to one previously observed in the Bacillus circulans CGTase structure [Lawson, C. L., et al. (1994) J. Mol. Biol. 236, 590-600], suggesting that the C-domain plays a genuine biological role in saccharide binding. In the acarbose-maltose complex, the tetrasaccharide inhibitor acarbose is found as an extended hexasaccharide species, bound in the -3 to +3 subsites. The transition state mimicking pseudosaccharide is bound in the -1 subsite of the enzyme in a 2H3 half-chair conformation, as expected. The active site of Novamyl lies in an open gully, fully consistent with its ability to perform internal cleavage via an endo as opposed to an exo activity.     

Crystal structures of amylosucrase from Neisseria polysaccharea in complex with D-glucose and the active site mutant Glu328Gln in complex with the natural substrate sucrose

The structure of amylosucrase from Neisseria polysaccharea in complex with beta-D-glucose has been determined by X-ray crystallography at a resolution of 1.66 A. Additionally, the structure of the inactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0 A. The D-glucose complex shows two well-defined D-glucose molecules, one that binds very strongly in the bottom of a pocket that contains the proposed catalytic residues (at the subsite -1), in a nonstrained (4)C(1) conformation, and one that binds in the packing interface to a symmetry-related molecule. A third weaker D-glucose-binding site is located at the surface near the active site pocket entrance. The orientation of the D-glucose in the active site emphasizes the Glu328 role as the general acid/base. The binary sucrose complex shows one molecule bound in the active site, where the glucosyl moiety is located at the alpha-amylase -1 position and the fructosyl ring occupies subsite +1. Sucrose effectively blocks the only visible access channel to the active site. From analysis of the complex it appears that sucrose binding is primarily obtained through enzyme interactions with the glucosyl ring and that an important part of the enzyme function is a precise alignment of a lone pair of the linking O1 oxygen for hydrogen bond interaction with Glu328. The sucrose specificity appears to be determined primarily by residues Asp144, Asp394, Arg446, and Arg509. Both Asp394 and Arg446 are located in an insert connecting beta-strand 7 and alpha-helix 7 that is much longer in amylosucrase compared to other enzymes from the alpha-amylase family (family 13 of the glycoside hydrolases).     

On the mechanism of alpha-amylase.

Two inhibitors, acarbose and cyclodextrins (CD), were used to investigate the active site structure and function of barley alpha-amylase isozymes, AMY1 and AMY2. The hydrolysis of DP 4900-amylose, reduced (r) DP18-maltodextrin and maltoheptaose (catalysed by AMY1 and AMY2) was followed in the absence and in the presence of inhibitor. Without inhibitor, the highest activity was obtained with amylose, kcat/Km decreased 103-fold using rDP18-maltodextrin and 10(5) to 10(6)-fold using maltoheptaose as substrate. Acarbose is an uncompetitive inhibitor with inhibition constant (L1i) for amylose and maltodextrin in the micromolar range. Acarbose did not bind to the active site of the enzyme, but to a secondary site to give an abortive ESI complex. Only AMY2 has a second secondary binding site corresponding to an ESI2 complex. In contrast, acarbose is a mixed noncompetitive inhibitor of maltoheptaose hydrolysis. Consequently, in the presence of this oligosaccharide substrate, acarbose bound both to the active site and to a secondary binding site. alpha-CD inhibited the AMY1 and AMY2 catalysed hydrolysis of amylose, but was a very weak inhibitor compared to acarbose.beta- and gamma-CD are not inhibitors. These results are different from those obtained previously with PPA. However in AMY1, as already shown for amylases of animal and bacterial origin, in addition to the active site, one secondary carbohydrate binding site (s1) was necessary for activity whereas two secondary sites (s1 and s2) were required for the AMY2 activity. The first secondary site in both AMY1 and AMY2 was only functional when substrate was bound in the active site. This appears to be a general feature of the alpha-amylase family.     

Barley malt-alpha-amylase. Purification, action pattern, and subsite mapping of isozyme 1 and two members of the isozyme 2 subfamily using p-nitrophenylated maltooligosaccharide substrates.

Isoforms AMY1, AMY2-1 and AMY2-2 of barley alpha-amylase were purified from malt. AMY2-1 and AMY2-2 are both susceptible to barley alpha-amylase/subtilisin inhibitor. The action of these isoforms is compared using substrates ranging from p-nitrophenylmaltoside through p-nitrophenylmaltoheptaoside. The kcat/Km values are calculated from the substrate consumption. The relative cleavage frequency of different substrate bonds is given by the product distribution. AMY2-1 is 3-8-fold more active than AMY1 toward p-nitrophenylmaltotrioside through p-nitrophenylmaltopentaoside. AMY2-2 is 10-50% more active than AMY2-1. The individual subsite affinities are obtained from these data. The resulting subsite maps of the isoforms are quite similar. They comprise four and six glucosyl-binding subsites towards the reducing and the non-reducing end, respectively. Towards the non-reducing end, the sixth and second subsites have a high affinity, the third has very low or even lack of affinity and the first (catalytic subsite) has a large negative affinity. The affinity declines from moderate to low for subsites 1 through 4 toward the reducing end. AMY1 has clearly a more negative affinity at the catalytic subsite, but larger affinities at both the fourth subsites, compared to AMY2. AMY2-1 has lower affinity than AMY2-2 at subsites adjacent to the catalytic site, and otherwise mostly higher affinities than AMY2-2. Theoretical kcat/Km values show excellent agreement with experimental values.     

Structure of a pancreatic alpha-amylase bound to a substrate analogue at 2.03 A resolution.

The structure of pig pancreatic alpha-amylase in complex with carbohydrate inhibitor and proteinaceous inhibitors is known but the successive events occurring at the catalytic center still remain to be elucidated. The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase (PPA, EC 3.2.1.1.) soaked with an enzyme-resistant substrate analogue, methyl 4,4'-dithio-alpha-maltotrioside, showed electron density corresponding to the binding of substrate analogue molecules at the active site and at the "second binding site." The electron density observed at the active site was interpreted in terms of overlapping networks of oligosaccharides, which show binding of substrate analogue molecules at subsites prior to and subsequent to the cleavage site. A weaker patch of density observed at subsite -1 (using a nomenclature where the site of hydrolysis is taken to be between subsites -1 and +1) was modeled with water molecules. Conformational changes take place upon substrate analogue binding and the "flexible loop" that constitutes the surface edge of the active site is observed in a specific conformation. This confirms that this loop plays an important role in the recognition and binding of the ligand. The crystal structure was refined at 2.03 A resolution, to an R-factor of 16.0 (Rfree, 18.5).     

Crystal structures of beta-amylase from Bacillus cereus var mycoides in complexes with substrate analogs and affinity-labeling reagents

The crystal structures of beta-amylase from Bacillus cereus var. mycoides in complexes with five inhibitors were solved. The inhibitors used were three substrate analogs, i.e. glucose, maltose (product), and a synthesized compound, O-alpha-D-glucopyranosyl-(1-->4)-O-alpha-D-glucopyranosyl-(1-->4)-D-xylopyranose (GGX), and two affinity-labeling reagents with an epoxy alkyl group at the reducing end of glucose. For all inhibitors, one molecule was bound at the active site cleft and the non-reducing end glucose of the four inhibitors except GGX was located at subsite 1, accompanied by a large conformational change of the flexible loop (residues 93-97), which covered the bound inhibitor. In addition, another molecule of maltose or GGX was bound about 30 A away from the active site. A large movement of residues 330 and 331 around subsite 3 was also observed upon the binding of GGX at subsites 3 to 5. Two affinity-labeling reagents, alpha-EPG and alpha-EBG, were covalently bound to a catalytic residue (Glu-172). A substrate recognition mechanism for the beta-amylase was discussed based on the modes of binding of these inhibitors in the active site cleft.     

Acarbose rearrangement mechanism implied by the kinetic and structural analysis of human pancreatic alpha-amylase in complex with analogues and their elongated counterparts

A mechanistic study of the poorly understood pathway by which the inhibitor acarbose is enzymatically rearranged by human pancreatic alpha-amylase has been conducted by structurally examining the binding modes of the related inhibitors isoacarbose and acarviosine-glucose, and by novel kinetic measurements of all three inhibitors under conditions that demonstrate this rearrangement process. Unlike acarbose, isoacarbose has a unique terminal alpha-(1-6) linkage to glucose and is found to be resistant to enzymatic rearrangement. This terminal glucose unit is found to bind in the +3 subsite and for the first time reveals the interactions that occur in this part of the active site cleft with certainty. These results also suggest that the +3 binding subsite may be sufficiently flexible to bind the alpha-(1-6) branch points in polysaccharide substrates, and therefore may play a role in allowing efficient cleavage in the direct vicinity of such junctures. Also found to be resistant to enzymatic rearrangement was acarviosine-glucose, which has one fewer glucose unit than acarbose. Collectively, structural studies of all three inhibitors and the specific cleavage pattern of HPA make it possible to outline the simplest sequence of enzymatic reactions likely involved upon acarbose binding. Prominent features incorporated into the starting structure of acarbose to facilitate the synthesis of the final tightly bound pseudo-pentasaccharide product are the restricted availability of hydrolyzable bonds and the placement of the transition state-like acarviosine group. Additional "in situ" experiments designed to elongate and thereby optimize isoacarbose and acarviosine-glucose inhibition using the activated substrate alphaG3F demonstrate the feasibility of this approach and that the principles outlined for acarbose rearrangement can be used to predict the final products that were obtained.     

Subsite mapping of the human pancreatic alpha-amylase active site through structural, kinetic, and mutagenesis techniques

We report a multifaceted study of the active site region of human pancreatic alpha-amylase. Through a series of novel kinetic analyses using malto-oligosaccharides and malto-oligosaccharyl fluorides, an overall cleavage action pattern for this enzyme has been developed. The preferred binding/cleavage mode occurs when a maltose residue serves as the leaving group (aglycone sites +1 and +2) and there are three sugars in the glycon (-1, -2, -3) sites. Overall it appears that five binding subsites span the active site, although an additional glycon subsite appears to be a significant factor in the binding of longer substrates. Kinetic parameters for the cleavage of substrates modified at the 2 and 4' ' positions also highlight the importance of these hydroxyl groups for catalysis and identify the rate-determining step. Further kinetic and structural studies pinpoint Asp197 as being the likely nucleophile in catalysis, with substitution of this residue leading to an approximately 10(6)-fold drop in catalytic activity. Structural studies show that the original pseudo-tetrasaccharide structure of acarbose is modified upon binding, presumably through a series of hydrolysis and transglycosylation reactions. The end result is a pseudo-pentasaccharide moiety that spans the active site region with its N-linked "glycosidic" bond positioned at the normal site of cleavage. Interestingly, the side chains of Glu233 and Asp300, along with a water molecule, are aligned about the inhibitor N-linked glycosidic bond in a manner suggesting that these might act individually or collectively in the role of acid/base catalyst in the reaction mechanism. Indeed, kinetic analyses show that substitution of the side chains of either Glu233 or Asp300 leads to as much as a approximately 10(3)-fold decrease in catalytic activity. Structural analyses of the Asp300Asn variant of human pancreatic alpha-amylase and its complex with acarbose clearly demonstrate the importance of Asp300 to the mode of inhibitor binding.     

Crystallographic evidence of a transglycosylation reaction: ternary complexes of a psychrophilic alpha-amylase

The psychrophilic Pseudoalteromonas haloplanctis alpha-amylase is shown to form ternary complexes with two alpha-amylase inhibitors present in the active site region, namely, a molecule of Tris and a trisaccharide inhibitor or heptasaccharide inhibitor, respectively. The crystal structures of these complexes have been determined by X-ray crystallography to 1.80 and 1.74 A resolution, respectively. In both cases, the prebound inhibitor Tris is expelled from the active site by the incoming oligosaccharide inhibitor substrate analogue, but stays linked to it, forming well-defined ternary complexes with the enzyme. These results illustrate competition in the crystalline state between two inhibitors, an oligosaccharide substrate analogue and a Tris molecule, bound at the same time in the active site region. Taken together, these structures show that the enzyme performs transglycosylation in the complex with the pseudotetrasaccharide acarbose (confirmed by a mutant structure), leading to a well-defined heptasaccharide, considered as a more potent inhibitor. Furthermore, the substrate-induced ordering of water molecules within a channel highlights a possible pathway used for hydrolysis of starch and related poly- and oligosaccharides.     

Crystal Structure of the Pig Pancreatic α-Amylase Complexed with ρ-Nitrophenyl-α-D-Maltoside-Flexibility in the Active Site

The X-ray structure analysis of a crystal of pig pancreatic alpha-amylase soaked with a rho-nitrophenyl-alpha-D-maltoside (pNPG2) substrate showed a pattern of electron density corresponding to the binding of a rho-nitrophenol unit at subsite -2 of the active site. Binding of the product to subsite -2 after hydrolysis of the pNPG2 molecules, may explain the low catalytic efficiency of the hydrolysis of pNPG2 by PPA. Except a small movement of the segment from residues 304-305 the typical conformational changes of the "flexible loop" (303-309), that constitutes the surface edge of the substrate binding cleft, were not observed in the present complex structure. This result supports the hypothesis that significant movement of the loop may depend on aglycone site being filled (Payan and Qian, J. Protein Chen. 22: 275, 2003). Structural analyses have shown that pancreatic alpha-amylases undergo an induced conformational change of the catalytic residue Asp300 upon substrate binding; in the present complex the catalytic residue is observed in its unliganded orientation. The results suggest that the induced reorientation is likely due to the presence of a sugar unit at subsite -1 and not linked to the closure of the flexible surface loop. The crystal structure was refined at 2.4 A resolution to an R factor of 17.55% (Rfree factor of 23.32%).     

Enzymatic analysis of an amylolytic enzyme from the hyperthermophilic archaeon Pyrococcus furiosus reveals its novel catalytic properties as both an alpha-amylase and a cyclodextrin-hydrolyzing enzyme

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcus furiosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90 degrees C. The substrate specificity of PFTA suggests that it possesses characteristics of both alpha-amylase and cyclodextrin-hydrolyzing enzyme. Like typical alpha-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and beta-cyclodextrin, which are resistant to alpha-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent alpha-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both alpha-amylase and cyclodextrin-hydrolyzing enzyme.     

Amylose chain behavior in an interacting context. III. Complete occupancy of the AMY2 barley alpha-amylase cleft and comparison with biochemical data.

In the first two papers of this series, the tools necessary to evaluate substrate ring deformations were developed, and then the modeling of short amylose fragments (maltotriose and maltopentaose) inside the catalytic site of barley alpha-amylase was performed. In this third paper, this docking has been extended to the whole catalytic cleft. A systematic approach to extend the substrate was used on the reducing side from the previous enzyme/pentasaccharide complex. However, due to the lack of an obvious subsite at the nonreducing side, an alternate protocol has been chosen that incorporates biochemical information on the enzyme and features on the substrate shape as well. As a net result, ten subsites have been located consistent with the distribution of Ajandouz et al. (E. H. Ajandouz, J. Abe, B. Svensson, and G. Marchis-Mouren, Biochimica Biophysica Acta, 1992, Vol. 1159, pp. 193-202) and corresponding binding energies were estimated. Among them, two extreme subsites (-6) and (+4), with stacking residues Y104 and Y211, respectively, have strong affinities with glucose rings added to the substrate. No other deformation has been found for the new glucose rings added to the substrate; therefore, only ring A of the DP 10 fragment has a flexible form when interacting with the inner stacking residues Y51. Global conservation of the helical shape of the substrate can be postulated in spite of its significant distortion at subsite (-1).     

Complex structures of Thermoactinomyces vulgaris R-47 alpha-amylase 1 with malto-oligosaccharides demonstrate the role of domain N acting as a starch-binding domain

The X-ray structures of complexes of Thermoactinomyces vulgaris R-47 alpha-amylase 1 (TVAI) with an inhibitor acarbose and an inactive mutant TVAI with malto-hexaose and malto-tridecaose have been determined at 2.6, 2.0 and 1.8A resolution, and the structures have been refined to R-factors of 0.185 (R(free)=0.225), 0.184 (0.217) and 0.164 (0.200), respectively, with good chemical geometries. Acarbose binds to the catalytic site of TVAI, and interactions between acarbose and the enzyme are very similar to those found in other structure-solved alpha-amylase/acarbose complexes, supporting the proposed catalytic mechanism. Based on the structure of the TVAI/acarbose complex, the binding mode of pullulan containing alpha-(1,6) glucoside linkages could be deduced. Due to the structural difference caused by the replaced amino acid residue (Gln396 for Glu) in the catalytic site, malto-hexaose and malto-tridecaose partially bind to the catalytic site, giving a mimic of the enzyme/product complex. Besides the catalytic site, four sugar-binding sites on the molecular surface are found in these X-ray structures. Two sugar-binding sites in domain N hold the oligosaccharides with a regular helical structure of amylose, which suggests that the domain N is a starch-binding domain acting as an anchor to starch in the catalytic reaction of the enzyme. An assay of hydrolyzing activity for the raw starches confirmed that TVAI can efficiently hydrolyze raw starch.     

The three-dimensional structure of acarbose bound to glycogen phosphorylase

Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.