Refined structure for the complex of <scp>d</scp>‐<i>gluco</i>‐dihydroacarbose with glucoamylase from <i>Aspergillus awamori</i> var. X100 to 2.2 Å resolution: dual conformations for extended inhibitors bound to the active site of glucoamylase

Stoffer, Bjarne; Aleshin, Alexander E.; Firsov, L. M.; Svensson, Birte; Honzatko, Richard B.

doi:10.1016/0014-5793(94)01354-4

Cited by 63 publications

(92 citation statements)

References 15 publications

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“…As long recognized (13,14), a water molecule is well positioned in the complex to make a nucleophilic attack at the incipient carbonium ionic centre. As indicated in both Figs.…”

Section: Glumentioning

confidence: 93%

“…The long known (9) industrially highly important amyloglucosidase (EC 3.2.1.3, AMG), referred to herein as glycoamylase, was chosen for this inquiry because the X-ray crystal structures are known for both the native protein (10, 1 1) and its complexes with inhibitors, namely, 1'-deoxynojirimycin (12) acarbose (13), and D-gluco-dihydroacarbose (13,14). The structures of the complexes with their natural substrates, maltose and isomaltose, remain unknown.…”

Section: Introductionmentioning

confidence: 99%

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Chemical mapping of the active site of the glucoamylase ofAspergillus niger

et al. 1996

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Abstract:A recently developed technique for the probing of the combining sites of lectins and antibodies, to establish the structure of the epitope that is involved in the binding of an oligosaccharide, is used to study the binding of methyl aisomaltoside by the enzyme glucoamylase. The procedure involved the determination of the effects on the kinetics of hydrolysis of both monodeoxygenation and mono-0-methylation at each of the seven hydroxyl groups in order to gain an estimate of the differential changes in the free energies of activation, AAG' . As expected, from previous publications, both deoxygenation and 0-methylation of OH-4 (reducing unit), OH-4', or OH-6' strongly hindered hydrolysis, whereas the kinetics were virtually unaffected by either the substitutions at OH-2 or structural changes at C-I. The substitutions at OH-3 caused increases of 2.1 and 1.9 kcallmol in the AAG'. In contrast, whereas deoxygenation of either OH-2' or OH-3' caused much smaller (0.96 and 0.52 kcallmol) increases in AAG', the mono-0-methylations resulted in severe steric hindrance to the formation of the activated complex. The relatively weak effects of deoxygenation suggest that the hydroxyl groups are replaced by water molecules and thereby participate in the binding by contributing effective complementarity. Methyl a-isomaltoside was docked into the combining site of the X-ray crystal structure at 2.4 A resolution of the complex with the inhibitor acarbose. A fit free of steric interactions with the protein was found that has the methyl a-glucopyranoside unit in the normal "C, conformation and the other glucose unit approachin a half-chair conformation with the interunit fragment defined by the torsion angles +IIJJ/W = 74°/1340/ % 166' (0-5'-C-l'h-64-6%-5-0-5).The model provides a network of hydrogen bonds that appears to well represent the activated complex formed by the glucoamylase with both maltose and isomaltose since the structures appear to provide a sound rationale for both the specificity and catalysis provided by the enzyme.Key war-ds: monodeoxy and mono-0-methyl derivatives of methyl a-isomaltoside, enzyme binding domain, functioning of glucoamylase, differential changes in free energy of activation, characteristics of hydrogen bonding networks. Resum6: Dans le but d'Ctudier la fixation de l'a-isomaltoside de mCthyle par l'enzyme glucoamylase, on a fait appel B une technique, dCveloppCe rCcemment pour I'examen des sites de fixation des lectines et des anticorps, pour Ctablir la structure de I'Cpitope impliqui dans la fixation d'un oligosaccharide. I,a procCdure implique la dktermination des effets, provoquCs sur la cinCtique d'hydrolyse, par la monodCsoxygCnation ainsi que la mono-0-methylation de chacun des sept groupes hydroxyles; on espitre ainsi obtenir une evaluation des changements diffkrentiels dans les Cnergies libres d'activation, AAG'. Tel qu'on peut s'y attendre sur la base des publications antkrieures, tant la dCsoxygCnation que la 0-mtthylation des groupes OH-4 (unit6 rkductrice), OH-4' ou OH-6' empsc...

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“…As long recognized (13,14), a water molecule is well positioned in the complex to make a nucleophilic attack at the incipient carbonium ionic centre. As indicated in both Figs.…”

Section: Glumentioning

confidence: 93%

Section: Introductionmentioning

confidence: 99%

Chemical mapping of the active site of the glucoamylase ofAspergillus niger

et al. 1996

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“…5 The three-dimensional structure of the Aspergillus awamori var. X100 GA catalytic domain has been obtained by x-ray crystallography at different pHs 6,7 and when complexed with the inhibitors 1-deoxinojirimycin, acarbose, D-gluco-dihydroacarbose, and D-glucono-1,5-lactone [8][9][10][11] and the monosaccharides D-glucose, D-mannose, and D-galactose (A. E. Aleshin and R. B. Honzatko, personal communication, 1996). The highly rigid GA active site is a well in the center of an (a/a) 6 barrel, with the two catalytic residues, the acid Glu179 and the base Glu400, at opposite sides of the cavity that also encloses a water molecule presumably required for catalysis.…”

Section: Introductionmentioning

confidence: 99%

Automated docking of monosaccharide substrates and analogues and methyl α-Acarviosinide in the glucoamylase active site

1997

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Glucoamylase is an important industrial glucohydrolase with a large specificity range. To investigate its interaction with the monosaccharides D-glucose, D-mannose, and D-galactose and with the substrate analogues 1-deoxynojirimycin, D-glucono-1,5-lactone, and methyl alpha-acarviosinide, MM3(92)-optimized structures were docked into its active site using AutoDock 2.1. The results were compared to structures of glucoamylase complexes obtained by protein crystallography. Charged forms of some substrate analogues were also docked to assess the degree of protonation possessed by glucoamylase inhibitors. Many forms of methyl alpha-acarviosinide were conformationally mapped by using MM3(92), characterizing the conformational pH dependence found for the acarbose family of glucosidase inhibitors. Their significant conformers, representing the most common states of the inhibitor, were used as initial structures for docking. This constitutes a new approach for the exploration of binding modes of carbohydrate chains. Docking results differ slightly from x-ray crystallographic data, the difference being of the order of the crystallographic error. The estimated energetic interactions, even though agreeing in some cases with experimental binding kinetics, are only qualitative due to the large approximations made by AutoDock force field.

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“…Structures of several u-amylases (Larson et al, 1994;Mizuno et al, 1993;Tao et al, 1989),/3-amylases (Cheong et al, 1995: Mikami et al, 1994, glucoamylase (Aleshin et al, 1992(Aleshin et al, , 1996 and complexes of these enzymes with inhibitors (Aleshin et aL, 1994;Bompard-gilles et al, 1996;Mikami et al, 1993: Stoffer et al, 1995 have provided valuable insights into their enzymatic mechanisms. However, there is no threedimensional structure of any enzyme cleaving the u-l,6-glycosidic linkage (MacGregor, 1993).…”

Section: Introductionmentioning

confidence: 99%

Preliminary X-ray crystallographic analysis of a novel maltogenic amylase from Bacillus stearothermophilus ET1

Cho

Cha

Park

et al. 1998

Acta Crystallogr D Biol Cryst

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A novel maltogenic amylase from Bacillus stearothermophilus ETI, which has a dual activity of u-l,4-and a-l,6-glycosidic bond cleavages and u-l,6-glycosidic bond formation, was crystallized by using the hanging-drop vapor-diffusion method. The best crystals were obtained by employing a high concentration of protein (56mgml '1) and a precipitant containing 22% glycerol, 1.6 M ammonium sulfate in 0.1 M Tris-HCI (pH 8.5). Native diffraction data to 2.66 A resolution have been obtained from crystals flash-frozen at 110 K. The crystals belong to the space group P2~212~ with unit-cell dimensions of a = 77.62, b = 121.23, c = 244.29 A, and contain three or four protomers per asymmetric unit. Structure determination by multiple isomorphous replacement is in progress.

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Refined structure for the complex of d‐gluco‐dihydroacarbose with glucoamylase from Aspergillus awamori var. X100 to 2.2 Å resolution: dual conformations for extended inhibitors bound to the active site of glucoamylase

Cited by 63 publications

References 15 publications

Chemical mapping of the active site of the glucoamylase ofAspergillus niger

Chemical mapping of the active site of the glucoamylase ofAspergillus niger

Automated docking of monosaccharide substrates and analogues and methyl α-Acarviosinide in the glucoamylase active site

Preliminary X-ray crystallographic analysis of a novel maltogenic amylase from Bacillus stearothermophilus ET1

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