Refined Crystal Structures of Glucoamylase from Aspergillus awamori var. X100

Aleshin, Alexander E.; Hoffman, Chad R.; Firsov, L. M.; Honzatko, Richard B.

doi:10.1006/jmbi.1994.1316

Cited by 172 publications

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“…Twelve of these a-helices fold into an a/a barrel, with the core of the barrel defining a pocket containing the general catalytic acid, Glul79 Svensson et al, 1990), and the catalytic base, Glu400 (Aleshin et al, , 1994b. Thus the catalytic domain of GA is not a trivial variant of the o/p-barrel, observed in a-and Pamylases (Matsuura et al, 1984;Buisson et al, 1987;Boel et al, 1990;Mikami et al, 1992Mikami et al, , 1993, cyclodextrin glucanosyltransferases (Hofinann et al, 1989;Kubota et al, 1990;Klein and Schulz, 1991), cellobiohydrolases (Rouvinen et al, 1990), and predicted for a large N-lcrin…”

Section: Table Of Contentsmentioning

confidence: 99%

“…The residues above either hydrogenbond to or make nonbonded contacts with substrate analogues, as revealed by X-ray difeaction studies (Aleshin et al, ,1994a(Aleshin et al, ,b, 1996. In addition, mutations have been made at Asp309, Glu400, and Gln401, residues directly involved in enzyme-Iigand interactions.…”

Section: Protein Engineering Of Gamentioning

confidence: 99%

“…have assigned Glul79 to the limit ing pKa value of 5.8 and suggested it as the acid catalyst Results of X-ray diffraction studies are consistent with Glul79 as the catalytic acid , but suggest instead that the ionizable group with pKa of 5.8 is a hydronium ion bound to the active site in the absence of the substrate and that Glul79 becomes protonated only in the presence of substrate (Aleshin et al, 1994a). Crystallographic studies of GA-ligand complexs assign Glu400 to the 20 role of catalytic base , a role confirmed by site-directed mutation (Frandsen er a/., 1994).…”

Section: Catalytic Properties Of Gamentioning

confidence: 99%

“…A water molecule, wtSOO, presumably the catalytic water required for general acid-base catalysis, is also found in the first subsite of the model with each complex (Aleshin et al, 1994a). The automated docking of different substrates in the GA active site has given us some insight into substrate binding, even though the results differ slightly from X-ray crystallographic data (Coutinho et al,I997a,b).…”

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confidence: 99%

“…Complexes of GA with different inhibitors suggest that the 6-OH group of the residue at subsite +1 projects into a void large enough to accommodate an additional glucosyl residue (Aleshin et aL, 1994a), especially when it is linked through an a-l,6-branch from the former residue (Aleshin and Honzatko, personal communication, 1995). This has been recently modeled by substrate docking (Coutinho et aL, 1997c).…”

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Mutational analysis of Aspergillus awamori glucoamylase selectivity to improve glucose yield

Liu

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This manuscript has been reproduced from the microfilm master. UME films the t®ct directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter fece, while others may be from any type of computer printer.The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction.In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletionOversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book.

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Section: Table Of Contentsmentioning

confidence: 99%

Section: Protein Engineering Of Gamentioning

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Section: Catalytic Properties Of Gamentioning

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Mutational analysis of Aspergillus awamori glucoamylase selectivity to improve glucose yield

Liu

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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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Automated docking of glucosyl disaccharides in the glucoamylase active site

Coutinho¹,

Dowd²,

Reilly³

1997

Proteins

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To better understand the molecular basis of glucomylase selectivity, low-energy conformers of glucosyl disaccharides obtained from relaxed-residue conformational mapping were flexibly docked into the glucoamylase active site using AutoDock 2.2. This procedure ensures that significant conformational space is searched and can produce bound structures comparable to those obtained by protein crystallography. alpha-Linked glucosyl disaccharides except alpha,alpha-trehalose dock easily into the active site while exclusively beta-linked disaccharides do not, explaining why only the former are glucoamylase substrates. The optimized docking modes are similar at the nonreducing end of the different substrates. Individual atomic energies of intermolecular interaction allow the definite identification of key hydroxyl groups for each substrate. This approach confirmed the versatility of the second subsite of the glucoamylase active site in binding different substrates.

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Refined Crystal Structures of Glucoamylase from Aspergillus awamori var. X100

Cited by 172 publications

References 0 publications

Mutational analysis of Aspergillus awamori glucoamylase selectivity to improve glucose yield

Mutational analysis of Aspergillus awamori glucoamylase selectivity to improve glucose yield

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

Automated docking of glucosyl disaccharides in the glucoamylase active site

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