Structure ofBacillus halmapalusα-amylase crystallized with and without the substrate analogue acarbose and maltose

Abstract

PDB References: -amylase complexed with acarbose and maltose, 2gjp, r2gjpsf; uncomplexed, 2gjr, r2gjrsf.Recombinant Bacillus halmapalus -amylase (BHA) was studied in two different crystal forms. The first crystal form was obtained by crystallization of BHA at room temperature in the presence of acarbose and maltose; data were collected at cryogenic temperature to a resolution of 1.9 Å . It was found that the crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 47.0, b = 73.5, c = 151.1 Å . A maltose molecule was observed and found to bind to BHA and previous reports of the binding of a nonasaccharide were confirmed. The second crystal form was obtained by pH-induced crystallization of BHA in a MES-HEPES-boric acid buffer (MHB buffer) at 303 K; the solubility of BHA in MHB has a retrograde temperature dependency and crystallization of BHA was only possible by raising the temperature to at least 298 K. Data were collected at cryogenic temperature to a resolution of 2.0 Å . The crystal belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 38.6, b = 59.0, c = 209.8 Å . The structure was solved using molecular replacement. The maltose-binding site is described and the two structures are compared. No significant changes were seen in the structure upon binding of the substrates.

Acarbose and the thermal aggregation ofBacillus amyloliquefaciensalpha-amylase (BAA): protective effect of an inhibitor

“…. Acarbose was found to interact with the active site residues, which is overall in accordance with the binding mode of acarbose in other alpha‐amylase types . The AGGRESCAN program detected several areas in the amino acid sequences which tend to aggregate.…”

Altered Large-Ring Cyclodextrin Product Profile Due to a Mutation at Tyr-172 in the Amylomaltase of Corynebacterium glutamicum

“…In CGTase, an enzyme that resembles AM in the catalytic reaction but that differs in structure, the aromatic amino acid residue involved in the reaction specificity was identified to be outside the catalytic-site region (17). A second substrate binding site has also been reported in the ␣-amylase family, such as in bacteria (20,25), archaea (24), and plants (32). The decrease in k cat and the modest decrease in the observed K m toward malto-oligosaccharides were observed in the Y380A barley R ␣-amylase I, whereby position 380 is the binding site outside the catalytic region (4,28).…”

“…Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) (7.5% [wt/vol]) was carried out on a Bio-Rad Mini-Protein III gel apparatus (Bio-Rad Laboratories, Hercules, MA) using the Laemmli buffer system (25 …”

Crystal Structure of the Polyextremophilic α-Amylase AmyB from Halothermothrix orenii: Details of a Productive Enzyme–Substrate Complex and an N Domain with a Role in Binding Raw Starch

“…An explanation for the different outcome has been offered by Davies et al: the transglycosylation product in BHA is suggested to result from the initial formation of a covalent intermediate from acarbose and a series of subsequent successive transglycosylation events with maltose, rather than with acarbose as in BA2. 33 In addition to the oligosaccharide bound in the A-B groove, AmyB ACR contains an unmodified acarbose molecule bound to subsites A-D formed on the C domain in a groove between the N domain and the C domain (i.e., the N-C groove) (Fig. 3f).…”