Structure of Bacillus halmapalus alpha-amylase crystallized with and without the substrate analogue acarbose and maltose.
Recombinant Bacillus halmapalus alpha-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 A. 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 A. 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 A. 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 A. 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.
Lyhne-Iversen
Louise
L
Department of Chemistry, Technical University of Denmark, Building 207, DK-2800 Kgs. Lyngby, Denmark.
Hobley
Timothy J
TJ
Kaasgaard
Svend G
SG
Harris
Pernille
P
D020909:Acarbose / Q000737:chemistry* / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D008320:Maltose / Q000737:chemistry* / Q000378:metabolism; D058619:Maltose-Binding Proteins; D013379:Substrate Specificity; D000516:alpha-Amylases / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D008320:Maltose; D000516:alpha-Amylases; D020909:Acarbose