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

Abstract

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.

Crystal Structure of a Maltogenic Amylase Provides Insights into a Catalytic Versatility

“…Contrary to our earlier prediction that the asymmetric unit of BSMA crystals would contain three to four molecules (13), only one dimer is present with a high solvent content of 70.1%. The BSMA structure is very similar to that of ThMA and is not discussed in detail in this report.…”

“…Crystallization-Initial effort was put into the structure determination of Bacillus stearothermophilus maltogenic amylase (BSMA) whose crystallization condition we have reported (13). We suffered because of fragility of the crystals, especially in the presence of a heavy metal compound, and subsequently switched to the crystallization of ThMA which led to the structure determination of the both enzymes.…”