Hwang K Y, Song H K, Chang C, Lee J, Lee S Y, Kim K K, Choe S, Sweet R M, Suh S W
Department of Chemistry, Seoul National University, Korea.
Mol Cells. 1997 Apr 30;7(2):251-8.
alpha-Amylases (alpha-1,4-glucan-4-glucanohydrolase, E.C.3.2.1.1) catalyze the cleavage of alpha-1, 4-glucosidic linkages of starch components, glycogen, and various oligosaccharides. Thermostable alpha-amylases from Bacillus species are of great industrial importance in the production of corn syrup or dextrose. Thermostable alpha-amylase from Bacillus licheniformis, a monomeric enzyme with molecular mass of 55,200 Da (483 amino acid residues), shows a remarkable heat stability. This enzyme provides an attractive model for investigating the structural basis for thermostability of proteins. The three-dimensional structure of thermostable alpha-amylase from Bacillus licheniformis has been determined by the multiple isomorphous replacement method of X-ray crystallography. The structure has been refined to a crystallographic R-factor of 19.9% for 58,601 independent reflections with F0 > 2 sigma F0 between 8.0 and 1.7 A resolution, with root mean square deviations of 0.013 A from ideal bond lengths and 1.72 degrees from ideal bond angles. The final model consists of 469 amino acid residues and 294 water molecules. Missing from the model are the N- and C-termini and the segment between Trp182 and Asn192. Like other alpha-amylases, the polypeptide chain folds into three distinct domains. The first domain (domain A), consisting of 291 residues (from residue 3 to 103 and 207 to 396), forms a (beta/alpha)8-barrel structure. The second domain (domain B), consisting of residues 104 to 206, is inserted between the third beta-strand and the third alpha-helix of domain A. The third C-terminal domain (domain C), consisting of residues 397 to 482, folds into an eight-stranded antiparallel beta-barrel. Neither calcium ion nor chloride ion is located near the active site. This study reveals the architecture of the thermostable alpha-amylase from Bacillus licheniformis. By homology with other alpha-amylases, important active site residues can be identified as Asp231, Glu261, and Asp328, which are all located at the C-terminal end of the central (beta/alpha)8-barrel. Since many of the stabilizing and destabilizing mutations obtained so far fall in domain B or at its border, this region of the enzyme appears to be important for thermostability. The factors responsible for the remarkable thermostability of this enzyme may be increased ionic interactions, reduced surface area, and increased packing interactions in the interior.
α-淀粉酶（α-1,4-葡聚糖-4-葡聚糖水解酶，E.C.3.2.1.1）催化淀粉成分、糖原和各种寡糖的α-1,4-糖苷键的裂解。芽孢杆菌属的耐热α-淀粉酶在玉米糖浆或葡萄糖的生产中具有重要的工业价值。地衣芽孢杆菌的耐热α-淀粉酶是一种分子量为55200道尔顿（483个氨基酸残基）的单体酶，具有显著的热稳定性。这种酶为研究蛋白质热稳定性的结构基础提供了一个有吸引力的模型。地衣芽孢杆菌耐热α-淀粉酶的三维结构已通过X射线晶体学的多重同晶置换法确定。该结构已针对58601个独立反射（F0>2σF0，分辨率在8.0至1.7埃之间）精修至晶体学R因子为19.9%，与理想键长的均方根偏差为0.013埃，与理想键角的偏差为1.72度。最终模型由469个氨基酸残基和294个水分子组成。模型中缺少N端和C端以及Trp182和Asn192之间的片段。与其他α-淀粉酶一样，多肽链折叠成三个不同的结构域。第一个结构域（结构域A）由291个残基组成（从第3个残基到第103个残基以及从第207个残基到第396个残基），形成一个（β/α）8桶状结构。第二个结构域（结构域B）由第104至206个残基组成，插入到结构域A的第三个β链和第三个α螺旋之间。第三个C端结构域（结构域C）由第397至482个残基组成，折叠成一个八链反平行β桶状结构。活性位点附近既没有钙离子也没有氯离子。这项研究揭示了地衣芽孢杆菌耐热α-淀粉酶的结构。通过与其他α-淀粉酶的同源性比较，可以确定重要的活性位点残基为Asp231、Glu261和Asp328，它们都位于中央（β/α）8桶状结构的C端。由于到目前为止获得的许多稳定和不稳定突变都位于结构域B或其边界，该酶的这一区域似乎对热稳定性很重要。导致这种酶具有显著热稳定性的因素可能是离子相互作用增加、表面积减小以及内部堆积相互作用增加。
