Cheong C G, Eom S H, Chang C, Shin D H, Song H K, Min K, Moon J H, Kim K K, Hwang K Y, Suh S W
Department of Chemistry, College of Natural Sciences, Seoul National University, Korea.
Proteins. 1995 Feb;21(2):105-17. doi: 10.1002/prot.340210204.
Sweet potato beta-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm x 0.4 mm x 1.0 mm within 2 weeks, belong to the tetragonal space group P4(2)2(1)2 with unit cell dimensions of a = b = 129.63 A and c = 68.42 A. The asymmetric unit contains 1 subunit of beta-amylase, with a crystal volume per protein mass (VM) of 2.57 A3/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric beta-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8-2.3 A (with 2 sigma cut-off) with good stereochemistry. The subunit structure of sweet potato beta-amylase (crystallized in the absence of alpha-cyclodextrin) is very similar to that of soybean beta-amylase (complexed with alpha-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent C alpha atoms of the two beta-amylases is 0.96 A. Each subunit of sweet potato beta-amylase is composed of a large (alpha/beta)8 core domain, a small one made up of three long loops [L3 (residues 91-150), L4 (residues 183-258), and L5 (residues 300-327)], and a long C-terminal loop formed by residues 445-493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (alpha/beta)8 barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (alpha/beta)8 barrel.
甘薯β-淀粉酶是由相同亚基组成的四聚体，这些亚基排列呈现出222分子对称性。其亚基由498个氨基酸残基组成（分子量55,880）。它已在室温下使用聚乙二醇1500作为沉淀剂进行结晶。晶体在2周内生长到尺寸为0.4毫米×0.4毫米×1.0毫米，属于四方晶系空间群P4(2)2(1)2，晶胞参数为a = b = 129.63 Å，c = 68.42 Å。不对称单元包含1个β-淀粉酶亚基，每蛋白质质量的晶体体积（VM）为2.57 ų/Da，溶剂含量为52%（体积）。甘薯四聚体β-淀粉酶的三维结构已通过分子置换法确定，使用大豆酶的单体结构作为起始模型。优化后的亚基模型包含3,863个非氢蛋白质原子（488个氨基酸残基）和319个水氧原子。对于8 - 2.3 Å分辨率范围内的数据（2σ截止），当前R值为20.3%，立体化学良好。甘薯β-淀粉酶（在没有α-环糊精的情况下结晶）的亚基结构与大豆β-淀粉酶（与α-环糊精复合）的亚基结构非常相似。两种β-淀粉酶487个等效Cα原子的均方根（RMS）差异为0.96 Å。甘薯β-淀粉酶的每个亚基由一个大的(α/β)8核心结构域、一个由三个长环[L3（残基91 - 150）、L4（残基183 - 258）和L5（残基300 - 327）]组成的小结构域以及由残基445 - 493形成的一个长的C末端环组成。保守的Glu 187被认为在催化中起重要作用，位于(α/β)8桶状核心与由三个长环（L3、L4和L5）组成的小结构域之间的裂隙处。保守的Cys 96在巯基试剂使酶活性失活中起重要作用，位于(α/β)8桶状结构的入口处。
