CRYSTALLIZATION, MOLECULAR REPLACEMENT SOLUTION, AND REFINEMENT OF TETRAMERIC BETA-AMYLASE FROM SWEET-POTATO
CRYSTALLIZATION, MOLECULAR REPLACEMENT SOLUTION, AND REFINEMENT OF TETRAMERIC BETA-AMYLASE FROM SWEET-POTATO
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DOI：
10.1002/prot.340210204
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发表时间：
1995-02-01
影响因子：
2.9
通讯作者：
SUH, SW
中科院分区：
文献类型：
--
作者：
CHEONG, CG;EOM, SH;SUH, SW
甘薯β-淀粉酶是一个由222个对称亚基组成的四聚体，其亚基由498个氨基酸残基组成，Mr为55，880。用聚乙二醇1500作沉淀剂，在室温下结晶。晶体在2周内生长到0.4 mm × 0.4 mm × 1.0 mm的尺寸，属于四面体空间群P4（2）2（1）2，晶胞尺寸为a = B = 129.63埃和c = 68.42埃。不对称单位包含β-淀粉酶的1个亚基，每蛋白质质量的晶体体积（V-M）为2.57埃（3）/Da，溶剂含量为52%（体积）。以大豆β-淀粉酶的单体结构为起始模型，采用分子置换法测定了甘薯β-淀粉酶四聚体的三维结构。改进的亚基模型包含3，863个非氢蛋白质原子（488个氨基酸残基）和319个水氧原子。对于分辨率范围为8-2.3埃（截止值为2 σ）的数据，当前R值为20.3%，具有良好的立体化学。甘薯β-淀粉酶（在不存在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-淀粉酶的487个当量C α原子的均方根（RMS）差为0.96埃。甘薯β-淀粉酶的每个亚基由大（α/β）（8）核心结构域、由三个长环[L3（残基91-150）、L4（残基183-258）和L5（残基300-327）]组成的小核心结构域和由残基445-493形成的长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）桶核心和由三个长环（L3，L4和L5）组成的小结构域之间的裂缝处。保守的Cys 96，在巯基试剂灭活酶活性中很重要，位于（α/β）（8）桶的入口处。(C)1995 Wiley-Liss，Inc.
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 Angstrom and c = 68.42 Angstrom. The asymmetric unit contains 1 subunit of beta-amylase, with a crystal volume per protein mass (V-M) Of 2.57 Angstrom(3)/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 Angstrom (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 Angstrom. 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. (C) 1995 Wiley-Liss, Inc.