Substrate recognition mechanism of α-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans
Substrate recognition mechanism of α-1,6-glucosidic linkage hydrolyzing enzyme, dextran glucosidase from Streptococcus mutans
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DOI：
10.1016/j.jmb.2008.03.016
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发表时间：
2008-05-09
影响因子：
5.6
通讯作者：
Kimura, Atsuo
中科院分区：
文献类型：
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作者：
Hondoh, Hironori;Saburi, Wataru;Kimura, Atsuo
我们已经确定了变形链球菌葡聚糖葡萄糖苷酶的晶体结构，该酶可从低聚异麦芽糖的非还原端水解 α-1,6-葡萄糖苷键，产生 α-葡萄糖。通过使用催化酸 Glu236 -> Gln 的突变体，其与异麦芽三糖（该酶的天然底物）的复合结构已被确定。该酶有 536 个氨基酸残基，分子量为 62,001 Da。通过分子置换方法确定天然结构和复杂结构，并精炼至 2.2 埃分辨率，最终导致天然结构显着反射的 R 因子为 18.3%，复杂结构显着反射的最终 R 因子为 18.4%。该酶由 A、B 和 C 三个结构域组成，并且在结构域 A 中具有 (β/α)(8)-桶，这是 α-淀粉酶家族酶所共有的。三个催化残基位于活性位点袋的底部，并且结合的异麦芽三糖占据子位点-1至+2。子位点-1处葡萄糖残基的环境与α-淀粉酶家族中该残基的环境相似。 Asp60 和 Arg398 之间的氢键以及子位点 -1 处葡萄糖单元的 O4 原子实现了对结合底物非还原端的识别。 Glu371 和 Lys275 的侧链原子与 +1 位点葡萄糖残基的 O2 和 O3 原子形成氢键。组成易剪 α-1,6-糖苷键（C1、O6 和 C6 原子）的原子位置与枯草芽孢杆菌 α-淀粉酶结构中麦芽五糖易剪 α-1,4 键（C1、O4 和 C4 原子）中的原子位置相同。与α-淀粉酶的比较表明，葡聚糖葡萄糖苷酶的Val195和α-1,6-水解酶的相应残基参与了这些酶的底物特异性的确定。 (c) 2008 Elsevier Ltd. 保留所有权利。
We have determined the crystal structure of Streptococcus mutans dextran glucosidase, which hydrolyzes the alpha-1,6-glucosidic linkage of isomaltooli-gosaccharides from their non-reducing ends to produce alpha-glucose. By using the mutant of catalytic acid Glu236 -> Gln, its complex structure with the isomaltotriose, a natural substrate of this enzyme, has been determined. The enzyme has 536 amino acid residues and a molecular mass of 62,001 Da. The native and the complex structures were determined by the molecular replacement method and refined to 2.2 angstrom resolution, resulting in a final R-factor of 18.3% for significant reflections in the native structure and 18.4% in the complex structure. The enzyme is composed of three domains, A, B and C, and has a (beta/alpha)(8)-barrel in domain A, which is common to the alpha-amylase family enzymes. Three catalytic residues are located at the bottom of the active site pocket and the bound isomaltotriose occupies subsites -1 to +2. The environment of the glucose residue at subsite -1 is similar to the environment of this residue in the alpha-amylase family. Hydrogen bonds between Asp60 and Arg398 and O4 atom of the glucose unit at subsite -1 accomplish recognition of the non-reducing end of the bound substrate. The side-chain atoms of Glu371 and Lys275 form hydrogen bonds with the O2 and O3 atoms of the glucose residue at subsite +1. The positions of atoms that compose the scissile alpha-1,6-glucosidic linkage (C1, O6 and C6 atoms) are identical with the positions of the atoms in the scissile alpha-1,4 linkage (C1, O4 and C4 atoms) of maltopentaose in the alpha-amylase structure from Bacillus subtilis. The comparison with the alpha-amylase suggests that Val195 of the dextran glucosidase and the corresponding residues of alpha-1,6-hydrolyzing enzymes participate in the determination of the substrate specificity of these enzymes. (c) 2008 Elsevier Ltd. All rights reserved.