Crystal structure of glycoside hydrolase family 78 α-L-rhamnosidase from Bacillus sp GL1
Crystal structure of glycoside hydrolase family 78 α-L-rhamnosidase from Bacillus sp GL1
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DOI：
10.1016/j.jmb.2007.09.003
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发表时间：
2007-11-23
影响因子：
5.6
通讯作者：
Murata, Kousaku
中科院分区：
文献类型：
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作者：
Cui, Zhongli;Maruyama, Yukie;Murata, Kousaku
α-L-鼠李糖苷酶(EC 3.2.1.40)催化从多糖和糖苷中分解释放鼠李糖。芽孢杆菌属GL1α-L-鼠李糖苷酶是糖苷水解酶(GH)家族78的成员之一，负责降解细菌生物膜结冷胶，并通过从植物糖苷-柚皮苷中释放鼠李糖，在食品和饮料工业中作为柑橘类水果的脱苦剂。用单波长反常衍射法测定了RhaB的X-射线晶体结构，用硒蛋氨酸衍生物，并在1.9埃分辨率下进行了精修，最终R因子为18.2%。从活性酶的同源二聚体中可以看出，RhaB在晶体堆积中的结构是一个同源二聚体，包含1908个氨基酸(残基3-956)、43个甘油分子、4个钙离子和1755个水分子。整个结构由5个结构域组成，其中4个结构域为β-夹心结构，命名为N、D1、D2和C结构域，以及一个(α/α)(6)桶结构，命名为A结构域。DALI的结构比较表明，RhaB与壳二糖磷酸化酶具有最高的结构相似性(Z值为25.3)。还测定了RhaB与反应产物鼠李糖(抑制常数，K-I=1.8 mm)形成的络合物的结构，并在2.1埃处进行了精制，最终R因子为19.5%。鼠李糖结合在(α/α)(6)桶结构域的深裂缝上，就像在L家族GHS中看到的那样。GH家族78酶中保守的几个带负电荷的残基，如Asp567、Glu572、Asp579和Glu841与鼠李糖相互作用，这些残基的RhaB突变体显著降低了酶的活性，表明这些残基对酶催化和/或底物结合是至关重要的。据我们所知，这是首次测定α-L-鼠李糖苷酶的晶体结构，并确定其族L(α/α)(6)桶为催化结构域。(C)2007爱思唯尔有限公司。保留所有权利。
alpha-L-Rhamnosidase (EC 3.2.1.40) catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides. Bacillus sp. GL1 alpha-L-rhamnosidase (RhaB), a member of glycoside hydrolase (GH) family 78, is responsible for degrading the bacterial biofilm gellan, and also functions as a debittering agent for citrus fruit in the food and beverage industries through the release of rhamnose from plant glycoside, naringin. The X-ray crystal structure of RhaB was determined by single-wavelength anomalous diffraction using a selenomethionine derivative and refined at 1.9 angstrom resolution with a final R-factor of 18.2%. As is seen in the homodimeric form of the active enzyme, the structure of RhaB in crystal packing is a homodimer containing 1908 amino acids (residues 3-956), 43 glycerol molecules, four calcium ions, and 1755 water molecules. The overall structure consists of five domains, four of which are beta-sandwich structures designated as domains N, D1, D2, and C, and an (alpha/alpha)(6)-barrel structure designated as domain A. Structural comparison by DALI showed that RhaB shares its highest level of structural similarity with chitobiose phosphorylase (Z score of 25.3). The structure of RhaB in complex with the reaction product rhamnose (inhibitor constant, K-i = 1.8 mM) was also determined and refined at 2.1 angstrom with a final R-factor of 19.5%. Rhamnose is bound to the deep cleft of the (alpha/alpha)(6)-barrel domain, as is seen in the clan-L GHs. Several negatively charged residues, such as Asp567, Glu572, Asp579, and Glu841, conserved in GH family 78 enzymes, interact with rhamnose, and RhaB mutants of these residues have drastically reduced enzyme activity, indicating that the residues are crucial for enzyme catalysis and/or substrate binding. To our knowledge, this is the first report on the determination of the crystal structure of alpha-L-rhamnosidase and identification of its clan-L (alpha/alpha)(6)-barrel as a catalytic domain. (c) 2007 Elsevier Ltd. All rights reserved.