Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase
Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase
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菊欧文氏菌 Cel5（内切葡聚糖酶 Z）和大肠杆菌推定几丁质酶的碳水化合物结合域的功能分析
DOI：
10.1128/jb.181.15.4611-4616.1999
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发表时间：
1999
影响因子：
3.2
通讯作者：
F. Barras
中科院分区：
文献类型：
--
作者：
H. Simpson;F. Barras
切氏欧文氏菌Cel5纤维素酶（以前称为内切葡聚糖酶Z）是一种多结构域酶，由催化结构域、连接区和纤维素结合结构域（CBD）组成。CBDCel 5的三维结构先前已通过核磁共振获得。为了确定单个残基在纤维素结合中的作用，进行定点诱变。三个芳香族残基（Trp18，Trp43和Tyr44）在纤维素结合中的作用被证明。暴露的潜在氢键供体，残基Gln22和Glu27，似乎没有发挥纤维素结合的作用，而残基Asp17被发现是重要的Cel5的稳定性。缺失突变体缺乏残基Asp17至Pro23仅弱结合纤维素。CBDCel 5的序列与来自大肠杆菌的推定的大蛋白质（称为Yheb）的一系列五个重复结构域具有同源性。其中一个重复结构域（Yheb1）由67个氨基酸组成，是从大肠杆菌中克隆的。大肠杆菌染色体，并通过金属螯合层析纯化。CBDCel 5与纤维素和几丁质结合，Yheb1与几丁质结合良好，但与纤维素结合很差。Yheb蛋白含有与几丁质酶的催化结构域具有序列同源性的区域，这与Yheb蛋白是几丁质酶的假设一致。
ABSTRACT The Cel5 cellulase (formerly known as endoglucanase Z) fromErwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBDCel5 has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBDCel5 exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBDCel5 bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.