The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.
The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.
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阿拉伯糖基转移酶结核分枝杆菌 EmbC 的 C 末端结构域是一种凝集素样碳水化合物结合模块。
DOI：
10.1371/journal.ppat.1001299
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发表时间：
2011-02
期刊：
影响因子：
6.7
通讯作者：
Besra GS
中科院分区：
文献类型：
--
作者：
Alderwick LJ;Lloyd GS;Ghadbane H;May JW;Bhatt A;Eggeling L;Fütterer K;Besra GS
含d-阿拉伯聚糖的聚合物阿拉伯半乳聚糖（AG）和脂阿拉伯甘露聚糖（LAM）是病原体结核分枝杆菌的独特细胞包膜的基本组分。AG和LAM的生物合成涉及一系列膜包埋的阿拉伯呋喃糖基（Araf）转移酶，其结构在很大程度上是未表征的，尽管事实上，它们中的一些是乙胺丁醇的药理学靶点，乙胺丁醇是结核病治疗的一线药物。在此，我们提出了乙胺丁醇敏感的Araf转移酶M的C-末端亲水结构域的晶体结构。结核EmbC，其对于LAM合成是必需的。EmbC的C-末端结构域（EmbCCT）的结构包括两个不同折叠的亚结构域，其中亚结构域II显示出与凝集素样碳水化合物结合模块（CBM）的明显相似性。与细胞壁衍生的二阿拉伯糖苷受体类似物的共结晶和与配体结合的CBM的结构比较表明，EmbCCT含有两个单独的碳水化合物结合位点，分别与亚结构域I和II相关。保守的色氨酸残基（Trp 868，Trp 985）在这些各自的网站单残基取代抑制EmbC催化的LAM的延伸。相同的取代差异废除的二-和五-阿拉伯呋喃糖苷受体类似物的结合EmbCCT，连接的活性损失受损受体底物结合，表明存在两个单独的碳水化合物结合位点，并证明亚结构域II确实作为一个碳水化合物结合模块的功能。这项工作提供了第一步解开的结构和功能的GT-C型糖基转移酶是必不可少的M。结核结核病（TB）是由结核分枝杆菌（Mycobacterium tuberculosis）引起的一种传染病，给世界上大部分人口造成负担。治疗活动性结核病通常需要在几个月内服用抗生素鸡尾酒，其中包括药物乙胺丁醇。这种一线化合物抑制一组阿拉伯糖基转移酶，称为EmbA、EmbB和EmbC，这些酶对阿拉伯聚糖的合成至关重要，阿拉伯聚糖是病原体独特细胞包膜中的重要多糖。乙胺丁醇如何精确地抑制阿拉伯糖基转移酶活性尚不清楚，部分原因是其药理学靶点的结构信息一直难以捉摸。在这里，我们报告的乙胺丁醇目标EmbC，一个390个氨基酸的片段，负责受体底物识别的C-末端结构域的高分辨率结构。结合X-射线晶体学分析与结构比较，定点诱变，活性和配体结合试验，我们确定了两个区域的C-末端结构域的EmbC能够结合受体底物模拟物，是全长酶的活性的关键。我们的研究结果开始定义结构不明的膜包埋糖基转移酶家族的结构-功能关系，这是结核病治疗的重要靶点。
The d-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbCCT) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbCCT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbCCT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis. Tuberculosis (TB), an infectious disease caused by the bacillus Mycobacterium tuberculosis, burdens large swaths of the world population. Treatment of active TB typically requires administration of an antibiotic cocktail over several months that includes the drug ethambutol. This front line compound inhibits a set of arabinosyltransferase enzymes, called EmbA, EmbB and EmbC, which are critical for the synthesis of arabinan, a vital polysaccharide in the pathogen's unique cell envelope. How precisely ethambutol inhibits arabinosyltransferase activity is not clear, in part because structural information of its pharmacological targets has been elusive. Here, we report the high-resolution structure of the C-terminal domain of the ethambutol-target EmbC, a 390-amino acid fragment responsible for acceptor substrate recognition. Combining the X-ray crystallographic analysis with structural comparisons, site-directed mutagenesis, activity and ligand binding assays, we identified two regions in the C-terminal domain of EmbC that are capable of binding acceptor substrate mimics and are critical for activity of the full-length enzyme. Our results begin to define structure-function relationships in a family of structurally uncharacterised membrane-embedded glycosyltransferases, which are an important target for tuberculosis therapy.
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影响因子：
4.3
作者：
Lee, RE;Brennan, PJ;Besra, GS
通讯作者：
Besra, GS
影响因子：
5.8
作者：
Gouet, P;Courcelle, E;Métoz, F
通讯作者：
Métoz, F
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
2.8
作者：
Amin, Anita G.;Goude, Renan;Parish, Tanya
通讯作者：
Parish, Tanya
影响因子：
5.7
作者：
Jamal-Talabani, S;Boraston, AB;Davies, GJ
通讯作者：
Davies, GJ