Functional significance of active site residues in the enzymatic component of the Clostridium difficile binary toxin.
Functional significance of active site residues in the enzymatic component of the Clostridium difficile binary toxin.
复制标题
DOI：
10.1016/j.bbrep.2016.08.011
复制
发表时间：
2016-12
影响因子：
2.7
通讯作者：
Acharya KR
中科院分区：
文献类型：
--
作者：
Davies AH;McGlashan J;Posner MG;Roberts AK;Shone CC;Acharya KR
艰难梭菌二元毒素（CDT）是一种ADP-核糖基转移酶，与C.艰难菌株CDT具有双重功能：结构域a（CDTa）催化肌动蛋白的ADP-核糖基化（酶组分），而结构域B（CDTb）将CDTa转运到胞质溶胶中（转运组分）。了解CDT的分子机制对于评估其在C.艰难感染鉴定对CDTa功能至关重要的氨基酸可能有助于药物抑制剂设计以控制C.艰难感染。在这里，我们报告了CDTa内的关键催化残基的突变及其对CDT细胞毒性的影响。CDTa突变体的活性随氨基酸取代的类型而变化，而不是全或无反应; S345 A保留细胞毒性，而S345 Y足以使CDT无细胞毒性。因此，CDTa细胞毒性水平与ADP-核糖基转移酶活性直接相关。ADP-核糖基转移酶活性决定来自艰难梭菌的CDTa的细胞毒性。CDT ADP-核糖基化遵循SN 1机制。单个氨基酸突变足以削弱CDTa细胞毒性。
Clostridium difficile binary toxin (CDT) is an ADP-ribosyltransferase which is linked to enhanced pathogenesis of C. difficile strains. CDT has dual function: domain a (CDTa) catalyses the ADP-ribosylation of actin (enzymatic component), whereas domain b (CDTb) transports CDTa into the cytosol (transport component). Understanding the molecular mechanism of CDT is necessary to assess its role in C. difficile infection. Identifying amino acids that are essential to CDTa function may aid drug inhibitor design to control the severity of C. difficile infections. Here we report mutations of key catalytic residues within CDTa and their effect on CDT cytotoxicity. Rather than an all-or-nothing response, activity of CDTa mutants vary with the type of amino acid substitution; S345A retains cytotoxicity whereas S345Y was sufficient to render CDT non-cytotoxic. Thus CDTa cytotoxicity levels are directly linked to ADP-ribosyltransferase activity. ADP-ribosyltransferase activity determines cytotoxicity of CDTa from Clostridium difficile. CDT ADP-ribosylation follows SN1 mechanism. A single amino acid mutation is sufficient to impair CDTa cytotoxicity.