Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum
Structure and mechanism of chitin deacetylase from the fungal pathogen Colletotrichum lindemuthianum
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DOI：
10.1021/bi0606694
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发表时间：
2006-08-08
期刊：
影响因子：
2.9
通讯作者：
van Aalten, Daan M. F.
中科院分区：
文献类型：
--
作者：
Blair, David E.;Hekmat, Omid;van Aalten, Daan M. F.
真菌病原菌炭疽杆菌（Colletotrichum lindemuthianum）在侵入和侵染植物过程中分泌一种内源性几丁质去n -乙酰化酶（ClCDA）来修饰暴露的菌丝几丁质。虽然真菌几丁质去n -乙酰化酶的生物化学数据大量可用，但没有结构数据存在。本文描述了ClCDA产物配合物的1.8埃晶体结构，并利用Hammett线性自由能关系、亚位探测和原子吸收光谱分析了反应机理。结构数据结合生化数据表明，ClCDA由一个单核金属酶组成，该金属酶利用与保守催化碱（天冬氨酸）和酸（组氨酸）密切相关的保守His-HisAsp锌结合三联体进行酸/碱催化。本文提供的数据表明，ClCDA具有高度保守的底物结合槽，其细微变化会影响底物特异性和亚位点亲和力。引人注目的是，该结构还表明，六组氨酸纯化标签似乎与活性位点凹槽形成紧密的相互作用。该酶需要（GlcNAc） 2至少占据0和+1亚位才能发挥活性，并通过四面体氧阴离子中间体进行。
The fungal pathogen Colletotrichum lindemuthianum secretes an endo-chitin de-N-acetylase (ClCDA) to modify exposed hyphal chitin during penetration and infection of plants. Although a significant amount of biochemical data is available on fungal chitin de-N-acetylases, no structural data exist. Here we describe the 1.8 angstrom crystal structure of a ClCDA product complex and the analysis of the reaction mechanism using Hammett linear free energy relationships, subsite probing, and atomic absorption spectroscopy studies. The structural data in combination with biochemical data reveal that ClCDA consists of a single domain encompassing a mononuclear metalloenzyme which employs a conserved His-HisAsp zinc-binding triad closely associated with the conserved catalytic base (aspartic acid) and acid (histidine) to carry out acid/base catalysis. The data presented here indicate that ClCDA possesses a highly conserved substrate-binding groove, with subtle alterations that influence substrate specificity and subsite affinity. Strikingly, the structure also shows that the hexahistidine purification tag appears to form a tight interaction with the active site groove. The enzyme requires occupancy of at least the 0 and +1 subsites by (GlcNAc) 2 for activity and proceeds through a tetrahedral oxyanion intermediate.