The Listeria innocua chitinase LinChi78 has a unique region that is necessary for hydrolytic activity
The Listeria innocua chitinase LinChi78 has a unique region that is necessary for hydrolytic activity
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无害李斯特菌几丁质酶 LinChi78 具有水解活性所必需的独特区域
DOI：
10.1007/s00253-018-9573-5
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发表时间：
2019
影响因子：
5
通讯作者：
Sakaguchi Masayoshi
中科院分区：
文献类型：
--
作者：
Honda Shotaro;Kimura Masahiro;Wakita Satoshi;Oka Yuji;Kawakita Masao;Oyama Fumitaka;Sakaguchi Masayoshi
几丁质酶一般由多个结构域组成；催化结构域和一个或多个并非绝对必需但可能改变几丁质分解活性的附加结构域。来自无害李斯特菌的 LinChi78 几丁质酶具有催化结构域 (CatD)、纤连蛋白 III 型样 (FnIII) 结构域、几丁质结合结构域 (ChBD) 以及位于 CatD 和 FnIII 结构域之间的未知功能区 (UFR)。 UFR 长度为 146 个氨基酸残基，在保守域数据库中没有同源域。我们使用 LinChi78 的几个 C 端和内部删除突变体对这些结构域和 UFR 进行了功能分析。人工底物的水解几乎不受 ChBD 和/或 FnIII 结构域缺失的影响，尽管 ChBD 缺失酶对胶体几丁质的活性比 LinChi78 低约 30%。另一方面，UFR 的缺失导致几丁质酶对人工底物和聚合物底物的活性大量丧失。经过进一步分析，我们发现 UFR 中第 567 个 (G) 和第 571 个 (I) 氨基酸残基之间的 GKQTI 延伸对于 LinChi78 活性至关重要，并证明 Gln569 和 Ile571 在引发该活性中发挥核心作用。总而言之，这些结果表明 LinChi78 具有由典型 CatD 和活性必需的附加区域组成的独特催化区域。 LinChi78 独特催化区域的表征将提高我们对 GH18 几丁质酶的理解。
Chitinases are generally composed of multiple domains; a catalytic domain and one or more additional domains that are not absolutely required but may modify the chitinolytic activity. The LinChi78 chitinase fromListeria innocuahas a catalytic domain (CatD), a fibronectin type III-like (FnIII) domain, a chitin-binding domain (ChBD), and an unknown-function region (UFR) located between the CatD and FnIII domains. The UFR is 146 amino acid residues in length and does not have a homologous domain in the Conserved Domain Database. We performed a functional analysis of these domains and the UFR using several C-terminally and internally deleted mutants of LinChi78. Hydrolysis of an artificial substrate was almost unaffected by deletion of the ChBD and/or the FnIII domain, although the ChBD-deleted enzymes were approximately 30% less active toward colloidal chitin than LinChi78. On the other hand, deletion of the UFR led to an extensive loss of chitinase activity toward an artificial substrate as well as polymeric substrates. Upon further analysis, we found that the GKQTI stretch, between the 567th (G) and 571th (I) amino acid residues, in the UFR is critical for LinChi78 activity and demonstrated that Gln569 and Ile571 play central roles in eliciting this activity. Taken together, these results indicated that LinChi78 has a unique catalytic region composed of a typical CatD and an additional region that is essential for activity. Characterization of the unique catalytic region of LinChi78 will improve our understanding of GH18 chitinases.