Chitinase D (designated as Pc-ChiD) was found in a hyperthermophilic archaeon, Pyrococcus chitonophagus (previously described as Thermococcus chitonophagus), that was isolated from media containing only chitin as carbon source. Pc-ChiD displays chitinase activity and is thermostable at temperatures up to 95°C, suggesting its potential for industrial use. Pc-ChiD has a secretion signal peptide and two chitin-binding domains (ChBDs) in the N-terminal domain. However, the C-terminal domain shares no sequence similarity with previously identified saccharide-degrading enzymes and does not contain the DXDXE motif conserved in the glycoside hydrolase (GH) 18 family chitinases. To elucidate its overall structure and reaction mechanism, we determined the first crystal structures of Pc-ChiD, both in the ligand-free form and in complexes with substrates. Structure analyses revealed that the C-terminal domain of Pc-ChiD, Pc-ChiD(ΔBD), consists of a third putative substrate-binding domain, which cannot be predicted from the amino acid sequence, and a catalytic domain structurally similar to that found in not the GH18 family but the GH23 family. Based on the similarity with GH23 family chitinase, the catalytic residues of Pc-ChiD were predicted and confirmed by mutagenesis analyses. Moreover, the specific C-terminal 100 residues of Pc-ChiD are important to fix the putative substrate-binding domain next to the catalytic domain, contributing to the structure stability as well as the long chitin chain binding. Our findings reveal the structure of a unique archaeal chitinase that is distinct from previously known members of the GH23 family.

中文翻译：

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古细菌几丁质酶ChiD及其配体复合物的晶体结构

几丁质酶D（指定为Pc- ChiD）存在于超嗜热古细菌Pyrococcus chitonophagus（以前称为Thermococcus chitonophagus）中，它是从仅含有几丁质作为碳源的培养基中分离出来的。Pc- ChiD显示几丁质酶活性，并且在高达95°C的温度下具有热稳定性，表明其具有工业用途的潜力。个人电脑-ChiD在N端结构域中具有分泌信号肽和两个几丁质结合结构域（ChBD）。但是，C末端域与先前鉴定的糖降解酶没有序列相似性，并且不包含糖苷水解酶（GH）18家族几丁质酶中保守的DXDXE基序。为了阐明其整体结构和反应机理，我们确定了Pc- ChiD的第一个晶体结构，既无配体形式，又与底物形成复合物。结构分析表明，Pc -ChiD，Pc的C末端结构域-ChiD（ΔBD）由无法从氨基酸序列预测的第三个推定的底物结合结构域和与在GH18家族而非GH23家族中发现的结构相似的催化结构域组成。基于与GH23家族几丁质酶的相似性，通过诱变分析预测并证实了Pc- ChiD的催化残基。此外，Pc- ChiD的特定C末端100个残基对于将推定的底物结合结构域固定在催化结构域附近非常重要，这有助于结构稳定性以及长的几丁质链结合。我们的发现揭示了独特的古细菌壳多糖酶的结构，该结构不同于GH23家族以前已知的成员。