State Key Laboratory of Fine Chemical Engineering, School of Life Science and Biotechnology and School of Software, Dalian University of Technology, Dalian 116024, China.
State Key Laboratory of Fine Chemical Engineering, School of Life Science and Biotechnology and School of Software, Dalian University of Technology, Dalian 116024, China; Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
J Biol Chem. 2018 Feb 23;293(8):2652-2660. doi: 10.1074/jbc.RA117.000119. Epub 2018 Jan 9.
Chitin is a linear homopolymer of -acetyl-β-d-glucosamines and a major structural component of insect cuticles. Chitin hydrolysis involves glycoside hydrolase family 18 (GH18) chitinases. In insects, chitin hydrolysis is essential for periodic shedding of the old cuticle ecdysis and proceeds via a pathway different from that in the well studied bacterial chitinolytic system. Group II chitinase (ChtII) is a widespread chitinolytic enzyme in insects and contains the greatest number of catalytic domains and chitin-binding domains among chitinases. In Lepidopterans, ChtII and two other chitinases, ChtI and Chi-h, are essential for chitin hydrolysis. Although ChtI and Chi-h have been well studied, the role of ChtII remains elusive. Here, we investigated the structure and enzymology of ChtII, a ChtII derived from the insect pest We present the crystal structures of two catalytically active domains of ChtII, ChtII-C1 and ChtII-C2, both in unliganded form and complexed with chitooligosaccharide substrates. We found that ChtII-C1 and ChtII-C2 both possess long, deep substrate-binding clefts with endochitinase activities. ChtII exhibited structural characteristics within the substrate-binding cleft similar to those in Chi-h and ChtI. However, ChtII lacked structural elements favoring substrate binding beyond the active sites, including an extra wall structure present in Chi-h. Nevertheless, the numerous domains in ChtII may compensate for this difference; a truncation containing one catalytic domain and three chitin-binding modules (ChtII-B4C1) displayed activity toward insoluble polymeric substrates that was higher than those of Chi-h and ChtI. Our observations provide the last piece of the puzzle of chitin hydrolysis in insects.
几丁质是一种由 N-乙酰-β-D-氨基葡萄糖组成的直链同聚物，是昆虫外骨骼的主要结构成分。几丁质水解涉及糖苷水解酶家族 18（GH18）几丁质酶。在昆虫中，几丁质水解对于旧外骨骼蜕皮的周期性脱落至关重要，其途径与在研究充分的细菌几丁质降解系统中不同。几丁质酶 II（ChtII）是昆虫中广泛存在的几丁质酶，在几丁质酶中包含最多的催化结构域和几丁质结合结构域。在鳞翅目昆虫中，ChtII 和另外两种几丁质酶，ChtI 和 Chi-h，对于几丁质水解是必不可少的。尽管 ChtI 和 Chi-h 已经得到了很好的研究，但 ChtII 的作用仍然难以捉摸。在这里，我们研究了 ChtII 的结构和酶学特性，ChtII 是一种源自昆虫害虫的几丁质酶。我们展示了两种催化活性结构域 ChtII-C1 和 ChtII-C2 的晶体结构，它们都处于未配体形式和与几丁寡糖底物复合物的形式。我们发现 ChtII-C1 和 ChtII-C2 都具有长而深的底物结合裂缝，具有内切几丁质酶活性。ChtII 在底物结合裂缝内表现出与 Chi-h 和 ChtI 相似的结构特征。然而，ChtII 缺乏有利于活性位点以外的底物结合的结构元件，包括 Chi-h 中存在的额外壁结构。尽管如此，ChtII 中的众多结构域可能弥补了这种差异；含有一个催化结构域和三个几丁质结合模块的截断体（ChtII-B4C1）对不溶性聚合底物的活性高于 Chi-h 和 ChtI。我们的观察结果提供了昆虫几丁质水解拼图的最后一块。
