Structural analysis of group II chitinase (ChtII) catalysis completes the puzzle of chitin hydrolysis in insects.
Structural analysis of group II chitinase (ChtII) catalysis completes the puzzle of chitin hydrolysis in insects.
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DOI：
10.1074/jbc.ra117.000119
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发表时间：
2018-02-23
期刊：
影响因子：
--
通讯作者：
Yang Q
中科院分区：
文献类型：
--
作者：
Chen W;Qu M;Zhou Y;Yang Q
甲壳素是N-乙酰基-β-d-葡糖胺的线性均聚物，是昆虫表皮的主要结构组分。几丁质水解涉及糖苷水解酶家族18（GH 18）几丁质酶。在昆虫中，几丁质水解是必不可少的定期脱落的旧角质层蜕皮和收益通过一个途径不同，在充分研究的细菌几丁质分解系统。Ⅱ类几丁质酶（Group II chitinase，ChtII）是一种广泛存在于昆虫体内的几丁质分解酶，是几丁质酶中催化结构域和几丁质结合结构域数目最多的酶。在鳞翅目昆虫中，ChtII和另外两种几丁质酶ChtI和Chi-h是几丁质水解所必需的。虽然ChtI和Chi-h已经被很好地研究，但ChtII的作用仍然难以捉摸。在这里，我们调查的结构和酶学OfChtII，ChtII来自昆虫害虫Ostrinia pesticalis。我们目前的晶体结构的两个催化活性域的OfChtII，OfChtII-C1和OfChtII-C2，无论是在unliganded形式和复杂的壳寡糖基板。我们发现，OfChtII-C1和OfChtII-C2都具有长，深底物结合裂缝内切几丁质酶活性。OfChtII表现出类似于OfChi-h和OfChtI的底物结合裂缝内的结构特征。然而，OfChtII缺乏有利于底物结合的结构元件超出活性位点，包括存在于OfChi-h中的额外壁结构。然而，OfChtII中的众多结构域可以补偿这种差异;含有一个催化结构域和三个几丁质结合模块（OfChtII-B4 C1）的截短物显示出对不溶性聚合物底物的活性高于OfChi-h和OfChtI。我们的观察提供了昆虫几丁质水解的最后一块拼图。
Chitin is a linear homopolymer of N-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 OfChtII, a ChtII derived from the insect pest Ostrinia furnacalis. We present the crystal structures of two catalytically active domains of OfChtII, OfChtII-C1 and OfChtII-C2, both in unliganded form and complexed with chitooligosaccharide substrates. We found that OfChtII-C1 and OfChtII-C2 both possess long, deep substrate-binding clefts with endochitinase activities. OfChtII exhibited structural characteristics within the substrate-binding cleft similar to those in OfChi-h and OfChtI. However, OfChtII lacked structural elements favoring substrate binding beyond the active sites, including an extra wall structure present in OfChi-h. Nevertheless, the numerous domains in OfChtII may compensate for this difference; a truncation containing one catalytic domain and three chitin-binding modules (OfChtII-B4C1) displayed activity toward insoluble polymeric substrates that was higher than those of OfChi-h and OfChtI. Our observations provide the last piece of the puzzle of chitin hydrolysis in insects.