Three-dimensional structure of the catalytic domain of chitinase Al from Bacillus circulars WL-12 at a very high resolution
Three-dimensional structure of the catalytic domain of chitinase Al from Bacillus circulars WL-12 at a very high resolution
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DOI：
10.2183/pjab.75.269
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发表时间：
1999
期刊：
影响因子：
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通讯作者：
Takuo Matsumoto;T. Nonaka;M. Hashimoto;Takeshi Watanabe;Y. Mitsui
中科院分区：
文献类型：
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作者：
Takuo Matsumoto;T. Nonaka;M. Hashimoto;Takeshi Watanabe;Y. Mitsui
来自Bacillus circulars WL-12的几丁质酶Al（18家族糖基水解酶之一）的成熟形式包括一个n端催化结构域，两个纤维连接蛋白III型结构域和一个c端几丁质结合结构域。通过对各结构域、组合结构域和完整成熟形态的结晶，我们获得了衍射达到真正原子分辨率（1.13A）的催化结构域晶体。催化结构域由（α/β）8-TIM-barrel组成。两个附着在TIM-barrel顶部的小β结构域为底物结合提供了一个深的间隙。失活（通过Glu204Gln突变）催化结构域与七聚n -乙酰氨基葡萄糖（7NAG (NI， NII， NIII， NIV， NV， NVI, NVII-hepta-acetyl-chitoheptaose， n -乙酰氨基葡萄糖的β- 1,4连接寡糖，聚合度为7)，这是一个真正的底物）络合的晶体，衍射分辨率高达1.5A。结合的7NAG底物在-1和+1位置的糖单元之间显示出明显的扭结。Glu204与可剪位点的接近性有力地支持了迄今为止的设想，即Glu204在所谓的“底物辅助催化”机制中起催化酸的作用，这与溶菌酶和相关糖基水解酶长期接受的一般酸碱机制不同。
The mature form of chitinase Al from Bacillus circulars WL-12 (one of the family 18 glycosyl hydrolases) comprises an N-terminal catalytic domain, two fibronectin type III domains and a C-terminal chitin-binding domain. In the efforts for crystallizing each domain, combined domains and the intact mature form, we have obtained crystals of the catalytic domain which diffract to a truly atomic resolution (1.13A). The catalytic domain consists of an (α/β)8-TIM-barrel. Two small β-domains attached on top of the TIM-barrel provide a deep cleft for substrate binding. Crystals of an inactivated (through Glu204Gln mutation) catalytic domain complexed with a heptameric N-acetyl glucosamme (7NAG (NI, NII, NIII, NIV, NV, NVI, NVII-hepta-acetyl-chitoheptaose which is a β-1, 4-linked oligosaccharide of N-acetylglucosamine with a polymerization degree of seven.), which is a true substrate) diffracted up to 1.5A resolution. The bound 7NAG substrate showed a marked kink between the sugar units at positions -1 and +1. The proximity of Glu204 to the scissile site strongly supports the hitherto conceived notion that Glu204 plays a role of catalytic acid in the so-called“substrate-assisted catalysis”mechanism which is distinct from the general acidbase mechanism long accepted for lysozyme and related glycosyl hydrolases.