The mechanism for crystalline chitin hydrolysis by chitinases

水解不溶性和结晶几丁质的能力是几丁质酶最内在和最有趣的特征。本研究基于三维结构研究了环状芽孢杆菌WL-12的几丁质酶A1 （ChiA）和粘质沙雷氏菌2170的几丁质酶A （ChiA）水解结晶几丁质的机理。(1) B. circulans WL-12暴露在ChiA1表面的芳香氨基酸残基的作用。ChiA1包括一个催化结构域、两个FnIII结构域和一个几丁质结合结构域。在催化结构域表面，鉴定出两个暴露的芳香残基。这些残基的定点突变表明它们在将几丁质链从结晶几丁质表面引入催化裂孔中起主要作用。(2) FnIII结构域的解结构。利用PET表达系统在大肠杆菌中产生并纯化了ChiA1的FnIII结构域。然后用核磁共振法确定了FnIII结构域的溶液结构。该结构与动物蛋白中的FnIII结构域非常相似，这支持了细菌FnIII结构域是通过水平基因转移从动物获得的观点。(3) S. marcesens 2170中ChiA水解结晶几丁质的机理。从粘多糖2170中鉴定出4个暴露的芳香残基，其中2个在甲基亚胺的n端结构域，2个在催化结构域。通过位点定向诱变研究了这些残基在结晶几丁质水解中的重要性。得到的结果使我们提出了甲基亚酸水解结晶甲壳素的模型。“ChiA通过三种芳香残基和GlcNAc残基之间的相互作用与结晶几丁质结合在晶体几丁质表面的单个几丁质链上。甲壳素链从链的还原端引入催化裂口。引入的几丁质链通过裂口滑动到催化位点，并逐渐裂解，连续释放（GlcNAc）2单元。”少

An ability to hydrolyze insoluble and crystalline chitin is the most intrinsic and interesting feature of chitinases. In this study, the mechanisms for crystalline chitin hydrolysis by chitinase A1 (ChiAl) from Bacillus circulans WL-12 and chitinase A (ChiA) from Serratia marcescens 2170 were studied on the basis of their 3D-structures.(1) Roles of the aromatic amino acid residues exposed on the surface of ChiA1 from B. circulans WL-12. ChiA1 comprises a catalytic domain, two FnIII domains and a chitin-binding domain. On the surface of the catalytic domain, two exposed aromatic residues were identified. Site-directed mutagenesis of these residues revealed that they play major role in introducing a chitin chain from the surface of crystalline chitin into the catalytic cleft.(2) Solution structure of the FnIII domain. The FnIII domain of ChiA1 was produced in E. coli by using PET expression system and purified. Then, solution structure of the FnIII domain was determined by NMR. The struc … More ture appeared to be very similar to those of the FnIII domains in animal proteins, supporting the idea that bacterial FnIII domains were acquired from animal through horizontal gene transfer.(3) The mechanism for crystalline chitin hydrolysis by ChiA from S. marcescens 2170. Four exposed aromatic residues, two in the N-terminal domain and two in the catalytic domain of ChiA from S. marcescens 2170 were identified. Importance of these residues in crystalline chitin hydrolysis was studied by site-directed mutagenesis of these residues. The results obtained lead us to propose following model for crystalline chitin hydrolysis by ChiA. "ChiA binds to crystalline chitin through interaction between three aromatic residues and the GlcNAc residues in a single chitin chain on the crystalline chitin surface. The chitin chain is introduced into the catalytic cleft from the reducing end side of the chain. The introduced chitin chain slide through the cleft to the catalytic site and progressively cleaved releasing (GlcNAc)2 units continuously." Less