The mechanism for crystalline chitin hydrolysis by chitinases

几丁质酶最本质和最有趣的特征是能够分解不溶性和结晶性的几丁质。本研究根据环状芽孢杆菌WL-12的几丁质酶A1(ChiA1)和粘质沙雷氏菌2170的几丁质酶A(Chia)的三维结构，研究了它们对结晶几丁质的作用机理：(1)环状芽孢杆菌WL-12几丁质酶A1表面的芳香族氨基酸残基的作用。ChiA1由一个催化结构域、两个FnIII结构域和一个甲壳素结合区组成。在催化区域的表面，发现了两个暴露的芳香族残基。这些残基的定点突变表明，它们在将几丁质链从结晶的几丁质表面引入催化裂隙中起主要作用。(2)FnIII结构域的溶液结构。利用PET表达系统在大肠杆菌中表达了ChiA1的FnIII结构域，并进行了纯化。然后用核磁共振确定了FnIII结构域的溶液结构。Struc…与动物蛋白中的FnIII结构域有更多的相似性，支持细菌的FnIII结构域是通过水平基因转移从动物中获得的观点。(3)粘质链霉菌CHIA对结晶甲壳素的水解机制。从粘质链霉菌2170中鉴定出四个暴露的芳香族残基，其中两个位于Chia的N-末端区域，两个位于Chia的催化区。通过对这些残基进行定点突变，研究了这些残基在结晶甲壳素水解中的重要性。根据所得结果，我们提出了CHIA对结晶甲壳素的以下水解模型。CHIA通过三个芳香族残基与结晶甲壳质表面单一甲壳素链上的GlcNAc残基相互作用与结晶甲壳素结合。甲壳素链从链的还原端侧引入催化裂隙。引入的甲壳素链通过裂隙滑动到催化位置，并不断地逐步裂解释放(GlcNAc)2个单元。较少

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