3D-structures of bacterial chitinases and the mechanism for crystaline chitin hyrrolysis

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。在这项研究中，以环状芽孢杆菌WL-12的几丁质酶A1（ChiA 1）和粘质沙雷氏菌2170.1的几丁质酶A（ChiA）和几丁质酶B（ChiB）为底物，从三维结构的角度研究了它们水解结晶几丁质的机理。芳香族氨基酸残基在B.circulans ChiA 1催化裂中的作用ChiA 1包含一个催化结构域、两个FnIII结构域和一个几丁质结合结构域。2）环状B.circulans ChiA 11的溶液结构。构建ChiA 1的每个结构域的三维结构已经确定，但该几丁质酶的整体结构是未知的。通过X射线散射，我们成功地估算了ChiA 1整个分子的溶液结构。3）环状B.circulans ChiA 1几丁质结合域（ChBD）的结合机制。ChBD中的Trp 687被揭示对于几丁质结合是最重要的。此外，还发现了粘质沙雷氏菌ChiB的环状结构沿着发生变化的可能性。两个Tyr和两个Trp线性排列向催化裂缝，被证明是必不可少的结合和水解的结晶β-甲壳素。Trp残基对结合活性的贡献大于Tyr残基。5）几丁质酶ChiA和ChiB对粘质沙雷氏菌结晶几丁质的水解。通过对ChiA和ChiB的三维结构分析，提出了甲壳素链在相反方向上的水解。通过还原末端标记技术和结晶β-甲壳素微纤维的倾斜衍射实验证明了这一点。

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 was studied on the basis of their 3D-structures by using chitinase A1(ChiA1) from Bacillus circulans WL-12 and chitinase A(ChiA) and chitinase B(ChiB) from Serratia marcescens 2170.1)Roles of the aromatic amino acid residues within the catalytic cleft of B.circulans ChiA1. ChiA1 comprises a catalytic domain, two FnIII domains and a chitin-binding domain. Two aromatic amino acid residues were shown to be essential determinants only for crystalline chitin hydrolysis.2)Solution structure of B.circulans ChiA1l. 3D-structure of each domain constructing ChiA1 has been determined previously but whole structure of this chitinases is unknown. By X-ray scattering, we succeeded to estimate solution structure of entire ChiA1 molecule.3)Binding mechanism of the chitin-binding domain(ChBD) of B.circulans ChiA1. Trp687 in ChBD was revealed to be most important for chitin binding. In addition, possibility of change in loop structure along with binding was strongly suggested.4)Roles of exposed aromatic amino acid residues of S.marcescens ChiB. Two Tyr and two Trp linearly aligned toward the catalytic cleft were shown to be essential for binding to and hydrolysis of crystalline β-chitin. Contribution of Trp residues to binding activity was larger than that of Tyr residues.5)Crystalline chitin hydrolysis by ChiA and ChiB from S.marcescens. Hydrolysis of chitin chain in opposite direction has been suggested by 3D-structural analysis of ChiA and ChiB. This was experimentally proved by reducing end labeling technique and tilt diffraction of crystalline β-chitin microfibrils.