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

几丁质酶水解不溶性和结晶性几丁质是几丁质酶最固有和最有趣的特征。本研究利用环状芽孢杆菌（Bacillus circulans）的几丁质酶A1（ChiA1）和粘质沙雷菌（Serratia marcescens）的几丁质酶A（ChiA）和几丁质酶B（ChiB）的三维结构，研究了结晶性几丁质水解的机理。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。两个芳香氨基酸残基被证明是晶体甲壳素水解的必要决定因素。2) B.circulans的解结构。构建ChiA1的每个结构域的三维结构已经被确定，但这种几丁质酶的整体结构是未知的。通过x射线散射，我们成功地估计了整个ChiA1分子的溶液结构。3)圆芽孢杆菌ChiA1几丁质结合域（ChBD）的结合机制。在ChBD中发现Trp687对几丁质结合最重要。此外，还强烈提示了环结构随结合变化的可能性。4)黏豆科植物暴露的芳香氨基酸残基的作用。两个Tyr和两个Trp沿催化裂口线性排列，对结晶β-几丁质的结合和水解至关重要。Trp残基对结合活性的贡献大于Tyr残基。5)大豆中ChiA和chb水解结晶几丁质。ChiA和ChiB的3d结构分析表明，甲壳素链的水解方向相反。通过还原末端标记技术和结晶β-几丁质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.