Jiang Zhengqiang, Hu Songqing, Ma Junwen, Liu Yuchun, Qiao Zhu, Yan Qiaojuan, Gao Yonggui, Yang Shaoqing
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China.
College of Food Science and Engineering, South China University of Technology, Guangzhou, Guangdong 510640, China.
Biochim Biophys Acta Proteins Proteom. 2021 Nov;1869(11):140709. doi: 10.1016/j.bbapap.2021.140709. Epub 2021 Aug 4.
A chitinase gene (RmChiA) encoding 445 amino acid (aa) residues from a fungus Rhizomucor miehei was cloned and overexpressed in Escherichia coli. Two kinds of RmChiA crystal forms, with space groups P3 2 1 and P1, were obtained by sitting-drop vapor diffusion and the structures were determined by X-ray diffraction. The overall structure of RmChiA monomer, which is the first structure of bacterial-type chitinases from nonpathogenic fungi, adopts a canonical triosephosphate isomerase (TIM) barrel fold with two protruding chitinase insertion domains. RmChiA exhibited a unique NxDxE catalytical motif and a real active site tunnel structure, which are firstly found in GH family 18 chitinases. The motif had high structural homolog with the typical DxDxE motif in other GH family 18 chitinases. The tunnel is formed by two unusual long loops, containing 15 aa and 45 aa respectively, linked by a disulfide bond across the substrate-binding cleft. Mutation experiments found that opening the roof of tunnel structure increased the hydrolysis efficiency of RmChiA, but the thermostability of the mutants decreased. Moreover, the tunnel structure endowed RmChiA with the exo-chitinase character.
从米黑根毛霉中克隆得到一个编码445个氨基酸残基的几丁质酶基因（RmChiA），并在大肠杆菌中进行了过表达。通过坐滴气相扩散法获得了两种空间群分别为P3 2 1和P1的RmChiA晶体形式，并通过X射线衍射确定了其结构。RmChiA单体的整体结构是来自非致病真菌的细菌型几丁质酶的首个结构，采用典型的磷酸丙糖异构酶（TIM）桶状折叠结构，带有两个突出的几丁质酶插入结构域。RmChiA展现出独特的NxDxE催化基序和一个实际的活性位点隧道结构，这在18家族糖基水解酶（GH family 18）几丁质酶中首次发现。该基序与其他18家族糖基水解酶几丁质酶中的典型DxDxE基序具有高度的结构同源性。该隧道由两个异常长的环形成，分别包含15个氨基酸和45个氨基酸，通过一个二硫键横跨底物结合裂隙相连。突变实验发现，打开隧道结构的顶部可提高RmChiA的水解效率，但突变体的热稳定性降低。此外，该隧道结构赋予RmChiA外切几丁质酶特性。
