Lü Yang, Yang Haitao, Hu Hongyan, Wang Ying, Rao Zihe, Jin Cheng
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Glycoconj J. 2009 Jul;26(5):525-34. doi: 10.1007/s10719-008-9203-z. Epub 2008 Oct 31.
Family 18 chitinases hydrolyze chitin through a substrate-assisted catalytic mechanism and are to a variable extent able to catalyze transglycosylation reactions. Previously Aspergillus fumigatus AfChiB1 was found to be able to catalyze transglycosylation reactions. Structural analysis reveals that AfChiB1 consists of an eight-stranded beta/alpha-barrel. Like other members of the family 18 hydrolases, AfChiB1 has conserved substrate binding site and catalytic acid, while a suitable nucleophile is missing. In this study, Trp137, Asp246, and Met243, which are close to the glycosidic cleavage site, were mutated to glutamate individually. As a result, the W137E remained its hydrolytic activity and was completely devoid of transglycosyl activity, while the D246E reduced its chitinolytic activity and increased its transglycosyl activity. And the M243E showed a remarkable reduction of chitinolytic activity and complete loss of transglycosyl activity. These results suggested that the transglycosyl reaction catalyzed by the AfChiB1 is due to lacking of nucleophile.
18 家族几丁质酶通过底物辅助催化机制水解几丁质，并在不同程度上能够催化转糖基化反应。此前发现烟曲霉 AfChiB1 能够催化转糖基化反应。结构分析表明，AfChiB1 由一个八链 β/α 桶组成。与 18 家族水解酶的其他成员一样，AfChiB1 具有保守的底物结合位点和催化酸，但缺少合适的亲核试剂。在本研究中，将靠近糖苷裂解位点的色氨酸 137、天冬氨酸 246 和甲硫氨酸 243 分别突变为谷氨酸。结果，W137E 保留了其水解活性，完全没有转糖基活性，而 D246E 降低了其几丁质分解活性并增加了其转糖基活性。并且 M243E 显示出几丁质分解活性显著降低和转糖基活性完全丧失。这些结果表明，AfChiB1 催化的转糖基反应是由于缺乏亲核试剂。
