文献类型：期刊文献

英文题名：Structural insights into the catalytic mechanism of a novel glycoside hydrolase family 113 β-1,4-mannanase from Amphibacillus xylanus

作者：You, Xin[1];Qin, Zhen[2];Yan, Qiaojuan[1];Yang, Shaoqing[3];Li, Yanxiao[1];Jiang, Zhengqiang[3]

机构：[1]China Agr Univ, Coll Engn, Beijing Adv Innovat Ctr Food Nutr & Human Hlth, Beijing 100083, Peoples R China;[2]East China Univ Sci & Technol, R&D Ctr Separat & Extract Technol Fermentat Ind, Sch Biotechnol, State Key Lab Bioreactor Engn, Shanghai 200237, Peoples R China;[3]China Agr Univ, Coll Food Sci & Nutrit Engn, Beijing 100083, Peoples R China

年份：2018

卷号：293

期号：30

起止页码：11746

外文期刊名：JOURNAL OF BIOLOGICAL CHEMISTRY

收录：;EI(收录号：20183105641522);WOS:【SCI-EXPANDED(收录号:WOS:000440134500008)】；

基金：This work was supported in part by the Key Program of the National Natural Science Foundation of China Grant 31630096 and Program for Changjiang Scholars Grant T2014055. The authors declare that they have no conflicts of interest with the contents of this article.

语种：英文

外文关键词：Binding sites - Hydrolases - Enzyme activity - Hydrolysis - Biochemical engineering - Catalysis

摘要：beta-1,4-Mannanase degrades beta-1,4-mannan polymers into manno-oligosaccharides with a low degree of polymerization. To date, only one glycoside hydrolase (GH) family 113 beta-1,4-mannanase, from Alicyclobacillus acidocaldarius (AaManA), has been structurally characterized, and no complex structure of enzyme-manno-oligosaccharides from this family has been reported. Here, crystal structures of a GH family 113 beta-1,4-mannanase from Amphibacillus xylanus (AxMan113A) and its complexes with mannobiose, mannotriose, mannopentaose, and mannahexaose were solved. AxMan113A had higher affinity for -1 and -1 mannoses, which explains why the enzyme can hydrolyze mannobiose. At least six subsites -4 to +2) exist in the groove, but mannose units preferentially occupied subsites -4 to -1 because of steric hindrance formed by Lys-238 and Trp-239. Based on the structural information and bioinformatics, rational design was implemented to enhance hydrolysis activity. Enzyme activity of AxMan113A mutants V139C, N237W, K238A, and W239Y was improved by 93.7, 63.4, 112.9, and 36.4%, respectively, compared with the WT. In addition, previously unreported surface-binding sites were observed. Site-directed mutagenesis studies and kinetic data indicated that key residues near the surface sites play important roles in substrate binding and recognition. These first GH family 113 beta-1,4-mannanase-manno-oligosaccharide complex structures may be useful in further studying the catalytic mechanism ofGH family 113 members, and provide novel insight into protein engineering of GHs to improve their hydrolysis activity.