文献类型：期刊文献

英文题名：An Energy Optimization Strategy Based on the Perfect Conformation of Prolyl Endopeptidase for Improving Catalytic Efficiency

作者：Li, Xiaolin[1];Cong, Yalong[1];Ma, Mingzhe[1];You, Zhi-Neng[4];Gao, Bei[4];Zhang, John Z. H.[1,3];Zhang, Lujia[1,2]

机构：[1]East China Normal Univ, Shanghai Engn Res Ctr Mol Therapeut & New Drug De, Shanghai Key Lab Green Chem & Chem Proc, Sch Chem & Mol Engn, Shanghai 200062, Peoples R China;[2]NYU, Dept Chem, New York, NY 10003 USA;[3]NYU Shanghai, NYU ECNU Ctr Computat Chem, Shanghai 200062, Peoples R China;[4]East China Univ Sci & Technol, Sch Biotechnol, Shanghai 200237, Peoples R China

年份：2020

卷号：68

期号：18

起止页码：5129

外文期刊名：JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY

收录：;EI(收录号：20202108674270);WOS:【SCI-EXPANDED(收录号:WOS:000535173900012)】；

基金：This work was supported by the National Key R&D Program of China (grant no. 2019YFA0905200), National Natural Science Foundation of China (grant nos. 31571786, U1805235, 21433004, and 91753103), and NYU-ECNU Center for Computational Chemistry at NYU Shanghai. We also thank the Supercomputer Center of East China Normal University (ECNU Public Platform for Innovation 001) for providing us computational time.

语种：英文

外文关键词：protein engineering; prolyl endopeptidase; molecular dynamics simulations; site-directed mutagenesis; catalytic efficiency

摘要：Prolyl endopeptidases (PEPs) hydrolyze proteins to yield bioactive peptides and are effective in the treatment of celiac disease. However, the catalytic efficiency of PEPs still has the potential to be improved, which could further strengthen their industrial and therapeutic applications. Herein, a novel rational design strategy based on a "near-attack conformation" of the catalytic state of PEP was adopted. Constrained dynamic simulations were applied, followed by the virtual screening of potentially favorable mutants according to their binding free energy. We redesigned Sphaerobacter thermophiles PEP with high-temperature activity/stability, a wide range of pH stabilities, and high proline specificity. As a result, the k(cat) value of two PEP mutants (I462W and Q560Y) increased by 208.2 and 150.1%, respectively, and the k(cat)/K-M increased by 32.7 and 6.3%, respectively. These data revealed that the PEP mutants had improved catalytic efficiency and that our strategy can be applied for enzyme engineering.