{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T15:55:27Z","timestamp":1747151727861,"version":"3.40.5"},"reference-count":31,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2022,7,18]],"date-time":"2022-07-18T00:00:00Z","timestamp":1658102400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"funder":[{"DOI":"10.13039\/501100012166","name":"National Key Research and Development Program of China","doi-asserted-by":"publisher","award":["2019YFA0905400","2021YFC2100600"],"award-info":[{"award-number":["2019YFA0905400","2021YFC2100600"]}],"id":[{"id":"10.13039\/501100012166","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32070041"],"award-info":[{"award-number":["32070041"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Proteins"],"published-print":{"date-parts":[[2022,12]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>The inversion from L\u2010 to D\u2010stereochemistry endows peptides improved bioactivity and enhanced resistance to many proteases and peptidases. To strengthen the biostability and bioavailability of peptide drugs, enzymatic epimerization becomes an important way to incorporate D\u2010amino acid into peptide backbones. Recently, a bifunctional thioesterase NocTE, which is responsible for the epimerization and hydrolysis of the C\u2010terminal (<jats:italic>p<\/jats:italic>\u2010hydroxyphenyl)glycine residue of \u03b2\u2010lactam antibiotic nocardicin A, exclusively directs to the generation of D\u2010diastereomers. Different from other epimerases, NocTE exhibits unique stereochemical selectivity. Herein, we investigated the catalytic mechanism of NocTE via molecular dynamic (MD) simulations and quantum mechanical\/molecular mechanics (QM\/MM) calculations. Through structural analyses, two key water molecules around the reaction site were found to serve as proton mediators in epimerization. The structural characteristics inspired us to propose a substrate\u2010assisted mechanism for the epimerization, where multi\u2010step proton transfers were mediated by water molecules and \u03b2\u2010lactam ring, and the free energy barrier was calculated to be 20.3\u00a0kcal\/mol. After that, the hydrolysis of D\u2010configured substrate was energetically feasible with the energy barrier of 14.3\u2009kcal\/mol. As a comparison, the energy barrier for the direct hydrolysis of L\u2010configured substrate was obtained to be 24.0\u00a0kcal\/mol. Our study provides mechanistic insights into catalytic activities of bifunctional thioesterase NocTE, uncovers more clues to the molecular basis for stereochemical selectivity and paves the way for the directed biosynthesis of novel peptide drugs with various stereostructural characteristics by enzyme rational design.<\/jats:p>","DOI":"10.1002\/prot.26395","type":"journal-article","created":{"date-parts":[[2022,7,7]],"date-time":"2022-07-07T06:57:59Z","timestamp":1657177079000},"page":"2035-2044","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Insight into substrate\u2010assisted catalytic mechanism and stereoselectivity of bifunctional nocardicin thioesterase"],"prefix":"10.1002","volume":"90","author":[{"given":"Qian","family":"Yu","sequence":"first","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental Sciences School of Life Sciences and Biotechnology, Shanghai Jiao Tong University  Shanghai China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wei","family":"Lu","sequence":"additional","affiliation":[{"name":"Department of Food Science and Engineering School of Agriculture and Biology, Shanghai Jiao Tong University  Shanghai China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daixi","family":"Li","sequence":"additional","affiliation":[{"name":"Institute of Biothermal Science and Technology, University of Shanghai for Science and Technology  Shanghai China"},{"name":"AI Research Center Peng Cheng National Laboratory  Shenzhen Guangdong China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3921-4412","authenticated-orcid":false,"given":"Ting","family":"Shi","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Microbial Metabolism, Joint International Research Laboratory of Metabolic and Developmental 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