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Insight into Substrate-assisted Catalytic Mechanism and Stereoselectivity of Bifunctional Nocardicin Thioesterase
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  • Ting Shi,
  • Qian Yu,
  • Wei Lu,
  • Daixi Li
Ting Shi
Shanghai Jiao Tong University State Key Laboratory of Microbial Metabolism

Corresponding Author:[email protected]

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Qian Yu
Shanghai Jiao Tong University State Key Laboratory of Microbial Metabolism
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Wei Lu
Shanghai Jiao Tong University School of Agriculture and Biology
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Daixi Li
University of Shanghai for Science and Technology
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The inversion from L- to D-stereochemistry 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-amino acid into peptide backbones. Recently, a bifunctional thioesterase NocTE, which is responsible for the epimerization and hydrolysis of the C-terminal (p-hydroxyphenyl)glycine residue of β-lactam antibiotic nocardicin A, exclusively directs to the generation of D-diastereomers. 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-assisted mechanism for the epimerization, where multi-step proton transfers were mediated by water molecules and β-lactam ring, and the free energy barrier was calculated to be 20.3 kcal/mol. After that, the hydrolysis of D-configured substrate was energetically feasible with the energy barrier of 14.3 kcal/mol. As a comparison, the energy barrier for the direct hydrolysis of L-configured substrate was obtained to be 24.0 kcal/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.
12 May 2022Submitted to PROTEINS: Structure, Function, and Bioinformatics
17 May 2022Submission Checks Completed
17 May 2022Assigned to Editor
17 May 2022Reviewer(s) Assigned
26 May 2022Review(s) Completed, Editorial Evaluation Pending
26 May 2022Editorial Decision: Revise Minor
31 May 20221st Revision Received
01 Jun 2022Submission Checks Completed
01 Jun 2022Assigned to Editor
01 Jun 2022Reviewer(s) Assigned
12 Jun 2022Review(s) Completed, Editorial Evaluation Pending
21 Jun 2022Editorial Decision: Accept