Function Switch of a Fungal Sesterterpene Synthase through Molecular Dynamics Simulation Assisted Alteration of an Aromatic Residue Cluster in the Active Pocket of PfNS

Author:

Zhang Weiyan1ORCID,Wang Xinye12,Zhu Guoliang1,Zhu Bin1,Peng Kaitong1,Hsiang Tom3,Zhang Lixin1,Liu Xueting1ORCID

Affiliation:

1. State Key Laboratory of Bioreactor Engineering East China University of Science of Technology 200237 Shanghai China

2. School of Life Sciences Ludong University 264025 Yantai Shandong China

3. School of Environmental Sciences University of Guelph 50 Stone Road East N1G 2W1 Guelph Ontario Canada

Abstract

AbstractTerpene synthases (TPSs) play pivotal roles in generating diverse terpenoids through complex cyclization pathways. Protein engineering of TPSs offers a crucial approach to expanding terpene diversity. However, significant potential remains untapped due to limited understanding of the structure‐function relationships of TPSs. In this investigation, using a joint approach of molecular dynamics simulations‐assisted engineering and site‐directed mutagenesis, we manipulated the aromatic residue cluster (ARC) of a bifunctional terpene synthase (BFTPS), Pestalotiopsis fici nigtetraene synthase (PfNS). This led to the discovery of previously unreported catalytic functions yielding different cyclization patterns of sesterterpenes. Specifically, a quadruple variant (F89A/Y113F/W193L/T194W) completely altered PfNS′s function, converting it from producing the bicyclic sesterterpene nigtetraene to the tricyclic ophiobolin F. Additionally, analysis of catalytic profiles by double, triple, and quadruple variants demonstrated that the ARC functions as a switch, unprecedently redirecting the production of 5/11 bicyclic (Type B) sesterterpenes to 5/15 bicyclic (Type A) ones. Molecular dynamics simulations and theozyme calculations further elucidated that, in addition to cation‐π interactions, C−H⋅⋅⋅π interactions also play a key role in the cyclization patterns. This study offers a feasible strategy in protein engineering of TPSs for various industrial applications.

Funder

National Key Research and Development Program of China

Higher Education Discipline Innovation Project

Science and Technology Commission of Shanghai Municipality

Publisher

Wiley

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