Molecular insights into the catalytic promiscuity of a bacterial diterpene synthase.

Zhong Li, Lilan Zhang, Kangwei Xu, Yuanyuan Jiang, Jieke Du, Xingwang Zhang, Ling-Hong Meng, Qile Wu, Lei Du, Xiaoju Li, Yuechan Hu, Zhenzhen Xie, Xukai Jiang, Ya-Jie Tang, Ruibo Wu, Rey-Ting Guo, Shengying Li

Journal: Nature communications 2023;14(1):4001

PMID: 37414771

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Abstract

Diterpene synthase VenA is responsible for assembling venezuelaene A with a unique 5-5-6-7 tetracyclic skeleton from geranylgeranyl pyrophosphate. VenA also demonstrates substrate promiscuity by accepting geranyl pyrophosphate and farnesyl pyrophosphate as alternative substrates. Herein, we report the crystal structures of VenA in both apo form and holo form in complex with a trinuclear magnesium cluster and pyrophosphate group. Functional and structural investigations on the atypical DSFVSD motif of VenA, versus the canonical Asp-rich motif of DDXX(X)D/E, reveal that the absent second Asp of canonical motif is functionally replaced by Ser116 and Gln83, together with bioinformatics analysis identifying a hidden subclass of type I microbial terpene synthases. Further structural analysis, multiscale computational simulations, and structure-directed mutagenesis provide significant mechanistic insights into the substrate selectivity and catalytic promiscuity of VenA. Finally, VenA is semi-rationally engineered into a sesterterpene synthase to recognize the larger substrate geranylfarnesyl pyrophosphate.

Address: State Key Laboratory of Microbial Technology, Shandong University, No. 72 Binhai Road, Qingdao, Shandong, 266237, China.; State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Hongshan Laboratory, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, 430062, China.; School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, 510006, China.; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Nanhai Road 7, Qingdao, Shandong, 266071, China.; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, 266237, China.; School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, 510006, China. [email protected].; State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Hongshan Laboratory, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, 430062, China. [email protected].; State Key Laboratory of Microbial Technology, Shandong University, No. 72 Binhai Road, Qingdao, Shandong, 266237, China. [email protected].; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, Shandong, 266237, China. [email protected].

DOI: 10.1038/s41467-023-39706-9

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MeSH Terms: Diphosphates, Alkyl and Aryl Transferases, Diterpenes, Computational Biology