Molecular insights into the catalytic promiscuity of a bacterial diterpene synthase

• Published in: Nature communications Vol. 14; no. 1; pp. 4001 - 15
• Main Authors: Li, Zhong, Zhang, Lilan, Xu, Kangwei, Jiang, Yuanyuan, Du, Jieke, Zhang, Xingwang, Meng, Ling-Hong, Wu, Qile, Du, Lei, Li, Xiaoju, Hu, Yuechan, Xie, Zhenzhen, Jiang, Xukai, Tang, Ya-Jie, Wu, Ruibo, Guo, Rey-Ting, Li, Shengying
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 06.07.2023; Nature Publishing Group; Nature Portfolio
• Subjects: 119/118; 38/23; 38/70; 631/535/1266; 639/638/45/173; 639/638/92/349/977; 639/638/92/60; 639/638/92/603; 82/80; 82/83; Alkyl and Aryl Transferases - genetics; Amino acids; Bioinformatics; Chemical synthesis; Chemists; Computational Biology; Crystal structure; Diphosphates; Diterpenes; Enzymes; Humanities and Social Sciences; Laboratories; Magnesium; Marine biology; Microorganisms; multidisciplinary; Mutagenesis; Phylogenetics; Science; Science (multidisciplinary); Structural analysis; Structure-function relationships; Substrates
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-39706-9

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 115 DSFVSD 120 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. Diterpene synthase VenA catalyses the synthesis of venezuelaene A with a unique 5-5-6-7 tetracyclic skeleton from geranylgeranyl pyrophosphate. Here, the authors report crystal structures of apo- and holo-VenA, provide mechanistic insights into its substrate selectivity and promiscuity, and engineer VenA into a sesterterpene synthase.