Engineering substrate channeling in a bifunctional terpene synthase

• 成果类型: Article
• 署名作者: Wenger, Eliott S.; Schultz, Kollin; Marmorstein, Ronen; Christianson, David W.
• 署名单位: University of Pennsylvania; University of Pennsylvania; University of Pennsylvania; University of Pennsylvania
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-12777
• DOI: 10.1073/pnas.2408064121
• 发表日期: 2024-10-08
• 关键词: genome mining approach; cryo-em; sesterterpene synthase; diterpene cyclase; fusion; enzyme; identification; biosynthesis; refinement; fusicoccin

摘要

Fusicoccadiene synthase from Phomopsis amygdala (PaFS) is a bifunctional terpene synthase. It contains a prenyltransferase (PT) domain that generates geranylgeranyl tenyl diphosphate, and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are connected by a flexible 69- residue linker. The PT domain mediates oligomerization to form predominantly octamers, with cyclase domains randomly splayed out around the PT core. Surprisingly, despite the random positioning of cyclase domains, substrate channeling is operative in catalysis since most of the GGPP generated by the PT remains on the enzyme for cyclization. Here, we demonstrate that covalent linkage of the PT and cyclase domains is not required for GGPP channeling, although covalent linkage may improve cyclases indicate that the PaFS PT and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryoelectron microscopy structure of the 600- kD linkerless construct, in which the 69- residue linker is spliced out and of the PT octamer and exhibit fascinating quaternary structural flexibility. These results suggest that optimal substrate channeling is achieved when a cyclase domain associates linked and regardless of whether this interaction is transient or locked in place.