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Crystal structure of 10-epi-cubebol synthase from Sorangium cellulosum (ScCubS) in complex with Pyrophosphate
Structural highlights
FunctionECUBS_SORC5 Catalyzes the cyclization of farnesyl diphosphate (FPP) to 10-epi-cubebol. Is also responsible for the formation of many other sesquiterpenes, mainly cadalanes and cubebanes, including 1,10-di-epi-cubebol and the cadalanes delta-cadinene, T-cadinol and alpha-cadinol.[1] Publication Abstract from PubMedTerpenes are the largest class of natural products and are attractive targets in the fuel, fragrance, pharmaceutical, and flavor industries. Harvesting terpenes from natural sources is environmentally intensive and often gives low yields and purities, requiring further downstream processing. Engineered terpene synthases (TSs) offer a solution to these problems, but the low sequence identity and high promiscuity among TSs are major challenges for targeted engineering. Rational design of TSs requires identification of key structural and chemical motifs that steer product outcomes. Producing the sesquiterpenoid 10-epi-cubebol from farnesyl pyrophosphate (FPP) requires many steps and some of Nature's most difficult chemistry. 10-epi-Cubebol synthase from Sorangium cellulosum (ScCubS) guides a highly reactive carbocationic substrate through this pathway, preventing early quenching and ensuring correct stereochemistry at every stage. The cyclizations carried out by ScCubS potentially represent significant evolutionary expansions in the chemical space accessible by TSs. Here, we present the high-resolution crystal structure of ScCubS in complex with both a trinuclear magnesium cluster and pyrophosphate. Computational modeling, experiment, and bioinformatic analysis identified residues important in steering the reaction chemistry. We show that S206 is crucial in 10-epi-cubebol synthesis by enlisting the nearby F211 to shape the active site contour and prevent the formation of early escape cadalane products. We also show that N327 and F104 control the distribution between several early-stage cations and whether the final product is derived from the germacrane, cadalane, or cubebane hydrocarbon scaffold. Using these insights, we reengineered ScCubS so that its main product was germacradien-4-ol, which derives from the germacrane, rather than the cubebane, scaffold. Our work emphasizes that mechanistic understanding of cation stabilization in TSs can be used to guide catalytic outcomes. How a 10-epi-Cubebol Synthase Avoids Premature Reaction Quenching to Form a Tricyclic Product at High Purity.,Whitehead JN, Leferink NGH, Komati Reddy G, Levy CW, Hay S, Takano E, Scrutton NS ACS Catal. 2022 Oct 7;12(19):12123-12131. doi: 10.1021/acscatal.2c03155. Epub, 2022 Sep 21. PMID:36249875[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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