ArticleJournal of the American Chemical Society2021
Engineering of Ancestors as a Tool to Elucidate Structure, Mechanism, and Specificity of Extant Terpene Cyclase.
Article in Journal of the American Chemical Society, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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21 citing papers in PubMed, 60 citations in OpenAlex.
- Reprogramming product selectivity and activity in promiscuous terpene synthases via substrate conformation engineering.Nature communications · 2026Article
- Deciphering the evolutionary origin of the enantioselectivity of short-chain dehydrogenases from plants toward 1-borneol.Nature communications · 2026Article
- Expanding the substrate scope of a bacterial monoterpene synthase for the production of sesquiterpenoid and diterpenoid products.The FEBS journal · 2026Article
- Progress and future outlook towards a safe and sustainable production and use of chemicals.Communications chemistry · 2025Review
- Ancestral sequence reconstruction as a tool for structural analysis of modular polyketide synthases.Nature communications · 2025Article
- Ancestral Sequence Reconstruction for Designing Biocatalysts and Investigating their Functional Mechanisms.JACS Au · 2024Review
- Ancestral Sequence Reconstruction to Enable Biocatalytic Synthesis of Azaphilones.Journal of the American Chemical Society · 2024Article
- Comparative analysis of reconstructed ancestral proteins with their extant counterparts suggests primitive life had an alkaline habitat.Scientific reports · 2024Article
- Confirmation of the stereochemistry of spiroviolene.Beilstein journal of organic chemistry · 2024Article
- Active Site Loop Engineering Abolishes Water Capture in Hydroxylating Sesquiterpene Synthases.ACS catalysis · 2023Article
- Decoding Catalysis by Terpene Synthases.ACS catalysis · 2023Review
- Molecular insights into the catalytic promiscuity of a bacterial diterpene synthase.Nature communications · 2023Article
- Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction.Proceedings of the National Academy of Sciences of the United States of America · 2023Article
- The Biosynthesis Related Enzyme, Structure Diversity and Bioactivity Abundance of Indole-Diterpenes: A Review.Molecules (Basel, Switzerland) · 2022Review
- Engineering functional thermostable proteins using ancestral sequence reconstruction.The Journal of biological chemistry · 2022Review
- Crystal Structure Based Mutagenesis of Cattleyene Synthase Leads to the Generation of Rearranged Polycyclic Diterpenes.Angewandte Chemie (International ed. in English) · 2022Article
- Thermoadaptation in an Ancestral Diterpene Cyclase by Altered Loop Stability.The journal of physical chemistry. B · 2022Article
- Hedycaryol - Central Intermediates in Sesquiterpene Biosynthesis, Part II.Chemistry (Weinheim an der Bergstrasse, Germany) · 2022Review
- Biotechnological potential and initial characterization of two novel sesquiterpene synthases from Basidiomycota Coniophora puteana for heterologous production of δ-cadinol.Microbial cell factories · 2022Article
- Mechanistic Insights into the Formation of the 6,10-Bicyclic Eunicellane Skeleton by the Bacterial Diterpene Synthase Bnd4.Angewandte Chemie (International ed. in English) · 2021Article
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Authors and funding
8 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Structural information is crucial for understanding catalytic mechanisms and to guide enzyme engineering efforts of biocatalysts, such as terpene cyclases. However, low sequence similarity can impede homology modeling, and inherent protein instability presents challenges for structural studies. We hypothesized that X-ray crystallography of engineered thermostable ancestral enzymes can enable access to reliable homology models of extant biocatalysts. We have applied this concept in concert with molecular modeling and enzymatic assays to understand the structure activity relationship of spiroviolene synthase, a class I terpene cyclase, aiming to engineer its specificity. Engineering a surface patch in the reconstructed ancestor afforded a template structure for generation of a high-confidence homology model of the extant enzyme. On the basis of structural considerations, we designed and crystallized ancestral variants with single residue exchanges that exhibited tailored substrate specificity and preserved thermostability. We show how the two single amino acid alterations identified in the ancestral scaffold can be transferred to the extant enzyme, conferring a specificity switch that impacts the extant enzyme's specificity for formation of the diterpene spiroviolene over formation of sesquiterpenes hedycaryol and farnesol by up to 25-fold. This study emphasizes the value of ancestral sequence reconstruction combined with enzyme engineering as a versatile tool in chemical biology.
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