Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase
| Autor: | Teresa A. Palazzo, Oliver Fiehn, Shen Tong, Bronwyn Lee Harrod, Prema Sambandaswami Karunanithi, Terrence E. O’Brien, Jörg Bohlmann, Philipp Zerbe, Sibongile Mafu, Iris Natalie Mollhoff, Dean J. Tantillo, Selina Marakana Rodriguez |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
DNA Complementary Stereochemistry Recombinant Fusion Proteins Larix Saccharomyces cerevisiae Pseudolarix Plant Roots Metabolic engineering 03 medical and health sciences chemistry.chemical_compound Biosynthesis Polyisoprenyl Phosphates Catalytic Domain Amino Acid Sequence Cloning Molecular Plant Proteins chemistry.chemical_classification Multidisciplinary Alkyl and Aryl Transferases biology Bicyclic molecule ATP synthase Mutagenesis Biological Sciences biology.organism_classification 030104 developmental biology Enzyme Biochemistry chemistry Agrobacterium tumefaciens biology.protein Mutagenesis Site-Directed RNA Interference Diterpene Diterpenes Sequence Alignment |
| Zdroj: | Proceedings of the National Academy of Sciences of the United States of America. 114(5) |
| ISSN: | 1091-6490 |
| Popis: | The diversity of small molecules formed via plant diterpene metabolism offers a rich source of known and potentially new biopharmaceuticals. Among these, the microtubule-destabilizing activity of pseudolaric acid B (PAB) holds promise for new anticancer agents. PAB is found, perhaps uniquely, in the coniferous tree golden larch ( Pseudolarix amabilis , Pxa). Here we describe the discovery and mechanistic analysis of golden larch terpene synthase 8 (PxaTPS8), an unusual diterpene synthase (diTPS) that catalyzes the first committed step in PAB biosynthesis. Mining of the golden larch root transcriptome revealed a large TPS family, including the monofunctional class I diTPS PxaTPS8, which converts geranylgeranyl diphosphate into a previously unknown 5,7-fused bicyclic diterpene, coined “pseudolaratriene.” Combined NMR and quantum chemical analysis verified the structure of pseudolaratriene, and co-occurrence with PxaTPS8 and PAB in P . amabilis tissues supports the intermediacy of pseudolaratriene in PAB metabolism. Although PxaTPS8 adopts the typical three-domain structure of diTPSs, sequence phylogeny places the enzyme with two-domain TPSs of mono- and sesqui-terpene biosynthesis. Site-directed mutagenesis of PxaTPS8 revealed several catalytic residues that, together with quantum chemical calculations, suggested a substantial divergence of PxaTPS8 from other TPSs leading to a distinct carbocation-driven reaction mechanism en route to the 5,7- trans -fused bicyclic pseudolaratriene scaffold. PxaTPS8 expression in microbial and plant hosts provided proof of concept for metabolic engineering of pseudolaratriene. |
| Databáze: | OpenAIRE |
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