Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase

Sibongile Mafu, Prema Sambandaswami Karunanithi, Teresa Ann Palazzo, Bronwyn Lee Harrod, Selina Marakana Rodriguez, Iris Natalie Mollhoff, Terrence Edward O'Brien, Shen Tong, Oliver Fiehn, Dean J. Tantillo, Jörg Bohlmann, Philipp Zerbe

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)974-979
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume114
Issue number5
DOIs
StatePublished - Jan 31 2017

Keywords

  • Chemotherapeutic drug
  • Diterpene biosynthesis
  • Plant natural products
  • Pseudolaric acid
  • Pseudolarix amabilis

ASJC Scopus subject areas

  • General

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    Mafu, S., Karunanithi, P. S., Palazzo, T. A., Harrod, B. L., Rodriguez, S. M., Mollhoff, I. N., O'Brien, T. E., Tong, S., Fiehn, O., Tantillo, D. J., Bohlmann, J., & Zerbe, P. (2017). Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase. Proceedings of the National Academy of Sciences of the United States of America, 114(5), 974-979. https://doi.org/10.1073/pnas.1612901114