title: Biosynthesis of the microtubule-destabilizing diterpene pseudolaric acid B from golden larch involves an unusual diterpene synthase.
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.
authors.affiliation: Department of Plant Biology, University of California, Davis, CA 95616.
authors.forename: Sibongile
authors.initials: S
authors.lastname: Mafu
authors.affiliation: Department of Plant Biology, University of California, Davis, CA 95616.
authors.forename: Prema Sambandaswami
authors.initials: PS
authors.lastname: Karunanithi
authors.affiliation: Department of Chemistry, University of California, Davis, CA 95616.
authors.forename: Teresa Ann
authors.initials: TA
authors.lastname: Palazzo
authors.affiliation: Department of Chemistry, University of California, Davis, CA 95616.
authors.forename: Bronwyn Lee
authors.initials: BL
authors.lastname: Harrod
authors.affiliation: Department of Plant Biology, University of California, Davis, CA 95616.
authors.forename: Selina Marakana
authors.initials: SM
authors.lastname: Rodriguez
authors.affiliation: Department of Plant Biology, University of California, Davis, CA 95616.
authors.forename: Iris Natalie
authors.initials: IN
authors.lastname: Mollhoff
authors.affiliation: Department of Chemistry, University of California, Davis, CA 95616.
authors.forename: Terrence Edward
authors.initials: TE
authors.lastname: O'Brien
authors.affiliation: West Coast Metabolomics Center, University of California, Davis, CA 95616.
authors.forename: Shen
authors.initials: S
authors.lastname: Tong
authors.affiliation: West Coast Metabolomics Center, University of California, Davis, CA 95616.|Biochemistry Department, King Abdulaziz University, Jeddah 23218, Saudi Arabia.
authors.forename: Oliver
authors.initials: O
authors.lastname: Fiehn
authors.affiliation: Department of Chemistry, University of California, Davis, CA 95616.
authors.forename: Dean J
authors.initials: DJ
authors.lastname: Tantillo
authors.affiliation: Michael Smith Laboratories, University of British Columbia, Vancouver, BC V6T 1Z4, Canada.
authors.forename: Jörg
authors.initials: J
authors.lastname: Bohlmann
authors.affiliation: Department of Plant Biology, University of California, Davis, CA 95616; pzerbe@ucdavis.edu.
authors.forename: Philipp
authors.initials: P
authors.lastname: Zerbe
mesh_terms: D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004224:Diterpenes / Q000378:metabolism*; D028221:Larix / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D018076:DNA, Complementary; D004224:Diterpenes; D010940:Plant Proteins; D011106:Polyisoprenyl Phosphates; D011993:Recombinant Fusion Proteins; C058391:pseudolaric acid B; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; C002963:geranylgeranyl pyrophosphate
keywords: Pseudolarix amabilis; chemotherapeutic drug; diterpene biosynthesis; plant natural products; pseudolaric acid