Enzymatic Total Synthesis of Gibberellin A<sub>4</sub>from Acetate

Sugai, Yoshinori; Miyazaki, Sho; Mukai, Shinichiro; Yumoto, Isamu; Natsume, Masahiro; Kawaide, Hiroshi

doi:10.1271/bbb.100733

Cited by 11 publications

(39 citation statements)

References 38 publications

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“…Recombinant enzymes for the labeled GGDP production were produced in Escherichia coli and purified with nickel-nitrilotriacetic acid resin (Qiagen, Valencia, CA) according to the manufacturer's protocol. An enzyme mixture for GGDP synthesis was prepared by the methods described previously (15).…”

Section: Methodsmentioning

confidence: 99%

“…After 1.5-h incubation at 28°C, the product was extracted with cyclohexane and analyzed by GC-MS. For synthesis of the fully 13 C-labeled product, SmMDS protein was mixed with GGDP synthetic enzyme mixture (15). The mixture contained six enzymes (1.3 mg of mevalonate kinase, 1.3 mg of phosphomevalonate kinase, 1.3 mg of diphosphomevalonate decarboxylase, 1.3 mg of isopentenyl diphosphate isomerase, 3.2 mg of GGDP synthase, and 11.5 mg of SmMDS), 5 mM MgCl 2 , 10 mM ATP, and 7.5 mg of [U- 13 C 6 ]mevalonate as the substrate in 32 ml of reaction buffer.…”

Section: Heterologous Expression Of Smmds Gene and Recombinant Proteimentioning

confidence: 99%

“…GC-MS spectral data were measured with JMS-Bu25 (JEOL, Tokyo, Japan). The capillary column DB-5 (0.25-mm inner diameter ϫ 15 m, 0.25-m film thickness; J&W Scientific, Folsom, CA) was used with GC-MS conditions as described previously (15). The NMR analysis was performed at the NMR facility at RIKEN Yokohama Institute.…”

Section: Heterologous Expression Of Smmds Gene and Recombinant Proteimentioning

confidence: 99%

“…We previously successfully synthesized in vitro entkaurene, taxadiene, and amorphadiene by combining the appropriate enzymes in a mixture (15). In this study, rSmMDS was added into the [ 13 C]GGDP synthetic mixture to synthesize 13 C-labeled miltiradiene.…”

Section: Journal Of Biological Chemistry 42845mentioning

confidence: 99%

“…In our previous work, fully 13 C-labeled gibberellins were synthesized from [U- 13 C 6 ]mevalonate via ent-kaurene. In that report, one-dimensional NMR analysis of fully 13 C-labeled ent-kaurene showed high sensitivity and carbon-carbon coupling (15). If such a fully 13 C-labeled compound is analyzed by homonuclear multidimensional 13 C NMR experiments, these spectra showing carbon-carbon connectivities would allow unambiguous determination of the chemical structures of various terpenoids with high sensitivity.…”

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confidence: 95%

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Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii

Sugai

Ueno

Hayashi

et al. 2011

Journal of Biological Chemistry

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Background:A model lycophyte, Selaginella moellendorffii, has a unique bifunctional terpene cyclase gene. Results: The bifunctional cyclase synthesized miltiradiene from geranylgeranyl diphosphate via (ϩ)-copalyl diphosphate. Conclusion: Fully 13 C-labeled miltiradiene was unambiguously elucidated by multidimensional NMR analyses. Significance: Enzymatic synthesis of highly enriched biosynthetic products enables one-dimensional and multidimensional 13 C NMR studies.

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Section: Methodsmentioning

confidence: 99%

Section: Heterologous Expression Of Smmds Gene and Recombinant Proteimentioning

confidence: 99%

Section: Heterologous Expression Of Smmds Gene and Recombinant Proteimentioning

confidence: 99%

Section: Journal Of Biological Chemistry 42845mentioning

confidence: 99%

mentioning

confidence: 95%

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Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii

Sugai

Ueno

Hayashi

et al. 2011

Journal of Biological Chemistry

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Genome-Based Discovery of an Unprecedented Cyclization Mode in Fungal Sesterterpenoid Biosynthesis

et al. 2016

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Sesterterpenoids are a group of terpenoid natural products that are primarily biosynthesized via cyclization of the C25 linear substrate geranylfarnesyl pyrophosphate (GFPP). Although the long carbon chain of GFPP in theory allows for many different cyclization patterns, sesterterpenoids are relatively rare species among terpenoids, suggesting that many intriguing sesterterpenoid scaffolds have been overlooked. Meanwhile, the recent identification of the first sesterterpene synthase has allowed the discovery of new sesterterpenoids by the genome mining approach. In this study, we characterized the unusual fungal sesterterpene synthase EvQS and successfully obtained the sesterterpene quiannulatene (1) with a novel and unique highly congested carbon skeleton, which is further oxidized to quiannulatic acid (2) by the cytochrome P450 Qnn-P450. A mechanistic study of its cyclization from GFPP indicated that the biosynthesis employs an unprecedented cyclization mode, which involves three rounds of hydride shifts and two successive C-C bond migrations to construct the 5-6-5-5-5 fused ring system of 1.

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Molecular evolution of the substrate specificity of ent-kaurene synthases to adapt to gibberellin biosynthesis in land plants

Shimane

Ueno

Morisaki

et al. 2014

Biochemical Journal

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ent-Kaurene is a key intermediate in the biosynthesis of the plant hormone gibberellin. In ent-kaurene biosynthesis in flowering plants, two diterpene cyclases (DTCs), ent-copalyl diphosphate (ent-CDP) synthase (ent-CPS) and ent-kaurene synthase (KS), catalyse the cyclization of geranylgeranyl diphosphate to ent-CDP and ent-CDP to ent-kaurene, respectively. In contrast, the moss Physcomitrella patens has a bifunctional ent-CPS/KS (PpCPS/KS) that catalyses both cyclization reactions. To gain more insight into the functional diversity of ent-kaurene biosynthetic enzymes in land plants, we focused on DTCs in the lycophyte Selaginella moellendorffii. The present paper describes the characterization of two S. moellendorffii DTCs (SmKS and SmDTC3) in vitro. SmDTC3 converted ent-CDP into ent-16α-hydroxykaurane and also used other CDP stereoisomers as substrate. Remarkably, SmKS, which produces ent-kaurene from ent-CDP, showed similar substrate selectivity: both SmKS and SmDTC3 synthesized sandaracopimaradiene from normal CDP. Therefore, the diversity of substrate recognition among KSs from other plants was investigated. PpCPS/KS could use normal CDP and syn-CDP as well as ent-CDP as substrate. In contrast, lettuce KS showed high specificity for ent-CDP, and rice KS recognized only ent-CDP. Our studies imply that ancient KS having low substrate specificity has evolved to be specific for ent-CDP to the biosynthesis of gibberellin.

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Enzymatic Total Synthesis of Gibberellin A₄from Acetate

Cited by 11 publications

References 38 publications

Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii

Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii

Genome-Based Discovery of an Unprecedented Cyclization Mode in Fungal Sesterterpenoid Biosynthesis

Molecular evolution of the substrate specificity of ent-kaurene synthases to adapt to gibberellin biosynthesis in land plants

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Enzymatic Total Synthesis of Gibberellin A4from Acetate

Cited by 11 publications

References 38 publications

Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii

Enzymatic 13C Labeling and Multidimensional NMR Analysis of Miltiradiene Synthesized by Bifunctional Diterpene Cyclase in Selaginella moellendorffii

Genome-Based Discovery of an Unprecedented Cyclization Mode in Fungal Sesterterpenoid Biosynthesis

Molecular evolution of the substrate specificity of ent-kaurene synthases to adapt to gibberellin biosynthesis in land plants

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Enzymatic Total Synthesis of Gibberellin A₄from Acetate