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 12 publications

(39 citation statements)

References 38 publications

(56 reference statements)

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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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An Isopentenol Utilization Pathway-Based “Deuterium-Scanning” Method for Mechanistic Investigations of Terpene Cyclases

Zhang,

Wang,

Liu

et al. 2024

ACS Catal.

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An isopentenol utilization pathway-based method for the investigation of the cyclization mechanism of terpene cyclases (TCs) is developed. By feeding deuterium-labeled prenols/isoprenols in combination with unlabeled ones to engineered E. coli hosts, terpene products with certain deuterium labeling patterns at hydrogen-bearing positions were obtained that can be used for deducing the cyclization processes, especially for those steps involving stereoselective hydride/proton shifts. Different types of TCs of varied origins for the biosynthesis of six known terpenes were used to test the scope and limitations of this method. Reliable results without significant deuterium dilution and scrambling are obtained by using this "deuterium-scanning" method and are consistent with those obtained previously. Limitations exist in the deuterium transfer process between those positions that are derived from the same labeled position of isoprenol, as exemplified by the failure of precisely tracking the origin of each deuterium in the labeled fusicocca-2,10(14)-diene obtained by feeding [2,2-2 H 2 ]-isoprenol. Nonetheless, the newly developed method could be used as an alternate to those using custom-labeled oligoprenyl diphosphates for probing the cyclization mechanism of TCs.

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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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Enzymatic Total Synthesis of Gibberellin A₄from Acetate

Cited by 12 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

An Isopentenol Utilization Pathway-Based “Deuterium-Scanning” Method for Mechanistic Investigations of Terpene Cyclases

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

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Enzymatic Total Synthesis of Gibberellin A4from Acetate

Cited by 12 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

An Isopentenol Utilization Pathway-Based “Deuterium-Scanning” Method for Mechanistic Investigations of Terpene Cyclases

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

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Enzymatic Total Synthesis of Gibberellin A₄from Acetate