Identification of a 3β-Hydroxysteroid Dehydrogenase/ 3-Ketosteroid Reductase Involved in α-Tomatine Biosynthesis in Tomato

Lee, Hyoung Jae; Nakayasu, Masaru; Akiyama, Ryota; Kobayashi, Midori; Miyachi, Haruka; Sugimoto, Yukihiro; Umemoto, Naoyuki; Saito, Kazuki; Muranaka, Toshiya; Mizutani, Masaharu

doi:10.1093/pcp/pcz049

Cited by 35 publications

(29 citation statements)

References 36 publications

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“…Heterologous expression of SlS5αR1 and SlS5αR2 proteins in insect cells was conducted as described by Ohnishi et al (2006). Similarly, the DNA fragments containing the Sl3βHSD1 gene ORF were ligated into pCold ProS2 vector (TaKaRa), and recombinant Sl3βHSD1 protein was prepared using bacterial expression system in E.coli strain BL21 (DE3) as described by Lee et al (2019).…”

Section: Heterologous Expression Of the Recombinant Sls5αr1 Sls5αr2 mentioning

confidence: 99%

“…GC-MS analysis of the reaction product was conducted as described previously (Lee et al 2019). Briefly, the residue was trimethylsilylated, and then subjected to a GC-MS-QP2010 Ultra (Shimadzu, Kyoto, Japan) with a DB-1MS (30 m×0.25 mm, 0.25 µm film thickness; J&W Scientific, CA, USA) capillary column.…”

Section: In Vitro Enzyme Activity Assaymentioning

confidence: 99%

“…Tomatidine possesses a single bond between C5 and C6 (dihydro type), while dehydrotomatidine has a Δ 5,6 double bond (dehydro type), and is biosynthesized from dehydrotomatidine by the four-step reaction process shown in Figure 1. We recently identified that the gene designated as Sl3βHSD1 is involved in the conversion of dehydrotomatidine to tomatidine (Lee et al 2019). Sl3βHSD1 is a multifunctional enzyme that possesses the activities of 3β-hydroxysteroid dehydrogenase (3βHSD) and 3-ketosteroid isomerase (3KSI) when acting on dehydrotomatidine to form tomatid-4-en-3-one and also shows the activity of 3-ketosteroid reductase (3KSR) when acting on tomatid-3-one to produce tomatidine (Lee et al 2019).…”

Section: Introductionmentioning

confidence: 99%

“…We recently identified that the gene designated as Sl3βHSD1 is involved in the conversion of dehydrotomatidine to tomatidine (Lee et al 2019). Sl3βHSD1 is a multifunctional enzyme that possesses the activities of 3β-hydroxysteroid dehydrogenase (3βHSD) and 3-ketosteroid isomerase (3KSI) when acting on dehydrotomatidine to form tomatid-4-en-3-one and also shows the activity of 3-ketosteroid reductase (3KSR) when acting on tomatid-3-one to produce tomatidine (Lee et al 2019). Therefore, Sl3βHSD1 is involved in the three reaction steps among the four reactions from dehydrotomatidine to tomatidine, and however, it has not yet been identified as the gene involved in a 5α-reduction step from tomatid-4-en-3-one to tomatid-3-one.…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, the presence of two isozymes was suggested by a crude enzyme assay performed on tomatoes (Rosati et al 2005). We recently isolated and characterized SlS5αR1, and co-incubation of SlS5αR1 with Sl3βHSD1 and dehydrotomatidine in vitro demonstrated that the four reaction steps converting dehydrotomatidine to tomatidine were reconstituted in the in vitro assay (Lee et al 2019). However, whether SlS5αR1 contributes to α-tomatine biosynthesis in vivo has remained unknown.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes

Akiyama

Lee

Nakayasu

et al. 2019

Plant Biotechnology

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α-tomatine and dehydrotomatine are steroidal glycoalkaloids (SGAs) that accumulate in the mature green fruits, leaves, and flowers of tomatoes (Solanum lycopersicum) and function as defensive compounds against pathogens and predators. The aglycones of α-tomatine and dehydrotomatine are tomatidine and dehydrotomatidine (5,6-dehydrogenated tomatidine), and tomatidine is derived from dehydrotomatidine via four reaction steps: C3 oxidation, isomerization, C5α reduction, and C3 reduction. Our previous studies (Lee et al. 2019) revealed that Sl3βHSD is involved in the three reactions except for C5α reduction, and in the present study, we aimed to elucidate the gene responsible for the C5α reduction step in the conversion of dehydrotomatidine to tomatidine. We characterized the two genes, SlS5αR1 and SlS5αR2, which show high homology with DET2, a brassinosteroid 5α reductase of Arabidopsis thaliana. The expression pattern of SlS5αR2 is similar to those of SGA biosynthetic genes, while SlS5αR1 is ubiquitously expressed, suggesting the involvement of SlS5αR2 in SGA biosynthesis. Biochemical analysis of the recombinant proteins revealed that both of SlS5αR1 and SlS5αR2 catalyze the reduction of tomatid-4-en-3-one at C5α to yield tomatid-3-one. Then, SlS5αR1-or SlS5αR2-knockout hairy roots were constructed using CRISPR/Cas9 mediated genome editing. In the SlS5αR2-knockout hairy roots, the α-tomatine level was significantly decreased and dehydrotomatine was accumulated. On the other hand, no change in the amount of α-tomatine was observed in the SlS5αR1-knockout hairy root. These results indicate that SlS5αR2 is responsible for the C5α reduction in α-tomatine biosynthesis and that SlS5αR1 does not significantly contribute to α-tomatine biosynthesis.

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Section: Heterologous Expression Of the Recombinant Sls5αr1 Sls5αr2 mentioning

confidence: 99%

Section: In Vitro Enzyme Activity Assaymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes

Akiyama

Lee

Nakayasu

et al. 2019

Plant Biotechnology

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Steroidal alkaloid biosynthesis is coordinately regulated and differs among tomatoes in the red‐fruited clade

2022

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The tomato (Solanum spp.) clade of Solanaceae features a unique assortment of cholesterol-derived steroidal alkaloids. However, little quantitative data exists reporting the profile and concentration of these alkaloids across diverse fruit germplasm. To address the lack of knowledge regarding the chemical diversity, concentration, and genetic architecture controlling tomato steroidal alkaloids, we quantitatively profiled and genotyped two tomato populations representing diversity in the red-fruited clade. We grew 107 genetically diverse fresh market, processing, landrace, and wild tomato in multiple environments. Nine steroidal alkaloid groups were quantified using ultrahigh performance liquid chromatography tandem mass spectrometry. The diversity panel and a biparental population segregating for high alpha-tomatine were genotyped to identify and validate quantitative trait loci (QTL) associated with steroidal alkaloids. Landraces and wild material exhibited higher alkaloid concentrations and more chemical diversity. Average total content of steroidal alkaloids, often dominated by lycoperoside F/G/esculeoside A, ranged from 1.9 to 23.3 mg 100 g −1 fresh wt. across accessions. Landrace and wild cherry accessions distinctly clustered based on elevated concentrations of early or late-pathway steroidal alkaloids. Significant correlations were observed among alkaloids from the early and late parts of the biosynthetic pathway in a species-dependent manner. A QTL controlling multiple, early steroidal alkaloid pathway intermediates on chromosome 3 was identified by genome-wide association studies (GWAS) and validated in a backcross population. Overall, tomato steroidal alkaloids are diverse in the red-fruited clade and their biosynthesis is regulated in a coordinated manner.

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Natural products: Potential therapeutic agents to prevent skeletal muscle atrophy

Yadav

Singh

et al. 2022

European Journal of Pharmacology

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Identification of a 3β-Hydroxysteroid Dehydrogenase/ 3-Ketosteroid Reductase Involved in α-Tomatine Biosynthesis in Tomato

Cited by 35 publications

References 36 publications

Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes

Characterization of steroid 5α-reductase involved in α-tomatine biosynthesis in tomatoes

Steroidal alkaloid biosynthesis is coordinately regulated and differs among tomatoes in the red‐fruited clade

Natural products: Potential therapeutic agents to prevent skeletal muscle atrophy

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