Two cytochromes P450 catalyze S-heterocyclizations in cabbage phytoalexin biosynthesis

Klein, Andrew P.; Sattely, Elizabeth S.

doi:10.1038/nchembio.1914

Cited by 46 publications

(46 citation statements)

References 36 publications

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“…Currently, the exact conversion mechanism of 6‐Cl‐IPA (IPA) into 6‐Cl‐THA (THA) is not clear; however, it has been narrowed to four genes, thus significantly facilitating further investigation. Recently, a P450 (CYP71CR2)‐mediated C−S bond formation was elucidated in cyclobrassinin biosynthesis . The S‐ hetero scaffold of cyclobrassinin is somewhat similar to that of THN.…”

Section: Figurementioning

confidence: 99%

Identifying the Minimal Enzymes for Unusual Carbon–Sulfur Bond Formation in Thienodolin Biosynthesis

Wang

et al. 2016

ChemBioChem

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Thienodolin (THN) features a tricyclic indole-S-hetero scaffold that encompasses two unique carbon-sulfur bonds. Although its biosynthetic gene cluster has been recently identified in Streptomyces albogriseolus, the essential enzymes for the formation of C-S bonds have been relatively unexplored. Here, we isolated and characterized a new biosynthetic gene cluster from Streptomyces sp. FXJ1.172. Heterologous expression, systematic gene inactivation, and in vitro biochemical characterization enable us to determine the minimum set of genes for THN synthesis, and an aminotransferase (ThnJ) for catalyzing the downstream conversion of tryptophan chlorination. In addition, we evaluated (and mainly excluded) a previously assumed pivotal intermediate by feeding experiments. With these results, we narrowed down four enzymes (ThnC-F) that are responsible for the two unprecedented C-S bond formations. Our study provides a solid basis for further unraveling of the unique C-S mechanisms.

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Section: Figurementioning

confidence: 99%

Identifying the Minimal Enzymes for Unusual Carbon–Sulfur Bond Formation in Thienodolin Biosynthesis

Wang

et al. 2016

ChemBioChem

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“…S8). The amount of brassinin that accumulates in this tobacco system (∼400 pmol·mg -1 dry weight) is on par with the levels measured (19) in the native producer B. rapa (SI Appendix, Table S4). …”

Section: A Dithiocarbamate S-methyltransferase (Dtc-mt) Catalyzes Thesupporting

confidence: 49%

“…2C) (19). Although the same filtering criteria yielded no candidates for the GST and PLP-dependent enzyme families, they did highlight MT and β-glucosidase (BGLU) candidates that are up-regulated in leaves following pathogen elicitation.…”

Section: Chemical Hypothesis and Bioinformatic Analysis To Identify Cmentioning

confidence: 99%

“…† White mustard and hairy rocket belong to the genera Sinapis and Erucastrum, respectively, which are each closely related to Brassica. (19). The threshold for gene up-regulation was >1.8 log 2 (fold change) for two pathogen elicitations compared with mock treatment (SI Appendix, SI Materials and Methods).…”

Section: Chemical Hypothesis and Bioinformatic Analysis To Identify Cmentioning

confidence: 99%

“…As an important step toward elucidating the biosynthesis of indole-sulfur phytoalexins, we recently identified the first two dedicated enzymes, which differentially convert brassinin into cyclobrassinin and spirobrassinol (19). Here, we leveraged three orthogonal approaches to discover the key upstream biosynthetic steps that link indole glucosinolate to brassinin, namely: (i ) chemical logic to predict biosynthetic enzyme families; (ii) pathogen-inducible pathway expression; and (iii) comparative genomics of Arabidopsis and Brassica rapa.…”

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Biosynthesis of cabbage phytoalexins from indole glucosinolate

Klein

Sattely

2017

Proc. Natl. Acad. Sci. U.S.A.

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Brassica crop species are prolific producers of indole-sulfur phytoalexins that are thought to have an important role in plant disease resistance. These molecules are conspicuously absent in the model plant Arabidopsis thaliana, and little is known about the enzymatic steps that assemble the key precursor brassinin. Here, we report the minimum set of biosynthetic genes required to generate cruciferous phytoalexins starting from the well-studied glucosinolate pathway. In vitro biochemical characterization revealed an additional role for the previously described carbon-sulfur lyase SUR1 in processing cysteine-isothiocyanate conjugates, as well as the S-methyltransferase DTCMT that methylates the resulting dithiocarbamate, together completing a pathway to brassinin. Additionally, the β-glucosidase BABG that is present in Brassica rapa but absent in Arabidopsis was shown to act as a myrosinase and may be a determinant of plants that synthesize phytoalexins from indole glucosinolate. Transient expression of the entire pathway in Nicotiana benthamiana yields brassinin, demonstrating that the biosynthesis of indole-sulfur phytoalexins can be engineered into noncruciferous plants. The identification of these biosynthetic enzymes and the heterologous reconstitution of the indole-sulfur phytoalexin pathway sheds light on an important pathway in an edible plant and opens the door to using metabolic engineering to systematically quantify the impact of cruciferous phytoalexins on plant disease resistance and human health.

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D₂O Labeling to measure active biosynthesis of natural products in medicinal plants

et al. 2018

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Plant natural products have served as a prominent source of medicines throughout human history, and are still used today as clinically-approved pharmaceuticals. However, many medicinal plants that produce useful compounds are slow-growing or recalcitrant to cultivation, making it difficult to investigate the underlying genetic/enzymatic machinery responsible for biosynthesis. To better understand the metabolism of bioactive natural products in slow-growing medicinal plants, we used D2O labeling and LC-MS-based metabolomics to explore the biosynthesis of medically-relevant alkaloids in three plant species. Our results provide evidence for sites of active biosynthesis for these alkaloids, and demonstrate that D2O labeling can be used as a general method to determine sites of active secondary metabolism over relatively short time scales. We anticipate that these results will facilitate discovery of complete metabolic pathways for plant natural products of medicinal importance, especially for approaches that rely upon transcriptomics and knowledge of active metabolism to identify biosynthetic enzymes.

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Two cytochromes P450 catalyze S-heterocyclizations in cabbage phytoalexin biosynthesis

Cited by 46 publications

References 36 publications

Identifying the Minimal Enzymes for Unusual Carbon–Sulfur Bond Formation in Thienodolin Biosynthesis

Identifying the Minimal Enzymes for Unusual Carbon–Sulfur Bond Formation in Thienodolin Biosynthesis

Biosynthesis of cabbage phytoalexins from indole glucosinolate

D₂O Labeling to measure active biosynthesis of natural products in medicinal plants

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Two cytochromes P450 catalyze S-heterocyclizations in cabbage phytoalexin biosynthesis

Cited by 46 publications

References 36 publications

Identifying the Minimal Enzymes for Unusual Carbon–Sulfur Bond Formation in Thienodolin Biosynthesis

Identifying the Minimal Enzymes for Unusual Carbon–Sulfur Bond Formation in Thienodolin Biosynthesis

Biosynthesis of cabbage phytoalexins from indole glucosinolate

D2O Labeling to measure active biosynthesis of natural products in medicinal plants

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Product

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D₂O Labeling to measure active biosynthesis of natural products in medicinal plants