The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Toporkova, Yana Y.; Смирнова, Е О; Iljina, Tatiana M.; Mukhtarova, Lucia S.; Gorina, Svetlana S.; Grechkin, Alexander N.

doi:10.1016/j.phytochem.2020.112512

Cited by 8 publications

(9 citation statements)

References 37 publications

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“…While many CYP74 isoforms display multifunctionality [ 91 ], catalyzing varying proportions of AOS, epoxy alcohol synthase (EAS), divinyl ether synthase (DES), and HPL products, the CYP74A and CYP74B clade members show predominantly AOS and HPL activities, respectively. To understand the potential function of C .…”

Section: Resultsmentioning

confidence: 99%

Oxylipin biosynthetic gene families of Cannabis sativa

et al. 2023

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Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are a group of chemically and functionally diverse fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism.

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Section: Resultsmentioning

confidence: 99%

Oxylipin biosynthetic gene families of Cannabis sativa

et al. 2023

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“…While many CYP74 isoforms display multifunctionality (91), catalyzing varying proportions of AOS, EAS, DES, and HPL products, the CYP74A and CYP74B clade members show predominantly AOS and HPL activities, respectively. To understand the potential function of C. sativa CYP74 family members, a phylogenetic analysis assessed their relationship within the relatively well-characterized CYP74 subclades from diverse plant species (Fig.…”

Section: Resultsmentioning

confidence: 99%

Cannabis sativa oxylipin biosynthesis: Genome-wide characterization of lipoxygenase, allene oxide synthase, allene oxide cyclase, hydroperoxide lyase, and 12-oxo-phytodienoic acid reductase gene families

Borrego

Robertson

Taylor

et al. 2022

Preprint

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Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are chemically and functionally diverse group of fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism.

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“…also proven from the docking analysis that these sites are located in the active pocket and close to the catalytic center. According to previous reports, [13][14] changes in the size and hydrophobicity of amino acids may affect the regio-and chemoselectivity of proteins, and then fluctuate the catalytic type and substrate affinity. Thus, we speculated that the replacement of L280 and M205 in CYP728B70 with more sterically hindered and hydrophilic amino acids would disrupt the active pocket and hinder the access of the substrate to the active center, thus affecting the interactions of the substrate and heme, leading to a sharp decline in yield.…”

Section: Substrate Docking and Site-directed Mutagenesis Of Cyp728b70mentioning

confidence: 96%

“…[12] CYP74B16 in Linum usitatissimum lost the divinyl ether synthase activity and recovered another two activities after substituting the phenylalanine at position 291 with a smaller and more hydrophobic valine. [13] Similarly, the chemical and regioselectivity of CYP72A63 is controlled by modifying the hydrophobicity and size of amino acids in the electron transport chain. [14] Until now, no futher functional studies on CYP728B70 and its isoenzymes have been reported.…”

Section: Introductionmentioning

confidence: 99%

Rational Design and Site‐Directed Mutagenesis Promote the Heterologous Production of the Antitumor Natural Product Dehydroabietic Acid

Ma,

Gao,

Qiu

et al. 2024

ChemistrySelect

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Cytochrome P450s participate in the biosynthesis of natural active ingredients and catalyze the formation of hydroxyl, carboxyl, epoxy and other groups from C−H bonds. CYP728B70 from Tripterygium wilfordii oxidizes the miltiradiene‐C18 methyl group to produce dehydroabietic acid, a diterpenoid intermediate with anticancer, antibacterial and anti‐inflammatory activities. To elevate the carboxylation product output, we excavated serveral homologous CYP728Bs and an N‐terminal truncated CYP728B70 transcript from T. wilfordii genome, of which CYP728B79 possessed a weak carboxylation effect towards miltiradiene through heterologous expression in Saccharomyces cerevisiae. We employed RoseTTAFold method and molecular dynamic simulations to predict its protein structure and docking pose of CYP728B70, and obtained several mutants with increased yield using Funclib and site‐directed mutagenesis methods. Our study confirmed the influence of peripheral residues on protein activity, improved the catalytic activity of CYP728B70, and would provide ideas for related enzyme modification.

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The CYP74B and CYP74D divinyl ether synthases possess a side hydroperoxide lyase and epoxyalcohol synthase activities that are enhanced by the site-directed mutagenesis

Cited by 8 publications

References 37 publications

Oxylipin biosynthetic gene families of Cannabis sativa

Oxylipin biosynthetic gene families of Cannabis sativa

Cannabis sativa oxylipin biosynthesis: Genome-wide characterization of lipoxygenase, allene oxide synthase, allene oxide cyclase, hydroperoxide lyase, and 12-oxo-phytodienoic acid reductase gene families

Rational Design and Site‐Directed Mutagenesis Promote the Heterologous Production of the Antitumor Natural Product Dehydroabietic Acid

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