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Cloning and Expression of a Perilla frutescens Cytochrome P450 Enzyme Catalyzing the Hydroxylation of Phenylpropenes.

ABSTRACT: Phenylpropanoid volatile components in plants are useful and valuable not only as flavorings, but also as medicines and food supplements. The pharmacological actions and toxicities of these compounds have been well studied but their synthetic pathways are generally unclear. In this study, we mined expressed sequence tag libraries of pure strains of perilla maintained for over 30 years for their oil type and conducted gas chromatography-mass spectrometry analyses of the perilla oils to confirm the presence of monohydrates speculated to be intermediates of the phenylpropene synthetics pathways. These putative monohydrate intermediates and their regioisomers were synthesized to identify the reaction products of assays of heterologously expressed enzymes. An enzyme involved in the synthesis of a phenylpropanoid volatile component was identified in perilla. Expression of this enzyme in Saccharomyces cerevisiae showed that it is a member of the cytochrome P450 family and catalyzes the introduction of a hydroxy group onto myristicin to form an intermediate of dillapiole. The enzyme had high sequence similarity to a CYP71D family enzyme, high regiospecificity, and low substrate specificity. This study may aid the elucidation of generally unexploited biosynthetic pathways of phenylpropanoid volatile components.

SUBMITTER: Baba M

PROVIDER: S-EPMC7284770 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Cloning and Expression of a <i>Perilla frutescens</i> Cytochrome P450 Enzyme Catalyzing the Hydroxylation of Phenylpropenes.

Baba Mariko M Yamada Ken-Ichi KI Ito Michiho M

Plants (Basel, Switzerland) 20200501 5

Phenylpropanoid volatile components in plants are useful and valuable not only as flavorings, but also as medicines and food supplements. The pharmacological actions and toxicities of these compounds have been well studied but their synthetic pathways are generally unclear. In this study, we mined expressed sequence tag libraries of pure strains of perilla maintained for over 30 years for their oil type and conducted gas chromatography-mass spectrometry analyses of the perilla oils to confirm th ...[more]

PMID: 32369997

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Project description:BACKGROUND:Polyunsaturated fatty acids such as linoleic acid (LA) and ?-linolenic acid (ALA) are abundant in vegetable oils and are important for human health. In the body, LA and ALA are respectively converted to the omega-6 fatty acid ?-linolenic acid (GLA) and the omega-3 fatty acid stearidonic acid (SDA) by ?6 desaturase (D6DES). Currently, dietary GLA and SDA are mainly obtained from marine organisms, but given their benefits to human health, many studies have aimed to enhance their accumulation in transgenic crops. Perilla frutescens (perilla) accumulates more ALA in its seed oil compared to other oilseed crops, making it a good candidate for the production of fatty acids via the fatty acid desaturase D6DES. RESULTS:In this study, we cloned the D6DES gene from Phytophthora citrophthora and confirmed its function in budding yeast. We then transformed the functional D6DES gene under the control of the seed-specific vicilin promoter into the perilla cultivar Yeobsil. The resulting transgenic perilla seeds accumulated significant levels of GLA and SDA, as well as putative C18:2?6,9 at minor levels. Developing seeds and leaves also accumulated GLA and SDA, although PcD6DES expression and GLA and SDA levels were much lower in leaves compared to developing seeds. GLA and SDA accumulated in both polar lipids and neutral lipids in mature perilla seeds expressing PcD6DES, especially in neutral lipids. Although the seed weight in PcD6DES perilla was 87-96% that of wild type, the total oil content per seed weight was similar between lines. The PcD6DES perilla plants contained very high content (over 45%) of both GLA and SDA in seed oil. CONCLUSIONS:Thus, PcD6DES perilla plants may represent a feasible alternative to traditional marine sources for the production of omega-3 oil capsules and to evening primrose seed oil for GLA as health food. In addition, these plants can be used to create other transgenic lines harboring additional genes to produce other desirable fish-oil like oils.

Project description:The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio- and stereospecific insertion of two hydroxyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1beta-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1beta(OH)EA versus 3alpha(OH)EA, indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified active-site residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1beta(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1beta(OH)EA, 2beta(OH)EA, and 3beta(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted approximately 1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6- and 5.7-fold increases in their Km values for the 1beta(OH)EA intermediate, respectively. A role for Ile486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1beta(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher Km for EA and a 4.4-fold slower turnover rate (kcat) for 1beta(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser368 and Ile486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.

Dataset Information

Cloning and Expression of a Perilla frutescens Cytochrome P450 Enzyme Catalyzing the Hydroxylation of Phenylpropenes.

Publications

Cloning and Expression of a <i>Perilla frutescens</i> Cytochrome P450 Enzyme Catalyzing the Hydroxylation of Phenylpropenes.

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