Biochemical Characterization of a Flavonoid O-methyltransferase from Perilla Leaves and Its Application in 7-Methoxyflavonoid Production.

Methylation is a common structural modification that can alter and improve the biological activities of natural compounds. O-Methyltransferases (OMTs) catalyze the methylation of a wide array of secondary metabolites, including flavonoids, and are potentially useful tools for the biotechnological production of valuable natural products. An OMT gene (PfOMT3) was isolated from perilla leaves as a putative flavonoid OMT (FOMT). Phylogenetic analysis and sequence comparisons showed that PfOMT3 is a class II OMT. Recombinant PfOMT3 catalyzed the methylation of flavonoid substrates, whereas no methylated product was detected in PfOMT3 reactions with phenylpropanoid substrates. Structural analyses of the methylation products revealed that PfOMT3 regiospecifically transfers a methyl group to the 7-OH of flavonoids. These results indicate that PfOMT3 is an FOMT that catalyzes the 7-O-methylation of flavonoids. PfOMT3 methylated diverse flavonoids regardless of their backbone structure. Chrysin, naringenin and apigenin were found to be the preferred substrates of PfOMT3. Recombinant PfOMT3 showed moderate OMT activity toward eriodictyol, luteolin and kaempferol. To assess the biotechnological potential of PfOMT3, the biotransformation of flavonoids was performed using PfOMT3-transformed Escherichia coli. Naringenin and kaempferol were successfully bioconverted to the 7-methylated products sakuranetin and rhamnocitrin, respectively, by E. coli harboring PfOMT3.

Isolation and characterization of isopiperitenol dehydrogenase from piperitenone-type Perilla.

Studying the biosynthesis of oil compounds in Perilla will help to elucidate regulatory systems for secondary metabolites and reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, isopiperitenol dehydrogenases 1 and 2 (ISPD1 and ISPD2), which are thought to participate the oxidation of isopiperitenol in the biosynthesis of perilla, were isolated from three pure lines of perilla. Both ISPD1 and ISPD2 oxidized isopiperitenol into isopiperitenone with an oxidized form of nicotinamide adenine dinucleotide (NAD(+)) cofactor. ISPD1 used both isopiperitenol diastereomers, whereas ISPD2 used cis-isomer as a substrate. However, only ISPD2 was isolated from piperitenone-type perilla. These results suggests that in perilla, ISPD2 is related to the biosynthesis of piperitenone, which was formed via (-)-cis-isopiperitenol.

Two types of alcohol dehydrogenase from Perilla can form citral and perillaldehyde.

Studies on the biosynthesis of oil compounds in Perilla will help in understanding regulatory systems of secondary metabolites and in elucidating reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, an aldo-keto reductase (AKR) and a geraniol dehydrogenase (GeDH), which are thought to participate in the biosynthesis of perilla essential oil components, such as citral and perillaldehyde, were isolated from three pure lines of perilla. These enzymes shared high amino acid sequence identity within the genus Perilla, and were expressed regardless of oil type. The overall reaction from geranyl diphosphate to citral was performed in vitro using geraniol synthase and GeDH to form a large proportion of citral and relatively little geraniol as reaction products. The biosynthetic pathway from geranyl diphosphate to citral, the main compound of citral-type perilla essential oil, was established in this study.

A domain swapping approach to elucidate differential regiospecific hydroxylation by geraniol and linalool synthases from perilla.

Geraniol and linalool are acyclic monoterpenes found in plant essential oils that have attracted much attention for their commercial use and in pharmaceutical studies. They are synthesized from geranyl diphosphate (GDP) by geraniol and linalool synthases, respectively. Both synthases are very similar at the amino acid level and share the same substrate; however, the position of the GDP to which they introduce hydroxyl groups is different. In this study, the mechanisms underlying the regiospecific hydroxylation of geraniol and linalool synthases were investigated using a domain swapping approach and site-directed mutagenesis in perilla. Sequences of the synthases were divided into ten domains (domains I to IV-4), and each corresponding domain was exchanged between both enzymes. It was shown that different regions were important for the formation of geraniol and linalool, namely, domains IV-1 and -4 for geraniol, and domains III-b, III-d, and IV-4 for linalool. These results suggested that the conformation of carbocation intermediates and their electron localization were seemingly to be different between geraniol and linalool synthases. Further, five amino acids in domain IV-4 were apparently indispensable for the formation of geraniol and linalool. According to three-dimensional structural models of the synthases, these five residues seemed to be responsible for the different spatial arrangement of the amino acid at H524 in the case of geraniol synthase, while N526 is the corresponding residue in linalool synthase. These results suggested that the side-chains of these five amino acids, in combination with several relevant domains, localized the positive charge in the carbocation intermediate to determine the position of the introduced hydroxyl group.

Geraniol and linalool synthases from wild species of perilla.

Geraniol and linalool synthases were isolated from three pure strains of Perilla hirtella and Perilla setoyensis, which are wild species of perilla. Their amino acid sequences were very similar to those of Perilla citriodora and Perilla frutescens that were reported previously. However, comparison of the sequences of the same functional synthases derived from different species of Perilla demonstrated that the similarities were high among P. citriodora, P. hirtella and P. frutescens, but low between P. setoyensis and any of the others. This result corresponds well with our previous results showing that P. setoyensis is remotely related to the other perilla species. Both geraniol and linalool synthases utilize geranyl diphosphate (GDP) as their catalytic substrate and they were expressed simultaneously in perilla. The linalool synthase is considered to be the enzyme whose metabolite seems not to be oxidized nor reduced in the plant body and the geraniol and limonene synthases are the initial-step-catalyzing enzymes for a variety of oil compounds. The regulation of the substrate flow between them would be interesting for further study.

[Effect of exogenous Ca2+ and NO donor SNP on seed germination and antioxidase activities of Perilla frutescens seedlings under NaCl stress].

OBJECTIVE: In order to find a method for improving the salt resistance of seeds and seedlings for Perilla frutescens under NaCl stress, seed germination and physiological characteristics of P. frutescens seedlings were studied. METHOD: Several physiological indexes of P. frutescens seeds treated by Ca2+ and sodium nitroprusside (SNP) under NaCl stress like the germination vigor, germination rate, germination index and vigor index were measured. And other indexes like the biomass of the seedlings, the content of malondialdehyde (MDA) in leaves, the activities of superoxide (SOD), peroxidase (POD) and catalase (CAT) were also measured. RESULT: The germination of P. frutescens seeds under NaCl stress was inhibited obviously. But after the treatment with Ca2+ and SNP, all of the germination indexes increased. And the seeds that treated with SNP + Ca2+ had the most significantly increase in all indexes. The germination vigor was 65.1%, the germination rate was 89.3%, the germination index and vigor index were 13.9 and 0.1109, respectively. The content of MDA decreased after the treatment. The activities of three enzymes include SOD, POD and CAT were increased by the treatment and get the maximin 0.84, 5.71 and 4.92 U x mg(-1) respectively. And the EGTA showed an obvious inhibition to the effect of SNP on P. frutescens. CONCLUSION: SNP (0.1 mmol x L(-1)) and Ca2+ (10 mmol x L(-1)) could significantly alleviate the damages to the seeds and seedlings of P. frutescens under NaCl stress, and promote the salt resistance of the seeds and seedlings. These results might suggest that exogenous NO might enhance P. frutescens salt resistance and alleviate salt injury possible by enhancing Ca2+ influx by activating Ca2+ channels and improving concentration of Ca2+ of P. frutescens seedlings.

Geraniol synthases from perilla and their taxonomical significance.

Geraniol synthases were isolated from five pure strains of Perilla citriodora and Perilla frutescens which vary in essential oil type, the main compounds of which were citral, elsholtziaketone, perillaketone, and perillene, respectively. This result supports the putative biosynthetic pathways of these three furylalkenes which are all produced by way of citral. Nucleotide sequences of geraniol synthases from three oil types of P. citriodora were identical, and almost the same as the sequence from P. frutescens, a species with twice the chromosome number of P. citriodora. This identity in sequence between P. citriodora and P. frutescens, together with other previous results, indicates that P. frutescens was formed as an amphidiploid of P. citriodora and an unknown wild species.

Limonene production in tobacco with Perilla limonene synthase cDNA.

Limonene synthase (LS) catalyses the stereo-specific cyclization of geranyl diphosphate (GPP) to form a monocyclic monoterpene, limonene. In an attempt to engineer monoterpene biosynthesis, three expression constructs of LS cDNA of Perilla frutescens, which were designed to be localized in either the plastid, the cytosol or the endoplasmic reticulum (ER), were introduced into tobacco in order to examine differences in enzyme activity and the productivity of limonene. High and moderate enzyme activity, respectively, was observed for plastid- and cytosol-localized LS, whereas no enzyme activity was seen for ER-localized LS, suggesting that the plastid is the preferred compartment for LS, while LS may also have an active form in the cytosol. The formation of limonene in vivo was confirmed by gas chromatography-mass spectrometry (GC-MS) in leaf extracts of both plastid- and cytosol-localized LS transgenic plants. The amount of limonene in plastid-localized LS transgenic plants was 143 ng g-1 fresh wt, whereas that in the cytosol-type was 40 ng g-1 fresh wt, and these limonene contents increased by 2.7-fold and 3.0-fold, respectively, with the addition of methyl jasmonate. The headspace analyses showed that the plastid- and the cytosol-localized LS transgenic plants (12 cm high) emitted 390 ng and 515 ng limonene per month, respectively. The possibility of genetically engineering monoterpene production is discussed.