Molecular Cloning, Functional Expression and Characterization of d-Limonene Synthase from Agastache rugosa.

Abstract

Monoterpenes are abundant in plant essential oils and a large part of the compounds have ring structures. These cyclic monoterpenes are biosynthesized from the acyclic precursor, geranyl pyrophosphate (GPP), through a sequence of reactions; isomerization to linalyl pyrophosphate (LPP), cyclization to a-terpinyl cation followed by skeleton modification through Wagner-Meerwein rearrangement and/or further cyclization by electrophilic attack of the cation to a double bond, and then termination by deprotonation or carbocation capture by nucleophiles such as water or pyrophosphate anion. 1)A series of reactions are usually catalyzed by a single enzyme termed monoterpene synthase. Recent development of molecular biological techniques allowed the cloning of various terpene synthase genes from plants. Comparison of the sequence data of the cloned genes clarified several common features as well as several amino acid residues conserved among mono-, sesqui-, and diterpene synthases.2) Functional expression of mutant or truncated genes prepared from the cloned genes 3,4) disclosed the relationship between these conserved amino acid residues and enzyme activities. X-Ray crystallographic analysis of 5-epi-aristolochene synthase 5) also provided important functional information. However, as for the stereochemical course of the reactions, only the classical observations using labeled substrates or substrate analogues have been reported to point out that the initial steps of the cyclization reaction proceeded through the conformations shown in Figs. 1A Pharmacognosy, Graduate School of Pharmaceutical Sciences, Kyoto University; 46-29 YoshidaShimoadachi, Sakyo, Kyoto 606-8501, Japan. Received December 10, 2001; accepted February 5, 2002 We cloned the gene of d-limonene synthase (ArLMS) from Agastache rugosa (Labiatae). The function of ArLMS was elucidated by the preparation of recombinant protein and subsequent enzyme assay. ArLMS consisted of 2077 nucleotides including 1839 bp of coding sequence that encodes a protein of 613 amino acids. This protein has a 60 kDa molecular weight, which is identical to that of d-limonene synthase from Schizonepeta tenuifolia (Labiatae). The deduced amino acid sequence of ArLMS shows high homology with the known d-and l-limonene synthases from Labiatae plants. Here, we discussed the amino acid residues responsible for the stereochemical regulation in limonene biosynthesis.

Biosynthesis of essential oils in aromatic plants: A review

“…Abies grandis AAB70707 [113] Abies grandis AAF61453 [113] Abies grandis AAF61454 [113] Abies grandis AAF61455 [113] Abies grandis AAK83564 [92] Abies grandis AAK83565 [92] Agastache rugosa AAL17636 [114] Antirrhinum majus AAO41727 [115] Antirrhinum majus AAO41726 [115] Antirrhinum majus AAO42614 [115] Antirrhinum majus ABR24418 [116] Arabidopsis thaliana AAO85533 [117] Arabidopsis thaliana AAN65379 [118] Arabidopsis thaliana AAG09310 [119] Arabidopsis thaliana AAU01970 [120] Arabidopsis thaliana AY691947 [120] Arabidopsis thaliana At1g61680 [117] Artemisia annua AAF13357 [121] Artemisia annua AAF13356…”

Phytochemistry and bioactivity of aromatic and medicinal plants from the genus Agastache (Lamiaceae)

“…Determination of volatile constituents:Essential oil hydrodistillation using Clevenger apparatus or pharmacopoeia distillation apparatus (Charles et al 1991; Mazza and Kiehn 1992; Svoboda et al 1995; Dung et al 1996; Tirillini et al 1997; Dapkevicius et al 1998; Kim et al 2001a; Maruyama et al 2002; Shin and Kang 2003; Omidbaigi and Sefidkon 2003, 2004; Estrada-Reyes et al 2004; Mallavarapu et al 2004; Shin and Pyun 2004; Bruni et al 2007; Omidbaigi et al 2008; Tian et al 2009; Ebadollahi et al 2010, 2011; Omidbaigi and Mahmoodi 2010; Skakovskii et al 2010; Gong et al 2012a, b; Li et al 2013);Essential oil distillation–extraction (Wang 2010);Extraction with organic solvents: hexane, hexane–EtOAc mixtures, EtOAc, EtOAc–MeOH mixtures, MeOH, dichloromethane (Kim et al 2001b; Shin et al 2001; Estrada-Reyes et al 2004);Extraction with diethyl ether and boiling methanol followed by cold storage (−20 °C) and steam distillation (Weyerstahl et al 1992);Headspace (Mazza and Kiehn 1992; Wilson et al 1992; Zielinska et al 2011);Glass microneedles used for the determination of secretory trichomes constituents (Tirillini et al 1997). …”

“…Essential oil hydrodistillation using Clevenger apparatus or pharmacopoeia distillation apparatus (Charles et al 1991; Mazza and Kiehn 1992; Svoboda et al 1995; Dung et al 1996; Tirillini et al 1997; Dapkevicius et al 1998; Kim et al 2001a; Maruyama et al 2002; Shin and Kang 2003; Omidbaigi and Sefidkon 2003, 2004; Estrada-Reyes et al 2004; Mallavarapu et al 2004; Shin and Pyun 2004; Bruni et al 2007; Omidbaigi et al 2008; Tian et al 2009; Ebadollahi et al 2010, 2011; Omidbaigi and Mahmoodi 2010; Skakovskii et al 2010; Gong et al 2012a, b; Li et al 2013);…”

“…The compounds were mostly purified using column chromatography on silica gel eluted with various solvents, sometimes preceded by liquid–liquid extraction:Chloroform–methanol mixture, methanol–water mixture, chloroform–methanol mixture, methanol (RA—Kim et al 1999; tilianin–Hong et al 2001; agastinol, agastenol—Lee et al 2002);Petroleum ether-acetone mixture (estragole, methyleugenol—Li et al 2013);Ethyl acetate/n-hexane gradient n-hexane/chloroform/methanol (for the elution of fractions) (agastaquinone—Lee et al 1995);Hexane–ethyl acetate ( l -pulegone—Maruyama et al 2002, Estrada-Reyes et al 2004; estragole, triterpenoids, flavonoids—Estrada-Reyes et al 2004). …”

“…Hexane–ethyl acetate ( l -pulegone—Maruyama et al 2002, Estrada-Reyes et al 2004; estragole, triterpenoids, flavonoids—Estrada-Reyes et al 2004). …”