ent-Kaurene Synthase from the FungusPhaeosphaeria sp. L487

Kawaide, Hiroshi; Imai, Ryozo; Sassa, Takeshi; Kamiya, Yûji

doi:10.1074/jbc.272.35.21706

Cited by 146 publications

(54 citation statements)

References 34 publications

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“…4B). The identity of the product was confirmed by GC-MS, using GGDP as a substrate in the presence of a recombinant CPS from pea (Ait-Ali et al, 1997; Kawaide et al, 1997; data not shown). ent-Kaurene was identified by full-scan GC-MS from an incubation mixture containing both CPS and His-T7-AtKS.…”

Section: Functional Studies Of the Atks Proteinmentioning

confidence: 94%

The GA2 Locus of Arabidopsis thalianaEncodes ent-Kaurene Synthase of Gibberellin Biosynthesis

et al. 1998

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The ga2 mutant of Arabidopsis thaliana is a gibberellin-deficient dwarf. Previous biochemical studies have suggested that the ga2 mutant is impaired in the conversion of copalyl diphosphate to ent-kaurene, which is catalyzed by ent-kaurene synthase (KS). Overexpression of the previously isolated KS cDNA from pumpkin (Cucurbita maxima) (CmKS) in the ga2 mutant was able to complement the mutant phenotype. A genomic clone coding for KS, AtKS, was isolated from A. thaliana using CmKS cDNA as a heterologous probe. The corresponding A. thaliana cDNA was isolated and expressed in Escherichia coli as a fusion protein. GAs are a group of diterpene compounds, some of which are plant-growth regulators that control many aspects of plant development, such as seed germination, shoot elongation, and flower development. A number of GAresponsive dwarf mutants that are deficient in the biosynthesis of active GAs have been characterized in various plant species (for a recent review, see Hedden and Kamiya, 1997;Ross et al., 1997). Biochemical characterization of these mutants has contributed to the elucidation of GA biosynthetic pathways. ent-Kaurene is an early intermediate in GA biosynthesis and is synthesized by the cyclization of GGDP via CDP. In plants, this two-step cyclization involves two distinct enzymes: CPS catalyzes the cyclization of GGDP to CDP, which is then converted to entkaurene by KS. CPS and KS were formally called entkaurene synthases A and B, respectively (Hedden and Kamiya, 1997). Biochemical studies suggested that CPS and KS may interact with each other (Duncan and West, 1981) and that these enzymes are localized in plastids (Railton et al., 1984;Sun and Kamiya, 1994; Aach et al., 1995). There is some evidence that ent-kaurene biosynthesis is controlled by environmental conditions such as photoperiod (Zeevaart and Gage, 1993) and temperature (Moore and Moore, 1991), as well as during plant development (Chung and Coolbaugh, 1986;Silverstone et al., 1997).From Arabidopsis thaliana six GA-responsive dwarf mutants (ga1 through ga6) have been isolated and characterized (Koornneef and van der Veen, 1980;Sponsel et al., 1997). Each mutant is blocked in a specific step in GA biosynthesis. The GA1 locus has been cloned (Sun et al., 1992) and shown to encode CPS (Sun and Kamiya, 1994). The GA4 and GA5 genes code for dioxygenases involved in later steps of the GA biosynthetic pathway (Chiang et al., 1995;Xu et al., 1995). The GA2, GA3, and GA6 loci may encode enzymes catalyzing steps in the GA biosynthetic pathway or regulators of these enzymes.The ga2-1 mutant is a nongerminating, extreme dwarf, which is phenotypically similar to strong alleles of the ga1 and ga3 mutants. (Koornneef and van der Veen, 1980). The biochemical characterization of the ga2 mutant was described by Zeevaart and Talon (1992). The ga2 mutant is responsive to exogenous ent-kaurene and is lacking in the ability to accumulate ent-kaurene when treated with an inhibitor of ent-kaurene metabolism. A cell-free extract prepared from siliques of the...

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Section: Functional Studies Of the Atks Proteinmentioning

confidence: 94%

The GA2 Locus of Arabidopsis thalianaEncodes ent-Kaurene Synthase of Gibberellin Biosynthesis

et al. 1998

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“…L487, and the function of the gene investigated. In contrast to plant enzymes, the fungal ent-kaurene synthase (FCPS/KS) catalyzes the two-step cyclization reaction from GGDP to ent-kaurene via CDP (11). This FCPS/KS is a bifunctional diterpene cyclase having both activities of plant CPS and KS.…”

Section: Ent-kaurenementioning

confidence: 99%

Functional Analysis of the Two Interacting Cyclase Domains in ent-Kaurene Synthase from the FungusPhaeosphaeria sp. L487 and a Comparison with Cyclases from Higher Plants

Kawaide

Sassa²,

Kamiya³

2000

Journal of Biological Chemistry

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We report here kinetic analysis and identification of the two cyclase domains in a bifunctional diterpene cyclase, Phaeosphaeria ent-kaurene synthase (FCPS/KS). Kinetics of a recombinant FCPS/KS protein indicated that the affinity for copalyl diphosphate is higher than that for geranylgeranyl diphosphate (GGDP). ent-Kaurene production from GGDP by FCPS/KS was enhanced by the addition of a plant ent-kaurene synthase (KS) but not by plant CDP synthase (CPS), suggesting that the rate of ent-kaurene production of FCPS/KS may be limited by the KS activity. Site-directed mutagenesis of aspartate-rich motifs in FCPS/KS indicated that the 318 DVDD motif near the N terminus and the 656 DEFFE motif near the C terminus may be part of the active site for the CPS and KS reactions, respectively. The other aspartate-rich 132 DDVLD motif near the N terminus is thought to be involved in both reactions. Functional analysis of the N-and C-terminal truncated mutants revealed that a N-terminal 59-kDa polypeptide catalyzed the CPS reaction and a C-terminal 66-kDa polypeptide showed KS activity. A 101-kDa polypeptide lacking the first 43 amino acids of the N terminus reduced KS activity severely without CPS activity. These results indicate that there are two separate interacting domains in the 106-kDa polypeptide of FCPS/KS. (Fig. 1, 3) is a tetracyclic diterpene hydrocarbon and one of the biosynthetic intermediates in the production of the gibberellin (GA) 1 plant hormones. Both higher plants and GA-producing fungi produce ent-kaurene from geranylgeranyl diphosphate (GGDP, 1) via (-)-copalyl diphosphate (CDP, 2) (1). In higher plants, two different diterpene cyclases, copalyldiphosphate synthase (CPS) and ent-kaurene synthase (KS), are involved in the biosynthesis of ent-kaurene; CPS catalyzes the cyclization of GGDP to CDP and KS catalyzes the cyclization of CDP to ent-kaurene (2). To date, several CPS and KS genes have been cloned from higher plants (3-8). These genes encode proteins with N-terminal transit peptides that the enzymes target to the plastid (3, 9, 10). Recently, an ent-kaurene synthase cDNA has been cloned from a GA-producing fungus Phaeosphaeria sp. L487, and the function of the gene investigated. In contrast to plant enzymes, the fungal ent-kaurene synthase (FCPS/KS) catalyzes the two-step cyclization reaction from GGDP to ent-kaurene via CDP (11). This FCPS/KS is a bifunctional diterpene cyclase having both activities of plant CPS and KS. ent-KaureneConversion of GGDP to ent-kaurene in fungi and plants involves two different cyclization reactions (12-14). According to cyclization mechanisms and aspartate-rich motifs, diterpene cyclases are classified into two groups by MacMillan and Beale (15). Type A cyclization reaction is initiated by ionization of the diphosphate, and type B cyclization reaction is initiated by protonation at the 14,15-double bond of GGDP. The reaction of CPS is initiated as a type B cyclization and proceeds without rearrangement of the carbon skeleton (Fig. 1). A similar cyclization reaction, ...

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“…2) On the other hand, the possibility of the presence of another cyclase, such as ent-CDP synthase, in the fungus 2) also existed in the formation of ent-13-epi-manoyl oxide. Possible biosynthetic relationships of these rare fungal metabolites are summarized as shown in Fig.…”

Section: 4)mentioning

confidence: 99%

Phaeoside, a Novel Galactoside of Hydroxymanoyl Oxide from the Gibberellin A₁-ProducingPhaeosphaeriasp. L487

Kenmoku

Sugai

Yajima

et al. 2004

Bioscience, Biotechnology, and Biochemistry

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ent-Kaurene Synthase from the FungusPhaeosphaeria sp. L487

Cited by 146 publications

References 34 publications

The GA2 Locus of Arabidopsis thalianaEncodes ent-Kaurene Synthase of Gibberellin Biosynthesis

The GA2 Locus of Arabidopsis thalianaEncodes ent-Kaurene Synthase of Gibberellin Biosynthesis

Functional Analysis of the Two Interacting Cyclase Domains in ent-Kaurene Synthase from the FungusPhaeosphaeria sp. L487 and a Comparison with Cyclases from Higher Plants

Phaeoside, a Novel Galactoside of Hydroxymanoyl Oxide from the Gibberellin A₁-ProducingPhaeosphaeriasp. L487

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ent-Kaurene Synthase from the FungusPhaeosphaeria sp. L487

Cited by 146 publications

References 34 publications

The GA2 Locus of Arabidopsis thalianaEncodes ent-Kaurene Synthase of Gibberellin Biosynthesis

The GA2 Locus of Arabidopsis thalianaEncodes ent-Kaurene Synthase of Gibberellin Biosynthesis

Functional Analysis of the Two Interacting Cyclase Domains in ent-Kaurene Synthase from the FungusPhaeosphaeria sp. L487 and a Comparison with Cyclases from Higher Plants

Phaeoside, a Novel Galactoside of Hydroxymanoyl Oxide from the Gibberellin A1-ProducingPhaeosphaeriasp. L487

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Phaeoside, a Novel Galactoside of Hydroxymanoyl Oxide from the Gibberellin A₁-ProducingPhaeosphaeriasp. L487