Comparison of the Enzymatic Properties of<i>ent</i>-Copalyl Diphosphate Synthases in the Biosynthesis of Phytoalexins and Gibberellins in Rice

Hayashi, Yutaka; Toyomasu, Tomonobu; Hirose, Yuko; Onodera, Yu; Mitsuhashi, Wataru; Yamane, Hisakazu; Sassa, Takeshi; Dairi, Tohru

doi:10.1271/bbb.70615

Cited by 13 publications

(14 citation statements)

References 33 publications

(48 reference statements)

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“…Substrate inhibition effects with GGPP ( 1a ) have also been reported previously for some, although not all, class II diterpene cyclases (14, 18–22). Upon kinetic analysis of the GGPP ( 1a ) concentration dependence of MtHPS activity, a clear substrate inhibition effect was observed.…”

Section: Resultssupporting

confidence: 74%

Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis

Mann

Prisic

et al. 2009

Journal of Biological Chemistry

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Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC 5.5.1.16). Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans.

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Section: Resultssupporting

confidence: 74%

Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis

Mann

Prisic

et al. 2009

Journal of Biological Chemistry

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“…Further, we characterized the enzymatic properties of recombinant OsCPS2/OsCyc2 and compared them with those of OsCPS1. 34) Several enzymatic properties of OsCPS2/OsCyc2, including the optimal pH (neutral), optimal temperature (30 C), and divalent cation requirement (Mg 2þ ), were very similar to those of OsCPS1. The K m and k cat values of OsCPS1 and OsCPS2/OsCyc2 were calculated to be 14:2 AE 1:5 mM and 4:5 Â 10 À2 /s, and 21:1 AE 5:6 mM and 7:8 Â 10 À2 /s, respectively.…”

Section: Labdane-related Diterpene Cyclase Gene Family In Ricementioning

confidence: 84%

“…OsCPS2/ OsCyc2 activity was not inhibited by 50-60 mM GGDP, which did inhibit OsCPS1 activity. 34) The ent-CDP synthases are localized in the plastid, as mentioned above. In the plastid, large amounts of carotenoid and phytol, which provide accessory pigments and the chlorophyll side chains for photosynthesis respectively, are synthesized from GGDP.…”

Section: Labdane-related Diterpene Cyclase Gene Family In Ricementioning

confidence: 92%

“…On the other hand, OsCPS1 activity exhibited approximately 70% inhibition with 100 mM Amo-1618, while OsCPS2/ OsCyc2 activity was inhibited by approximately 10%. 34) Amo-1618 is a quaternary ammonium compound that inhibits CPS activity, a step in GA biosynthesis. 35) It is noteworthy that Amo-1618, which can suppress fungal ent-CDP synthase activity, 35) inhibits OsCPS2/OsCyc2 activity much less effectively than it does OsCPS1 activity.…”

Section: Labdane-related Diterpene Cyclase Gene Family In Ricementioning

confidence: 99%

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Recent Advances Regarding Diterpene Cyclase Genes in Higher Plants and Fungi

Toyomasu

2008

Bioscience, Biotechnology, and Biochemistry

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100

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Cyclic diterpenoids are commonly biosynthesized from geranylgeranyl diphosphate (GGDP) through the formation of carbon skeletons by specific cyclases and subsequent chemical modifications, such as oxidation, reduction, methylation, and glucosidation. A variety of diterpenoids are produced in higher plants and fungi. Rice produces four classes of diterpene phytoalexins, phytocassanes A to E, oryzalexins A to F, oryzalexin S, and momilactones A and B. The six diterpene cyclase genes involved in the biosynthesis of these phytoalexins were identified and characterized. Fusicoccin A was produced by the phytopathogenic Phomopsis amygdali and served as a plant H þ -ATPase activator. A PaFS, encoding a fungal diterpene synthase responsible for fusicoccin biosynthesis, was isolated. The PaFS is an unusual chimeric diterpene synthase that possesses not only terpene cyclase activity (the formation of fusicoccadiene, a biosynthetic precursor of fusicoccin A), but also prenyltransferase activity (the formation of GGDP). Thus, we identified a unique multifunctional diterpene synthase family in fungi.

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“…Furthermore, in a phylogenetic tree based on amino acid sequence information, OsCPS2/OsCyc2 and OsCPS4/OsCyc1 were grouped in a different clade from the clade including OsCPS1 and ent-CDP synthases in other plant species. 4) This information prompted us to question whether homologs of OsCPS2/OsCyc2 and OsCPS4/OsCyc1 are present in plant species other than rice. To address the question, we isolated and characterized CPS-type genes in wheat (Triticum aestivum), a member of the Gramineae family.…”

mentioning

confidence: 99%

Cloning and Characterization of cDNAs Encodingent-Copalyl Diphosphate Synthases in Wheat: Insight into the Evolution of Rice Phytoalexin Biosynthetic Genes

Toyomasu

Kagahara

Hirose

et al. 2009

Bioscience, Biotechnology, and Biochemistry

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Comparison of the Enzymatic Properties ofent-Copalyl Diphosphate Synthases in the Biosynthesis of Phytoalexins and Gibberellins in Rice

Cited by 13 publications

References 33 publications

Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis

Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis

Recent Advances Regarding Diterpene Cyclase Genes in Higher Plants and Fungi

Cloning and Characterization of cDNAs Encodingent-Copalyl Diphosphate Synthases in Wheat: Insight into the Evolution of Rice Phytoalexin Biosynthetic Genes

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