Abietadiene synthase

RAVN, M. M., COATES, R. M., FLORY, J. E., PETERS, R. J., CROTEAU, R., Stereochemistry of the cyclization-rearrangement of (+)-copalyl diphosphate to (-)-abietadiene catalyzed by recombinant abietadiene synthase from Abies grandis, Org. Lett., 2000, 2,573-576. 

VOGEL, B. S WILDUNG, M. R., VOGEL, G., CROTEAU, R Abietadiene synthase from grand fir (Abies grandis) - cDNA isolation, characterization, and bacterial expression of a bifunctional diterpene cyclase involved in resin acid biosynthesis, J. Biol. Chem., 1996,271, 23262-23268. 

To date over 30 plant terpenoid synthases have been cloned as cDNAs, and many of these were found to encode enzymes of secondary metabolism (43). Isolation and analysis of six genomic clones encoding monoterpene ((—)-pinene and (—)-limonene), sesquiterpene ((E)-a-bisabolene and S-selinene) and diterpene (abietadiene) synthases from Abies grandis, and a diterpene (taxadiene) synthase from Taxus brevifolia have been reported (44). Overexpression of a cotton farnesyl diphosphate synthase (EPPS) in transgenic Artemesia annua has resulted in 3- to 4-fold increase in the yield of the sesquiterpenoid anti-malarial drug, artemisinin, in hairy roots (45). 

Abietic acid, a common component of conifer rosin, is formed from (-)-abieta-7(8),13(14)-diene ((-)-abietadiene) (Scheme 18 A) [144]. The cyclization of GGPP to abietadiene requires several discrete steps and enzyme-bound intermediates, yet is catalyzed by a single protein, abietadiene synthase. The mechanism of this enzyme involves protonation-induced cyclization of GGPP to enzyme-bound (-i-)-CPP, followed by typical ionization of (-i-)-CPP and cyclization to the transient olefin intermediate, pimaradiene, which undergoes reprotonation at the vinyl substituent, methyl migration and deprotonation to (-)-abietadiene [145]. Similarly, the route to (-)-kaurene involves protonation-initiated cyclization of GGPP to yield (-)-CPP, followed by ionization-initiated cyclization of (-)-CPP to a pimarenyl cation, methyl migration and deprotonation to (-)-kaurene (Scheme 18 B) [20]. 

Characterization of the constitutive and wound-inducible abietadiene synthase from Pmws contorta (lodgepole pine) said Abies grandis (grand fir), respectively, showed a pH optimum of 7.8, a preference for Mg + over Mn + and Fe as the required metal ion, and no monovalent cation requirement [145]. Studies with alternate substrates demonstrated that (+)-CPP was efficiently cyclized whereas (-)-CPP was ineffectual [146]. The pimaradiene intermediate, when... 

The abietadiene synthase from fir was purified and internal amino-acid sequence information was employed to isolate the corresponding clone from a wound-induced stem cDNA library [147]. The cDNA encodes an 868 residue protein (99,536 Da) bearing a plastidial transit peptide and exhibiting the greatest sequence similarity to the only other known gymnosperm diterpene cyclase, taxadiene synthase from Taxus brevifolia [130]. Preliminary studies have identified a highly active truncation starting at V85 which approximates the preprotein-mature protein junction. 

The bifunctional terpene cyclases with type I and type II activity are restricted to fungal and plant diterpene cyclases. These enzymes like the monofunctional type I or type II plant enzymes exhibit the apy-domain architecture, but in contrast to the monofunctional terpene cyclases, both the a-domain and the p-domain contain the highly conserved DDXXD and DXDD motifs, respectively, thus establishing the catalytic activities for type I and type II cyclizations within one enzyme. A model system is the recently structurally characterized abietadiene synthase from A. grandis [202] that catalyzes first the class II conversion of... 

A(3 (1-42), 2287 ABA biosynthesis, 1736 A(3 aggregation, 2286 Abdominal troubles, 3477 Aberrant behavior, 1375 Aberrant estrous cycles, 2421 Abies alba, 2980 Abietadiene synthase, 2725 Abiotic and biotic elicitors, 2783 Abiotic factor, 1697 Abiotic stresses, 2930 AP neuropathology, 2285 AP oligommzation, 2285 AP oligomCTs, 2283 AP peptides, 1308, 2285 AP polymaization, 2287 Abrin, 296... 

The abietadiene synthase from grand fir (Abies grandis) catalyzes two cyclization reactions at separate but interdependent active sites. The enzyme first converts GGPP in a type-B cyclization at one active site to (+)-CPP, which then undergoes a type A cyclization and additional reactions at the second active site to form a mixture of abietadiene isomers [26, 32]. 

Peters, R.J., Ravn, M.M., Coates, R.M. and Croteau, R.B. (2001) Bifunctional abietadiene synthase Free diffusive transfer ofthe (+)-copalyl diphosphate intermediate between two distinct active sites. /. Am. Chem. Soc., 123, 8974-8978. 

Abietadiene synthase, an enzyme which catalyzes the cyclization of geranylgeranyl pyrophosphate to 7,13-abietadiene. (fig. 1) has been partially purified from the lOO.OOOg supernatant of young needle extract by a combination of PEG precipitation, ion exchange, affinity color and gel filtration chromatographies. 

Our data suggest the existence of a singie soiubie abietadiene cyclase catalyzing the cyciisation of GGPP in a singie product. The purification of abietadiene synthase from conifer needies is complicated because of the high amounts of polyphenols and resin and the poor development of the secretory tissues. 

A series of ionization and proton-induced cycUzations 16 20 catalyzed by the taxadiene synthase convert GGPP 1 into the taxadienes 21 and 22 for en mute synthesis of biological important natural products such as taxol and analogs (Scheme 8.2) [7]. Abietanes andkauranes are important classes of diterpenes. In the presence of abietadiene synthase, 1 undergoes a proton-induced cascade cyclization to produce the copalyl diphosphate intermediate 23 (Scheme 8.2) [8]. The latter is converted to 24 via an ionic cyclization and then to 25 through a methyl 1,2-shift between C13 and C14 [9]. In contrast, diterpene mt-kaur-16-ene 29 is not formed via the same cyclase enzyme. Consequently, it was proposed that two enzymes, mt-copalyl diphosphate and cnt-kaurene... 

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