Abietadiene cyclase

PURIFICATION AND CHARACTERIZATION OF ABIETADIENE CYCLASE OF MARITIME PINE PINUS PINASTER Ait.)... 

This procedure not only sen/ed to concentrate the desired protein but also to remove competing phosphatases and it provided a roughly 2 fold purification of the abietadiene cyclase activities (table 1). The precipitate obtained was suspended in Tris buffer A and applied to a DEAE Sephacel column that was developped with a gradient of increasing NaCI content. Abietadiene cyclase activity was eluted at about 0,18M NaCI. 

Table 1 Protein content, abietadiene cyclase activity of the different chromatographic steps during the purification procedure.

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. 

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. 

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 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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