Abietadiene Synthase from Grand Fir (Abies grandis):  Characterization and Mechanism of Action of the “Pseudomature” Recombinant Enzyme

Abstract

The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abietic acid. A cDNA encoding abietadiene synthase has been isolated from grand fir and the heterologously expressed bifunctional enzyme shown to catalyze both the protonation-initiated cyclization of geranylgeranyl diphosphate to the intermediate (+)-copalyl diphosphate and the ionization-dependent cyclization of (+)-copalyl diphosphate, via a pimarenyl intermediate, to the olefin end products. Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombinant protein expressed in Escherichia coli proved to be unsuitable for detailed structure-function studies. Since the transit peptide-mature protein cleavage site could not be determined directly, a truncation series was constructed to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic properties. Both the native synthase and the pseudomature synthase having 84 residues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene, levopimaradiene, and neoabietadiene, as well as to three minor products, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir. Kinetic evaluation of abietadiene synthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provided evidence for two functionally distinct active sites, the first for the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end products, and demonstrated that the rate-limiting step of the coupled reaction sequence resides in the second cyclization process. The structural implications of these findings are discussed in the context of primary sequence elements considered to be responsible for binding the substrate and intermediate and for initiating the respective cyclization steps.

16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: Potent Mechanism-Based Inhibitors of Recombinant ent-Kaurene Synthase from Arabidopsis thaliana

“…11 Reactions were initiated by addition of [15-3 H]ent-copalyl PP (5), 14e and [14-3 H]ent-kaurene was isolated by extraction and analyzed by liquid scintillation counting. 11,43 (Table 3) Studies with native KS partially purified from wild cucumber (Marah macrocarpus) seeds demonstrated that ring rearrangement occurs during the enzyme catalyzed cyclization of entcopalyl PP. 10a Stereo-electronic considerations dictate that the ring D rearrangement cannot be concerted with the preceding cyclization 6 → 7 to the beyeranyl + intermediate.…”

“…43 The production of ent-kaurene was quantified by extracting the incubation solutions with hexanes and passing the organic extracts through short columns of MgSO 4 and silica gel to dry them and to remove any oxygenated diterpenoids resulting from non-specific solvolysis prior to liquid scintillation counting. 43 Inhibition studies were carried out with saturating amounts of substrate (1.5 μM [15-3 H]ent-copalyl PP) and varying amounts of inhibitor, and in the case of 13 it was necessary to add substrate directly after inhibitor to detect any enzymatic activity. The resulting data were fit to an exponential equation using KaleidaGraph 4.0 (Synergy Software) to determine IC 50 values as previously described.…”

The Primary Diterpene Synthase Products of Picea abies Levopimaradiene/Abietadiene Synthase (PaLAS) Are Epimers of a Thermally Unstable Diterpenol

“…Interestingly, the products of AgAS were subsequently demonstrated to be sensitive to the assay workup conditions; when passed through silica gel and MgSO 4 prior to GC-MS analysis, abietadiene was the major product (9). However, without this treatment step, levopimaradiene, abietadiene, and neoabietadiene were seen in nearly equal amounts, along with minor amounts of palustradiene (6). This inconsistency was hypothesized to be the result of isomerization of levopimaradiene and neoabietadiene to abietadiene with the silica gel and MgSO 4 treatment (6) but was not investigated further until now.…”

“…This inconsistency was hypothesized to be the result of isomerization of levopimaradiene and neoabietadiene to abietadiene with the silica gel and MgSO 4 treatment (6) but was not investigated further until now. Consequently, AgAS was described as a multi-product diterpene synthase (6).…”

A Single Residue Switch Converts Abietadiene Synthase into a Pimaradiene Specific Cyclase

“…4 In addition, AgAS has been shown to produce sandaracopimara-8(14),15-diene (7) as a minor component (∼2%) of its product output, 8 suggesting 7 as a potential stable intermediate. However, labeling studies demonstrated intramolecular proton transfer, 9 and 7 is not converted to abietadienes by AgAS, 2 indicating that if 7 is a true intermediate it is only transiently formed en route to abietadienes in the AgAS active site. The catalyzed intramolecular proton transfer entails a shift from tertiary (5a + ) to secondary carbocation that is driven, at least in part, by ion pairing of the resulting isopimar-8(14)-en-15-yl + (5b + ) with the pyrophosphate anion released by initiating ionization of CPP (6).…”