Abietadiene synthase from grand fir (Abies grandis): Characterization and mechanism of action of the "pseudomature" recombinant enzyme

The oleoresin secreted by grand fir (Abies grandis) is composed of resin ac ids derived largely from the abietane family of diterpene olefins as precur sors which undergo subsequent oxidation of the C18-methyl group to a carbox yl function, for example, in the conversion of abieta-7,13-diene to abietic acid. A cDNA encoding abietadiene synthase has been isolated from grand fi r and the heterologously expressed bifunctional enzyme shown to catalyze bo th the protonation-initiated cyclization of geranylgeranyl diphosphate to t he intermediate (+)-copalyl diphosphate and the ionization-dependent cycliz ation of (+)copalyl diphosphate, via a pimarenyl intermediate, to the olefi n end products. Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombina nt protein expressed in Escherichia coli proved to be unsuitable for detail ed structure-function studies. Since the transit peptide-mature protein cle avage site could not be determined directly, a truncation series was constr ucted to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic prope rties. Both the native synthase and the pseudomature synthase having 84 res idues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abiet adiene, levopimaradiene, and neoabietadiene, as well as to three minor prod ucts, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir. Kinetic evaluation of abietadiene s ynthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provide d evidence for two functionally distinct active sites, the first for the cy clization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end prod ucts, and demonstrated that the rate-limiting step of the coupled reaction sequence resides in the second cyclization process. The structural implicat ions of these findings are discussed in the context of primary sequence ele ments considered to be responsible for binding the substrate and intermedia te and for initiating the respective cyclization steps.