Bifunctional abietadiene synthase: Free diffusive transfer of the (+)-copalyl diphosphate intermediate between two distinct active sites

Abietadiene synthase (AS) catalyzes two sequential, mechanistically distinc t cyclizations in the conversion of geranylgeranyl diphosphate to a mixture of abietadiene double bond isomers as the initial step of resin acid biosy nthesis in grand fir (Abies grandis). The first reaction converts geranylge ranyl diphosphate to the stable bicyclic intermediate (+)-copalyl diphospha te via protonation-initiated cyclization. In the second reaction, diphospha te ester ionization-initiated cyclization generates the tricyclic perhydrop henanthrene-type backbone, and is directly coupled to a 1,2-methyl migratio n that generates the C13 isopropyl group characteristic of the abietane fam ily of diterpenes. Using the transition-state analogue inhibitor 14,15-dihy dro-15-azageranylgeranyl diphosphate, it was demonstrated that each reactio n of abietadiene synthase is carried out at a distinct active site. Mutatio ns in two aspartate-rich motifs specifically delete one or the other activi ty and the location of these motifs suggests that the two active sites resi de in separate domains. These mutants effectively complement each other, su ggesting that the copalyl diphosphate intermediate diffuses between the two active sites in this monomeric enzyme. Free copalyl diphosphate was detect ed in steady-state kinetic reactions, thus conclusively demonstrating a fre e diffusion transfer mechanism. In addition, both mutant enzymes enhance th e activity of wild-type abietadiene synthase with geranylgeranyl diphosphat e as substrate. The implications of these results for the kinetic mechanism of abietadiene synthase are discussed.