cyclization mechanism catalyzed by KSs and the Ile mutants, via ent-pimar-15-en-8-yl, ent-beyeran-16-yl, and ent-kauran-16-yl intermediates, detailed overview
production of ent-kaurene as a precursor for important signaling molecules such as the gibberellins seems to have arisen early in plant evolution, with corresponding cyclase(s) present in all land plants (i.e., embryophyta). The relevant enzymes seem to represent fusion of the class II diterpene cyclase that produces the intermediate ent-copalyl diphosphate (ent-CPP) and the subsequently acting class I diterpene synthase that produces ent-kaurene, although the bifunctionality of the ancestral gene is only retained in certain early diverging plants, with gene duplication and sub-functionalization leading to distinct ent-CPP synthases and ent-kaurene synthases (KSs) generally observed
substitution of threonine for a conserved isoleucine has been shown to short-circuit the complex bicyclization and rearrangement reaction catalyzed by ent-kaurene synthases (KSs) after initial cyclization, leading to predominant production of ent-pimaradiene, at least in KSs from angiosperms. This effect extends to KSs from earlier diverging plants (i.e., bryophytes), including a bifunctional/KS. A dramatic effect of this single residue switch on product outcome to electrostatic stabilization of the ent-pimarenyl carbocation intermediate formed upon initial cyclization by the hydroxyl introduced by threonine substitution is paralleled by similar effects from substitution of alanine
the enzyme produces the diterpene ent-kaur-16-ene, which in vascular plants (i.e., tracheophytes) serves as an intermediate in biosynthesis of the gibberellin phytohormones. Production of ent-kaur-16-ene from the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) proceeds via two distinct bicyclization reactions. The first is catalyzed by copalyl diphosphate synthases (CPSs, EC 5.5.1.13) that are representative of class II diterpene cyclases and produce ent-copalyl diphosphate. This is then subsequently further cyclized and rearranged by ent-kaurene synthases (KSs)
the enzyme produces the diterpene ent-kaur-16-ene, which in vascular plants (i.e., tracheophytes) serves as an intermediate in biosynthesis of the gibberellin phytohormones
the mutant produces ent-pimara-8(14),15-diene, ent-kaur-16-ene, 8alpha-hydroxy-ent-pimara-15-ene, and ent-pimara-7,15-diene from ent-copalyl diphosphate
site-directed mutagenesis, the mutant shows altered product spectrum compared to the wild-type enzyme, the mutant produces a mixture of four products, pedominantly ent-pimara-8(14),15-diene, and a small amount of the double bond isomer ent-pimara-7,15-diene, and variable amounts of ent-kaur-16-ene, as well as substantial amounts of 8a-hydroxy-ent-pimar-15-ene
site-directed mutagenesis, the mutant shows altered product spectrum compared to the wild-type enzyme, the mutant produces a mixture of four products, pedominantly ent-pimara-8(14),15-diene, and a small amount of the double bond isomer ent-pimara-7,15-diene, and variable amounts of ent-kaur-16-ene, as well as substantial amounts of 8a-hydroxy-ent-pimar-15-ene
site-directed mutagenesis, the mutant shows altered product spectrum compared to the wild-type enzyme, the mutant produces a mixture of four products, pedominantly ent-pimara-8(14),15-diene, and small amounts of the double bond isomer ent-pimara-7,15-diene and 8a-hydroxy-ent-pimar-15-ene, and variable amounts of ent-kaur-16-ene
construction of transgenic Arabidopsis thaliana plants, using the Agrobacterium tumefaciens infection system, overexpressing KS leads to increased ent-kaurene production but not to an increase in bioactive gibberellins, no altered morphology or phenotype compared to wild-type plants