Purification and Properties of ent-Kaurene Synthase B from Immature Seeds of Pumpkin

Abstract

enf-Kaurene synthase B (KSB) was purified 291-fold from a crude enzyme preparation from endosperm of pumpkin (Cucurbifa maxima L.). Separation of ent-kaurene synthase A and KSB was achieved by hydrophobic interaction chromatography. The fractions containing KSB activity were further purified by diethylaminoethyl, phenyl, and hydroxyapatite column chromatography. Using sodium dodecyl phosphate-polyacrylamide gel electrophoresis, the purest enzyme preparation showed a major band at an apparent molecular m a s of 81 kD. The amount of protein in this band was correlated with KSB activity after diethylaminoethyl and hydroxyapatite chromatography. The N terminus of the 81-kD protein was blocked. Therefore, the protein was partially digested with protease and the amino acid sequences of the resulting major peptide fragments were analyzed. A polyclonal antibody was raised against a synthetic peptide based on the longest peptide fragment combined with a keyhole limpet hemocyanin. The antibody recognized only the 81 -kD denatured protein and not the native KSB. The properties of KSB were examined using the phenyl-purified enzyme preparation. The K,,, value for copalyl pyrophosphate was 0.35 p~, and the optimal pH was 6.8 to 7.5. The KSB activity required divalent cations such as Mg2+, Mn2+, and Co2+, whereas Cu2+, Ca2+, and BaZ+ inhibited the activity.ent-Kaurene is an important intermediate in GA biosynthesis and is synthesized from GGPP via CPP (Fig. 1). These steps are catalyzed by KSA and KSB, respectively (Coolbaugh, 1983). Other terpenoids, such as carotenoids and phytol, also have GGPP as a precursor, whereas CPP is a precursor of macrocyclic diterpenes. Hence, KSA and KSB are important enzymes in the early stage of GA biosynthesis (Coolbaugh, 1983; Chung and Coolbaugh, 1986;Graebe, 1987). Duncan and West (1981) separated KSA and KSB from wild cucumber (Mavah macrocarpus L.) using a quaternalyaminoethyl column and suggested that the conversion of GGPP to ent-kaurene is catalyzed by the two different enzymes. These authors showed that KSA and KSB associated with each other during ent-kaurene synthesis and that KSB preferentially utilized endogenous CPP This study was performed through special coordination funds of the Science and Technology Agency of the Japanese government, and the Shorai Foundation for Science and Technology.* Corresponding author; e-mail ykamiya@postman.riken.go.jp; fax 81-48 -462-4691.produced by KSA rather than exogenous CPP. Both KSA and KSB were suggested to be localized in plastids (Aach et al., 1995). Sun et al. (1992) cloned the GAZ locus of Arabidopsis by genomic subtraction. Recently, Sun and Kamiya (1994) showed that the GA1 locus encodes KSA. A putative KSA has also been cloned from maize by transposon tagging (Bensen et al., 1995). Castor bean (Xicinus communis) produces kaurene and the related diterpenes beyerene, trachylobane, and sandaracopimaradiene, which are formed by alternative cyclization of the common intermediate CPP, and KSB was partially purified (Spickett et al., 199...

Contribution of the Mevalonate and Methylerythritol Phosphate Pathways to the Biosynthesis of Gibberellins inArabidopsis

“…DL-MVL and [2-13 C]MVL (99% labeled) were purchased from Aldrich, and mevastatin was from Sigma. ent- [1,7,12,18-13 C 4 ] Kaurene was produced from [2-13 C]MVA by a cell-free system prepared from C. maxima endosperm as previously reported (27,28).…”

“…This indicates the loss of one 13 C label in ring D and is consistent with the predicted labeling pattern through the MEP pathway from [2-13 C]DX. To confirm this result, we analyzed ent-kaurene produced from [2-13 C]MVA by a cell-free system from C. maxima endosperm (27,28), where ring D would not contain 13 C-labels (Fig. 3, Route 3).…”

Abietadiene synthase catalysis: Mutational analysis of a prenyl diphosphate ionization-initiated cyclization and rearrangement

“…AS is bifunctional in catalyzing two sequential, mechanistically different cyclizations at separate active sites occurring in structurally distinct domains (6). Protonation across the terminal 14-15 double bond of GGPP, followed by bicyclization and deprotonation, produces the stable intermediate (ϩ)-copalyl diphosphate (CPP; 2), in a reaction similar to that catalyzed by (Ϫ)-copalyl diphosphate synthase (kaurene synthase A) of gibberellin biosynthesis (7,8). This reaction occurs in an N-terminal active site with an acid͞base catalytic mechanism that may serve as a general model for such protonation-initiated cyclizations (9).…”

“…This reaction occurs in an N-terminal active site with an acid͞base catalytic mechanism that may serve as a general model for such protonation-initiated cyclizations (9). CPP diffuses from the N-terminal domain to a C-terminal active site (6) where, in a reaction similar to that mediated by kaurene synthase B (7,8), AS ionizes the diphosphate ester (CPP) to promote cyclization to the tricyclic perhydrophenanthrene backbone. However, this cyclization by AS is further coupled to a 1,2-methyl migration, by means of intramolecular proton transfer within a pimarenyl intermediate (10), to generate the C13 isopropyl group characteristic of the abietane skeleton.…”

The First Step of Gibberellin Biosynthesis in Pumpkin Is Catalyzed by at Least Two Copalyl Diphosphate Synthases Encoded by Differentially Regulated Genes

“…This precursor is produced in two steps: (a) the cyclization of GGDP to CDP and (b) the cyclization of CDP to ent-kaurene. In plants these reactions are catalyzed by two distinct enzymes that have been purified separately (Duncan and West, 1981;Saito et al, 1995). Formerly, the enzymes were known as kaurene synthetase A and B (Duncan and West, 1981).…”