title: Membrane-bound geranylgeranyl diphosphate phosphatases: purification and characterization from Croton stellatopilosus leaves.
abstract: Geranylgeranyl diphosphate phosphatase is an enzyme catalyzing the dephosphorylation of geranylgeranyl diphosphate (GGPP) to form geranylgeraniol (GGOH). The enzyme activity of GGPP phosphatase was detected in leaves of Croton stellatopilosus, a Thai medicinal plant containing plaunotol, a commercial anti-peptic acyclic diterpenoid. Enzymological studies of GGPP phosphatase in C. stellatopilosis leaves revealed that the enzyme is a membrane-bound protein that could be removed from 20,000g pellet by 0.1% Triton X-100 without significant loss of enzyme activity. The solubilized enzyme preparation was separated into two activity peaks, PI and PII, by BioGel A gel filtration chromatography. PI and PII were both partially purified and characterized. PI appeared to be a tetrameric enzyme with its native molecular mass of 232kDa and subunit size of 58kDa, whereas PII was a monomeric enzyme with a molecular mass of 30-34kDa. Both phosphatases utilized GGPP as the preferred substrate over farnesyl and geranyl diphosphates. The apparent K(m) values for GGPP of PI and PII appeared to be 0.2 and 0.1mM, respectively. Both activities were Mg(2+) independent and exhibited slightly acidic pH optima, 6.0-6.5 for PI and 6.5-7.0 for PII. The catalytic activities of PII was strongly inhibited by 1.0mM of Zn(2+), Mn(2+) and Co(2+), whereas that of PI was not affected. Both enzyme preparations were very stable upon storage at -20 degrees C for 45 days without significant loss of phosphatase activity. The presence of GGPP phosphatase enzymes in C. stellatopilosus is consistent with its putative involvement in the biosynthetic pathway of plaunotol although whether PI or PII is the actual enzyme involved in the pathway remains to be clarified.
authors.affiliation: Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Pyathai Road, Pathumwan, Bangkok 10330, Thailand.
authors.forename: Natsajee
authors.initials: N
authors.lastname: Nualkaew
authors.forename: Wanchai
authors.initials: W
authors.lastname: De-Eknamkul
authors.forename: Toni M
authors.initials: TM
authors.lastname: Kutchan
authors.forename: Meinhart H
authors.initials: MH
authors.lastname: Zenk
mesh_terms: D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D029802:Croton / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D010744:Phosphoric Monoester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D018515:Plant Leaves / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D010744:Phosphoric Monoester Hydrolases