Geranyl pyrophosphate synthase: characterization of the enzyme and evidence that this chain-length specific prenyltransferase is associated with monoterpene biosynthesis in sage (Salvia officinalis).
Cell-free homogenates from sage (Salvia officinalis) leaves convert dimethylallyl pyrophosphate and isopentenyl pyrophosphate to a mixture of geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate, with farnesyl pyrophosphate predominating. These prenyltransferase activities were localized primarily in the soluble enzyme fraction, and separation of this preparation on Sephadex G-150 revealed the presence of a partially resolved, labile geranyl pyrophosphate synthase activity. The product of the condensation reaction between [1-14C]dimethylallyl pyrophosphate and [1-3H]isopentenyl pyrophosphate was verified as [14C,1-3H]geranyl pyrophosphate by TLC isolation, enzymatic hydrolysis to geraniol, degradative studies, and the preparation of the crystalline diphenylurethane. The cis-isomer, neryl pyrophosphate, was not a product of the enzymatic reaction. By employing a selective tissue extraction procedure, the geranyl pyrophosphate synthase activity was localized in the leaf epidermal glands, the site of monoterpene biosynthesis, suggesting that the role of this enzyme is to supply the C10 precursor for the production of monoterpenes. Glandular extracts enriched in geranyl pyrophosphate synthase were partially purified by a combination of hydrophobic interaction chromatography on phenyl-Sepharose and gel permeation chromatography on Sephadex G-150. Substrate and product specificity studies confirmed the selective synthesis of geranyl pyrophosphate by this enzyme, which was also characterized with respect to molecular weight, pH optimum, cation requirement, inhibitors, and kinetic parameters, and shown to resemble other prenyltransferases.
Croteau
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Institute of Biological Chemistry, Washington State University, Pullman 99164-6340.
Purkett
P T
PT
D002474:Cell-Free System; D002845:Chromatography; D002855:Chromatography, Thin Layer; D004122:Dimethylallyltranstransferase / Q000302:isolation & purification / Q000378:metabolism*; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D009943:Organophosphorus Compounds / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012717:Sesquiterpenes; D013729:Terpenes / Q000378:metabolism*; D014166:Transferases / Q000378:metabolism*
D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D013729:Terpenes; C004809:isopentenyl pyrophosphate; C015234:geranyl pyrophosphate; C004808:farnesyl pyrophosphate; D014166:Transferases; D004122:Dimethylallyltranstransferase