Farnesol is utilized for isoprenoid biosynthesis in plant cells via farnesyl pyrophosphate formed by successive monophosphorylation reactions

Nicotiana 0301 basic medicine 0303 health sciences Phosphotransferases (Phosphate Group Acceptor) Farnesol Catalysis Plants, Toxic 03 medical and health sciences Polyisoprenyl Phosphates Microsomes Phosphorylation Sesquiterpenes Cells, Cultured Plant Proteins
DOI: 10.1073/pnas.96.23.13080 Publication Date: 2002-07-26T14:35:07Z
ABSTRACT
The ability of Nicotiana tabacum cell cultures to utilize farnesol (F-OH) for sterol and sesquiterpene biosynthesis was investigated. [ 3 H]F-OH was readily incorporated into sterols by rapidly growing cell cultures. However, the incorporation rate into sterols was reduced by greater than 70% in elicitor-treated cell cultures whereas a substantial proportion of the radioactivity was redirected into capsidiol, an extracellular sesquiterpene phytoalexin. The incorporation of [ 3 H]F-OH into sterols was inhibited by squalestatin 1, suggesting that [ 3 H]F-OH was incorporated via farnesyl pyrophosphate (F-P-P). Consistent with this possibility, N. tabacum proteins were metabolically labeled with [ 3 H]F-OH or [ 3 H]geranylgeraniol ([ 3 H]GG-OH). Kinase activities converting F-OH to farnesyl monophosphate (F-P) and, subsequently, F-P-P were demonstrated directly by in vitro enzymatic studies. [ 3 H]F-P and [ 3 H]F-P-P were synthesized when exogenous [ 3 H]F-OH was incubated with microsomal fractions and CTP. The kinetics of formation suggested a precursor–product relationship between [ 3 H]F-P and [ 3 H]F-P-P. In agreement with this kinetic pattern of labeling, [ 32 P]F-P and [ 32 P]F-P-P were synthesized when microsomal fractions were incubated with F-OH and F-P, respectively, with [γ- 32 P]CTP serving as the phosphoryl donor. Under similar conditions, the microsomal fractions catalyzed the enzymatic conversion of [ 3 H]GG-OH to [ 3 H]geranylgeranyl monophosphate and [ 3 H]geranylgeranyl pyrophosphate ([ 3 H]GG-P-P) in CTP-dependent reactions. A novel biosynthetic mechanism involving two successive monophosphorylation reactions was supported by the observation that [ 3 H]CTP was formed when microsomes were incubated with [ 3 H]CDP and either F-P-P or GG-P-P, but not F-P. These results document the presence of at least two CTP-mediated kinases that provide a mechanism for the utilization of F-OH and GG-OH for the biosynthesis of isoprenoid lipids and protein isoprenylation.
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