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

• DOI: 10.1073/pnas.96.23.13080
• Publication Date: 2002-07-26T14:35:07Z
• Authors: Long Thai; Jeffrey S. Rush; Jude E. Maul; Timothy Devarenne; Dana L. Rodgers; Joseph Chappell; Charles J. Waechter
• Keywords: 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

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.