phytosterols and 1-deoxy-2-pentulose

Topics: Butadienes; Erythritol; Euphorbia; Fungi; Glucose; Hemiterpenes; Latex; Metabolic Networks and Pathways; Mevalonic Acid; Phosphates; Phytosterols; Triterpenes; Xylulose

The cooperation of the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, operating in parallel in plants to generate isoprenoid precursors, has been studied extensively. Elucidation of the isoprenoid metabolic pathways is indispensable for the rational design of plant and microbial systems for the production of industrially valuable terpenoids. Here, we describe a new method, based on numerical modeling of mass spectra of metabolically labeled dolichols (Dols), designed to quantitatively follow the cooperation of MVA and MEP reprogrammed upon osmotic stress (sorbitol treatment) in Arabidopsis (

Topics: Arabidopsis; Carbon Isotopes; Chromatography, Gas; Dolichols; Erythritol; Isotope Labeling; Metabolic Networks and Pathways; Mevalonic Acid; Models, Theoretical; Osmotic Pressure; Phytosterols; Sorbitol; Spectrometry, Mass, Electrospray Ionization; Sugar Phosphates; Terpenes; Xylulose

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate, the universal precursor for isoprenoid biosynthesis. The key enzyme of the cytoplasmic mevalonic acid (MVA) pathway is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). Treatment of Tobacco Bright Yellow-2 (TBY-2) cells by the HMGR-specific inhibitor mevinolin led to growth reduction and induction of apparent HMGR activity, in parallel to an increase in protein representing two HMGR isozymes. Maximum induction was observed at 24 h. 1-Deoxy-d-xylulose (DX), the dephosphorylated first precursor of the plastidial 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway, complemented growth inhibition by mevinolin in the low millimolar concentration range. Furthermore, DX partially re-established feedback repression of mevinolin-induced HMGR activity. Incorporation studies with [1,1,1,4-2H4]DX showed that sterols, normally derived from MVA, in the presence of mevinolin are synthesized via the MEP pathway. Fosmidomycin, an inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase, the second enzyme of the MEP pathway, was utilized to study the reverse complementation. Growth inhibition by fosmidomycin of TBY-2 cells could be partially overcome by MVA. Chemical complementation was further substantiated by incorporation of [2-13C]MVA into plastoquinone, representative of plastidial isoprenoids. Best rates of incorporation of exogenous stably labeled precursors were observed in the presence of both inhibitors, thereby avoiding internal isotope dilution.

Topics: Cytosol; Erythritol; Fosfomycin; Hydroxymethylglutaryl CoA Reductases; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Mevalonic Acid; Nicotiana; Phytosterols; Plastids; Plastoquinone; Signal Transduction; Sugar Phosphates; Xylulose