Enzyme Inhibitor Studies Reveal Complex Control of Methyl-D-Erythritol 4-Phosphate (MEP) Pathway Enzyme Expression in Catharanthus roseus

Abstract

In Catharanthus roseus, the monoterpene moiety exerts a strong flux control for monoterpene indole alkaloid (MIA) formation. Monoterpene synthesis depends on the methyl-D-erythritol 4-phosphate (MEP) pathway. Here, we have explored the regulation of this pathway in response to developmental and environmental cues and in response to specific enzyme inhibitors. For the MEP pathway entry enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a new (type I) DXS isoform, CrDXS1, has been cloned, which, in contrast to previous reports on type II CrDXS, was not transcriptionally activated by the transcription factor ORCA3. Regulation of the MEP pathway in response to metabolic perturbations has been explored using the enzyme inhibitors clomazone (precursor of 5-ketochlomazone, inhibitor of DXS) and fosmidomycin (inhibitor of deoxyxylulose 5-phosphate reductoisomerase (DXR)), respectively. Young leaves of non-flowering plants were exposed to both inhibitors, adopting a non-invasive in vivo technique. Transcripts and proteins of DXS (3 isoforms), DXR, and hydroxymethylbutenyl diphosphate synthase (HDS) were monitored, and protein stability was followed in isolated chloroplasts. Transcripts for DXS1 were repressed by both inhibitors, whereas transcripts for DXS2A&B, DXR and HDS increased after clomazone treatment but were barely affected by fosmidomycin treatment. DXS protein accumulated in response to both inhibitors, whereas DXR and HDS proteins were less affected. Fosmidomycin-induced accumulation of DXS protein indicated substantial posttranscriptional regulation. Furthermore, fosmidomycin effectively protected DXR against degradation in planta and in isolated chloroplasts. Thus our results suggest that DXR protein stability may be affected by substrate binding. In summary, the present results provide novel insight into the regulation of DXS expression in C. roseus in response to MEP-pathway perturbation.

Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments

“…Degradation of a key metabolic enzyme, deoxyxylulose 5-phosphate synthase (DXS), in the methylerythritol 4-phosphate pathway for isoprenoid biosynthesis is regulated by metabolite levels (Guevara-García et al, 2005;Han et al, 2013). Furthermore, inactive DXS is likely recognized by a J protein, J20, which functions as an adaptor delivering misfolded or damaged client proteins to Hsp70 chaperone for either refolding or proteolysis depending on intracellular contexts (Pulido et al, 2013).…”

Quantitative Analyses

“…In this species, the MEP pathway provides the building blocks for the monoterpene moiety of the monoterpene indolealkaloids, a group of medically important secondary metabolites. Using specific inhibitors to enzymes of the MEP pathway, we could unravel novel aspects of MEP pathway regulation (Han et al, 2013). -The discovery of key amino acids in R2R3MYB transcriptions factors, which determine promoter target specificity (Heppel et al, 2013), and the identification of R2R3MYB transcription factors specifically regulating stilbene synthesis (Höll et al, 2013), are the fruit of a collaboration with Jochen Bogs (Bingen), who deserves most of the credit.…”

Determination of Alkaloids inCatharanthus roseusandVinca minorby High-Performance Liquid Chromatography–Tandem Mass Spectrometry

“…Secologanin is derived from the terpenoid (isoprenoid) biosynthetic pathway, while tryptamine is derived from the indole biosynthetic pathway (Chung et al 2007;Guirimand et al 2011;Verma, Khan, et al 2014). Starting from the amino acid tryptophan and the monoterpenoid geraniol, the biosynthesis of bisindole alkaloids in C. roseus and V. minor involves at least thirty-five intermediates and thirty enzymes (Echevarria-Machado et al 2004;Huang et al 2012;Han et al 2013). Aglycon leads to a branch point, tabersonine.…”