Cymbopogon flexuosus known as lemongrass is an eminent aromatic grass which produces lemon scented essential oils through the 2C-methyl-D-erythritol-4-phosphate (MEP) pathway. In the second step which is believed to be the first committed step of this pathway, 1-deoxy-D-xylulose-5-phosphate (DXP) simultaneously undergoes an intra-molecular rearrangement and reduction by an enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) to form 2-C-methyl-D-erythritol-4-phosphate (MEP). In the present work we have measured the activities of DXR enzyme accompanying leaf development in C. flexuosus. Also, the DXR enzyme was purified and characterized and referred as CfDXR. The CfDXR activities markedly fluctuated in 1-5th leaf position of one month old tiller which represents gradient increase in leaf age. The CfDXR activity was recorded maximal in the 1st and 2nd leaf position which represents early (immature) developmental stages and declined rapidly in subsequent leaf positions (3rd-5th). The CfDXR was purified to homogeneity by three step procedure: ammonium sulfate fractionation, followed by ion-exchange chromatography on DEAE-cellulose and gel exclusion chromatography using sephadex G-75. The purified CfDXR showed a specific activity of 52U/mg protein. It is consists of two identical polypeptides with Mr of 45 KDa as detected by SDS-PAGE. The maximum activity (Vmax) of the purified CfDXR with DXP as substrate was 8.56 µM x min-1 whereas for NADPH 14.99 µM x min-1. The purified CfDXR had Km = 3.71 µM for the DXP and 5.99 µM for NADPAH as substrates. The optimum temperature and pH of the CfDXR was 40-60 C and pH 7.5-8.0, respectively. The CfDXR required bivalent cations (Co2+, Mn2+ and Mg2+) for activity. It showed the highest activity in presence of Co2+ (1 mM) followed by Mn2+ and Mg2+. The enzyme when stored at 4 C in 100mM Tris-HCl buffer (pH 7.5) for one month, was quite stable retaining more than 80% of the initial activity.
Cite this article:
Ashish Kumar Gupta, Deepak Ganjewala. Purification and Characterization of the 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase From Cymbopogon Flexuosus Leaves. Research J. Pharm. and Tech. 8(3): Mar., 2015; Page 320-327. doi: 10.5958/0974-360X.2015.00053.0