Figure 1.
Schematic representation of metabolic pathway of tropane alkaloid biosynthesis. TR-I, Tropinone reductase I; TR-II, Tropinone reductase II.
Figure 2.
Comparison of tropinone reductase from W. somnifera (WsTR-I) with known tropinone reductases by multiple sequence alignment.
Asterisks indicate identical amino acid positions. Arrows denote the regions selected to design degenerate primers. Residues highlighted in grey are involved in co-factor binding. Box designates the signature YXXXK motif of short chain dehydrogenases/reductases (SDRs) and sequence highlighted in black denotes catalytic tetrad. Green highlighted residues participate in tropinone binding. Yellow highlight indicates the residues conserved in SDRs.
Figure 3.
SDS-PAGE analysis of His-tagged recombinant tropine forming tropinone reductase of W. somnifera (WsTR-I).
A, induced expression of WsTR-I in E. coli: lane 1, un-induced culture extract; lane 2, induced soluble fraction; lane 3, induced insoluble fraction; lane 4, protein molecular weight marker; B, lane 1, enzyme eluted from affinity Column (Ni++-NTA); lane 2, crude soluble fraction; lane 3, protein molecular weight marker; C, lane 1, protein molecular weight marker; lane 2, crude soluble fraction; lane 3 and 4, enzyme eluted from gel filtration column.
Figure 4.
Identification of the reaction product of recombinant WsTR-I with tropinone as substrate.
TLC: I, tropine standard; II, WsTR-I reaction mixture after catalytic reaction termination; III, NaBH4 reduction products (tropine and pseudotropine) of tropinone; IV, tropinone standard. GC: A, authentic tropinone; B, authentic tropine; C, control (enzyme minus) assay mixture of WsTR-I; D, experimental (complete) assay mixture of WsTR-I assay; E, NaBH4 aided reduction products (tropine and pseudotropine) of tropinone.
Figure 5.
Gas chromatography-mass spectrometry analysis (GC-MS) of the enzymatic reaction mixture with tropinone as substrate.
A, MS-MS spectra of first GC peak and their matching with NIST and Wiley library leading to identification of the sample (enzymatic reaction mixture) peak (Rt14.3 min) as tropinone (substrate of the enzymatic reaction) and peak (Rt14.9 min) as tropine (product of the enzymatic reaction). B, MS-MS spectra of the second GC peak and their matching with NIST and Wiley library.
Figure 6.
Effect of pH on the catalytic activity of tropine forming tropinone reductase (WsTR-1) of W. somnifera.
Table 1.
Comparative account of catalytic parameters (pH optima and Km) of W. somnifera tropine forming tropinone reductase (WsTR-I) with other plant TRs reported from plants.
Table 2.
Kinetic parameters of Withania somnifera tropine forming tropinone reductase (WsTR-I) catalyzed reaction.
Figure 7.
Comparative catalytic activity of tropine forming tropinone reductase of W. somnifera (WsTR-I) with different substrate analogues (at 5 mM concentration).
Activity was measured relative to tropinone.
Figure 8.
Phylogenetic relationship of tropine forming tropinone reductase of W. somnifera (WsTR-I) with other plant TRs.
The tree was constructed with maximum likelihood method MEGA5 using sequences of Solanum tuberosum TRI (CAC34420.1), Solanum nigrum TRI (CAQ19734.1), Datura stramonium TRI (AAA33281.1), Anisodus acutangulus TRI (ACB71202.1), Hyoscyamus niger TRI (BAA85844.1), Datura stramonium TRII (AAA33282.1), Anisodus acutangulus TRII (ACB71203.1), Hyoscyamus niger TRII (AAB09776.1), Solanum tuberosum TRII (CAB52307.1), Solanum dulcamara TRII (CAQ19732.1), Calystegia sepium TRI (CAD20555.1), Medicago truncatula (ACJ84726.1), Populus trichocarpa (EEE95485.1), Oryza sativa (ABF95192.1), Hordeum vulgare (BAJ87177.1), Zea mays (ACG34080.1), Dendrobium nobile TRI (AFD23287.1), Glycine max (XP_003552795.1), Vitis vinifera (XP_002282554.1), Arabidopsis lyrata (ABW81053.1), Cochlearia officinalis (CAO02390.1), Arabidopsis thaliana (AAM10204.1), Boechera divaricarpa (ABW74581.1), Arabidopsis cebennensis (ABW81184.1), Zea mays TRII (NP_001147765.1), Dendrobium nobile TRII (AFD23289.1), Ectocarpus siliculosus (CBJ29615.1) along with the WsTR-I sequence.
Figure 9.
Homology based 3D model of tropine forming tropinone reductase of W. somnifera (WsTR-I).
A, WsTR-I superimposed on to DsTR-I and DsTR-II. The model was constructed on Swiss-model workspace taking DsTRI (1ae1.pdb) as a template. Comparative representation was performed by UCSF Chimera package. WsTR-I, DsTR-I and DsTR-II are depicted as gray, purple and blue, respectively. NADPH and tropinone are visible in cleft of active site; B, Tropinone binding pocket of WsTR-I. A model was prepared by alignment of WsTR-I, DsTR-I and DsTR-II following energy minimization in Swiss-PDBviewer. Tropinone binding site was visualized by Ligand Explorer. Amino acids close to tropinone are labeled; C, Three dimensional (3-D) models of WsTR-I, DsTR-I and DsTR-II were aligned and analyzed in Pymol. Residues are lebelled in green (WsTR-I), cyan (DsTR-I) and magenta (DsTR-II). Tropinone is shown in orange.
Figure 10.
Real time PCR based comparative pattern of expression of tropine forming tropinone reductase gene (WsTR-I) in different tissues of W.
somnifera (A) and real time PCR based assessment of effect signal molecules on WsTR-I expression (B). MeJa, 1 h, methyl jasmonate, 1 h treatment, MeJa, 6 h, methyl jasmonate, 6 h treatment, SA, 1 h, Salicylic acid, 1 h treatment, SA, 6 h, Salicylic acid, 6 h treatment, W, 1 h, Wounding, 1 h treatment, W, 6 h, Wounding, 6 h treatment.
Table 3.
Gas chromatography-based quantification of tropinone and tropine in different tissues of Withania somnifera.
Table 4.
Quantitative results of incorporation of radiolabel (14C) from U-[14C]-sucrose into tropanes (tropine and pseudotropine) by rootless shoots (twigs) of W. somnifera.