Tropine Forming Tropinone Reductase Gene from <i>Withania somnifera</i> (Ashwagandha): Biochemical Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-Wide Gene Expression Patterns

<div><p><i>Withania somnifera</i> is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and <i>E. coli</i> over-expression of a tropinone reductase gene (<i>WsTR-I</i>) from <i>W. somnifera</i>, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ∼60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of <i>WsTR-I</i> transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of <i>WsTR-I</i> expression matched those of tropine levels. The data suggest that, in <i>W. somnifera</i>, aerial tissues as well possess tropane alkaloid biosynthetic competence. <i>In vivo</i> feeding of U-[¹⁴C]-sucrose to orphan shoot (twigs) and [¹⁴C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the <i>de novo</i> synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones.</p></div

Real time PCR based comparative pattern of expression of tropine forming tropinone reductase gene (<i>WsTR-I</i>) in different tissues of <i>W.</i>

<p><i> somnifera</i> (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.</p

Gas chromatography-based quantification of tropinone and tropine in different tissues of <i>Withania somnifera</i>.

#<p>DW: Dry weight.</p

Comparison of tropinone reductase from <i>W. somnifera</i> (WsTR-I) with known tropinone reductases by multiple sequence alignment.

<p>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.</p

Schematic representation of metabolic pathway of tropane alkaloid biosynthesis. TR-I, Tropinone reductase I; TR-II, Tropinone reductase II.

<p>Schematic representation of metabolic pathway of tropane alkaloid biosynthesis. TR-I, Tropinone reductase I; TR-II, Tropinone reductase II.</p

Gas chromatography-mass spectrometry analysis (GC-MS) of the enzymatic reaction mixture with tropinone as substrate.

<p>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 (R_t14.3 min) as tropinone (substrate of the enzymatic reaction) and peak (R_t14.9 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.</p

Homology based 3D model of tropine forming tropinone reductase of <i>W. somnifera</i> (WsTR-I).

<p>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.</p

Quantitative results of incorporation of radiolabel (¹⁴C) from U-[¹⁴C]-sucrose into tropanes (tropine and pseudotropine) by rootless shoots (twigs) of <i>W. somnifera</i>.

<p>Quantitative results of incorporation of radiolabel (¹⁴C) from U-[¹⁴C]-sucrose into tropanes (tropine and pseudotropine) by rootless shoots (twigs) of <i>W. somnifera</i>.</p

SDS-PAGE analysis of His-tagged recombinant tropine forming tropinone reductase of <i>W. somnifera</i> (WsTR-I).

<p>A, induced expression of WsTR-I in <i>E. coli</i>: 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.</p

Kinetic parameters of <i>Withania somnifera</i> tropine forming tropinone reductase (WsTR-I) catalyzed reaction.

<p>Kinetic parameters of <i>Withania somnifera</i> tropine forming tropinone reductase (WsTR-I) catalyzed reaction.</p