Molecular Cloning of a Functional Tropinone Reductase from Leaves of Withania coagulans

1. Tropane Alkaloids Withaniacoagulans Isolation of a WcTR-I cDNA Tropane Alkaloid Biosynthesis • Three major classes: N-methylpyrrolinium-derived nicotine alkaloids, tropine-derived true tropane alkaloids, and pseudotropine-derived nortropane alkaloids, the calystegines. • The tropane alkaloids hyoscyamine and scopolamine are anticholinergic agents that act on the parasympathetic nervous system to treat spasms, to sedate patients and for dilation of the pupil (mydriasis). • Another new group of nortropane alkaloids, calystegines, resemble monosaccharides in structure, and it is not surprising that they have proved to be strong glycosidase inhibitors. Kingdom :Plantae Subkingdom :Tracheobionta Superdivision :Spermatophyta Division :Magnoliophyta Class :Magnoliopsida Subclass :Rosidae Order :Solanales Family :Solanaceae Genus :Withania Species :coagulans • Popularly known as vegetable rennet, paneerbandh, paneerdoddi. • Native to arid parts of Indian subcontinent. • Commercially exploited for its milk coagulatiing properties. • Application of the herb is recommended in nervous exhaustion, debility, insomnia, wasting diseases and impotence, dyspepsia, flatulent colic and other intestinal infections. • Berries are used as a blood purifier. • The twigs and smoke of the plant are beneficial for oral health and hygiene. • Its fruits are used for liver complaints, asthma and biliousness. Flowers are used in the management of diabetes. • Rich in steroidal lactones, the withanolides. A) Degenerate PCR; 1, DNA molecular weight marker, 2, PCR product of TrRdNterF and TrRdCterR; B) 3’ RACE; 1, DNA molecular weight marker, 2, PCR product of TrRdCterF and 3’AP; C) PCR to isolate full length WcTR-I; 1, DNA molecular weight marker, 2; PCR product of TRIFLF and TRIFLR from leaf. Substrate Specificity and Effect of Cations on WcTR-I Activity Authentication of Reaction Product Effect of pH and Temperature at WcTR-I activity Multiple sequence alignment of WcTR-I with known TR-I Heterologous Expression of recombinant tropinone reductase Molecular Cloning of a Functional Tropinone Reductase from Leaves of Withaniacoagulans Superimposition of three-dimensional (3D) models of tropinonereductases (TRs) Comparative catalytic activity of W. coagulanstropinone reductase I (WcTR-I) with different ketonic substrates. Activity was measured relative to tropinone as substrate (5 mM each). A) Effect of pH at enzyme activity: Standard assay mix was prepared including potassium phosphate buffer of different pH. Activity was recorded as change in A340. B) Effect of heat at stability of enzyme activity: Protein was incubated at different temperatures for 30 min. The incubated protein was used in assay to record the activity. A) GC of tropinone (standard); B) GC of tropine (standard) ; C) GC of control reaction (enzyme minus) of WcTR-I; D, GC of experimental (complete assay mix) of WcTR-I assay. Analysis of induced over-expressed recombinant WcTR-I. A) Comparative SDS-PAGE of crude bacterial extracts. 1. Induced insoluble fraction. 2. Induced soluble fraction. 3. Uninduced insoluble fraction. 4. Uninduced soluble fraction. B) Protein immunoblot. M. Protein size marker. E. Induced crude soluble fraction. C) SDS-PAGE analysis of affinity purified fractions. M. Protein size markers. 1, 2 and 3. affinity purified fractions. Comparative catalytic activity of WcTR-I in presence of cations/salts (5 mM). Amit K. Kushwaha1, Neelam S. Sangwan1, Sandhya Tripathi1, Rajender S. sangwan1,2* 1Central Institute of Medicinal and Aromatic Plants, Lucknow 2Bio-Processing Unit (DBT), Mohali *e-mail: sangwan.lab@gmail.com A) Comparative representation was performed by UCSF Chimera package. WcTR-I, DsTR-I and DsTR-II are depicted as blue, purple and gray, respectively. NADPH and tropinone (highlighted in green) are visible in cleft of the active site. B) Neighbouring residues involved in tropinone binding are depicted in WcTR-I active site. Asterick indicates the identical amino acids. Residues highlighted in yellow are involved in co-enzyme binding. Box designate the signature YXXXK motif of SDRs and sequence highlighted in black denotes catalytic tetrad. Green highlighted residues participate in tropinone binding. Blue highlight indicates residues conserved in SDRs. Conclusions Substrate Saturation Curves for WcTR-I Forward Reaction Substrate Saturation Kinetics of WcTR-I for Reverse Reaction • A full length cDNA fragment of 822 bp was isolated from the leaves of Withaniacoagulans. • Sequence analysis revealed the presence of signature motifs of SDR family of proteins at homologous positions in WcTR-I sequence. • Cloned cDNA was induced over-expressed as recombinant His-tagged tropinone reductase and purified from soluble fraction. • GC based assay of WcTR-I identified tropine as reaction product. • Physical properties and kinetic parameters of recombinant Tropinone reductase were elucidated with spectrophotometric assays @ A340. • Enzyme showed catalytic activity in a narrow pH range with pH maxima at 6.6. • Substrate saturation curves were normal hyperbolic. • Oxidation of tropine was also detected in reverse reaction. • Higher thermostability of the enzyme was catalytic novelity in consonance with desert habitat of the plant. Km values for substrate and cofactors for W. coagulans Tropinone reductase I Acknowledgement The assay mixtures were prepared by sequentially increasing concentration of test compound. A, Tropinone; B, NADPH. DBT- Govt. of India NMITLI-CSIR Director, CSIR-CIMAP Activity was recorded by gradually increasing the concentration of test compound. A) Tropine,B) NADP+.