Amit K. Kushwaha & Rajender S. Sangwan Metabolic and structural Biology

1. 3’ RACE fragment encoding Putative TRI from the root of Withaniasomnifera Ashwagandha (Withaniasomnifera) 1 aa 273 aa Kingdom : Plantae Subkingdom : Tracheobionta Superdivision : Spermatophyta Division : Magnoliophyta Class : Magnoliopsida Subclass : Rosidae Order: Solanales Family : Solanaceae Genus : Withania Species: somnifera TAA DaturastramoniumP50162 368 bp UTR M P M P UI II IS M • Popularly considered as the Indian ginseng. • Described as rasayana (rejuvenation) and expected to promote physical and mental health, rejuvenate the body in debilitated conditions and increase longevity. • These pharmacological properties are attributed to its secondary metabolites: Alkaloids, Withanolides etc. • Alkaloids of tropane class are the first group of secondary metabolites isolated from Ashwagandha (Withaniasomnifera) long before the isolation of withanolides but hardly attended for further or deeper research, except for its use in the trade of the herb. Rather, there is a pride in possession of ‘alkaloid rich’ Ashwagandha in commercial market of the plant. A1 A2 M Tropinone reductase and biosynthesis of tropane alkaloids Tropane Alkaloids and TRs:The Background Alignment and selection of conserved regions to design Degenerate primers M: 1 Kb plus ladder P: PCR product of TrRdCterF and 3’AP • Bicyclic amines specifically produced in solanaceousroots. • 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). • However, a new group of nortropane alkaloids, calystegines, resemble monosaccharides in structure, and it is not surprising that they have proved to be strong glycosidase inhibitors. • TR-I (EC 1.1.1.206), produces tropine with a 3α-hydroxyl group, whereas the other, TR-II (EC 1.1.1.236), produces pseudotropine (ψ-tropine) with a β-hydroxyl group. Tropine and ψ-tropine are not interconvertedin vivo and are further metabolized. • The amino acid sequences indicate that the TRs belong to the short-chain dehydrogenase/reductase family. Tropinone TR Tropane Alkaloids Cloning, Heterologous Expression and Catalytic Kinetics of aRecombinant Tropinone Reductase from Ashwagandha (Withaniasomnifera) 5’ RACE fragment encoding Putative TRI from the SMARTercDNA library from the root of Withaniasomnifera A partial cDNA fragment encoding Tropinone reductase I from the root of Withaniasomnifera 1 aa 273 aa 273aa TAA AnisodusacutangulusACB71202.1 Daturastramonium(P50162) 1aa 105 aa Putative WsTRI (471 bp) 13 aa 569 bp UTR M: 1 Kb plus ladder P: PCR product of TRIF2 and TrRdCterR A1: CDS II A/ TRIUR3 A2: CDS II A/ TRIUR4 M: 1 Kb plus ladder Complete cDNA encoding Putative TRI from the root of Withaniasomnifera Heterologous Expression and Purification of Recombinant Tropinone reductase Metabolic profiling of TropaneAlakaloids in plantarelated to Tropinone reductase 1 aa 273 aa A B C D E TAA AnisodusacutangulusACB71202.1 M A B C A B M Leaves were powdered in liq. N2. Liquid-Solid and Liquid-Liquid Extractions were performed with Hexane, Methanol and Chloroform. Extracts were evaporated to dryness and dissolved in methanol for further analysis. 822 bp UTR UTR P M Amit K. Kushwaha& Rajender S. Sangwan Metabolic and structural Biology CSIR - Central Institute of Medicinal and Aromatic Plants, Lucknow A: Hexane Extract B: Methanol Extract C: Tropine standard D: Tropinone standard E: Chloroform Extract M: Protein mol weight marker A: Induced soluble fraction B & C: Eluate from gel filteration column UI: Uninducedculture II: Induced insoluble fraction IS: Induced soluble fraction M: Protein mol weight marker A: Eluate from Affinity Column (Ni++-NTA) B: Induced soluble fraction M: Protein mol weight marker M: 100 bp ladder P: PCR product of TRIFLF and TRIFLR Physical Characteristics of Recombinant Tropinone reductase Kinetic Parameters for Recombinant Tropinone reductase Conclusions • A full length cDNA fragment of 822 bp was isolated from the Withaniasomnifera. • Sequence analysis revealed 87% similarity to the Tropine forming Tropinone reductase of Anisodusacutangulus, of the SDR family of proteins. • His-TRI recombinant protein was over-expressed in bacteria, and Tropinone reductase activity was detected in soluble fraction. • A ~30 Kda recombinant protein was purified by Ni++-NTA affinity chromatography followed by gel filtration, as shown in SDS-PAGE. • Physical properties and kinetic parameters of recombinant Tropinone reductase were elucidated with Spectrophotometric assays @ A340. • Substrate saturation curves were normal hyperbolic. • Oxidation of tropine was also detected in reverse reaction, not reported earlier. Acknowledgement DBT- Govt. of India NMITLI-CSIR Prof. Ram Rajasekharan, Director, CSIR-CIMAP Dr. (Mrs.) N. S. Sangwan, Senior Principle Scientist, CSIR-CIMAP