Cloning And Characterization Of Tropinone Reductase Ⅰ From Brugmansia Arborea

Tropane alkaloids(TAs) which mainly extracted from Solanaceae plants have been known to produce including hyoscyamine, anisodamine and scopolamine, among which hyoscyamine and scopolamine as anticholinergics are widely used in the parasympathetic nervous system, thus they Commonly were used in clinicalthe for pain relief, anesthesia, refraining drug addiction, control of motion sickness and cure of Parkinson’s disease. Likewise, anisodamine is also a non-specific choligergic antagonist exhibiting similar spectrum of pharmacological effects of hyoscyamine. Compared to hyoscyamine,scopolamine shows more commercial value due to higher pharmacological activity and fewer side-effects in clinicalthe, whereas it has commonly lower yields than hyoscyamine in plants and due to the difficulty in industrial synthesis and expensive cost, thus in Solanaceae plants, through the metabolic engineering to obtain a high yield of scopolamine plant will be our direction of future research.Brugmansia arborea is a woody plant species producing tropane alkaloids. Through previous reports found that D. arborea contains a high scopolamine and belongs scopolamine type, So we take this as materials research, by tropinone reductase I(TRI) gene sequence of the homologous species(Datura stramonium and Datura innoxia), we cloned the tropinone reductase I(TRI) gene sequence from B.arborea using the RACE method, the full-length c DNA of Ba TRI of 1138-bp consists of a coding sequence of 819 bp, 5’UTR of 41-bp, 3’UTR of 278-bp. The full-length c DNA of Ba TRI encoded a 272-amino-acid polypeptide that was highly similar with tropinone reductase I from TAs-producing herbal plant species. By in vitro recombination, we Can purified a protein of approximately 30 k Da which was consistent with the predicted 29.7k Da after efficient expressing in Rosstar. Simultaneously, in order to compare with Ds TRI, We cloned Ds TRI from the D.stramonium. Enzyme kinetics Assaying of Ba TRI display a maximum reduction activity at p H 6.8 to 8.0 when tropinone was used as substrate, it also had the maximum oxidation activity at p H 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of Ba TRI for tropinone were respectively 2.65 m M, 88.3 nkat.mg-1 and 2.93 S-1, at p H 6.4(physiological activity of plants). Simultaneously, Ba TRI had a significantly higher activity than Ds TRI. In addition to, 4-methylcyclohexanoneand 3-quinuclidinone hydrochloride,which are typical structural analogues of tropinone representing monocyclic and bi-cyclic substrates were widely used to test the substrate specificity of TRs. Moreover,The Km, Vmax and Kcat values of Ba TRI for tropine were 0.56 m M, 171.62 nkat.mg-1 and 5.69 S-1, respectively, at p H 9.6,the results show that the Km values of Ba TRI for tropine less than the Km values of Ba TRI for tropinone, however, the two reactions are not the same p H, the reaction to Ba TRI for tropinone in weakly acidic while the reaction to Ba TRI for tropine in strongly alkaline, and the reaction to Ba TRI for tropine was very low in weakly acidic, so in vivo of the B.arborea the reduction is mainly accounted for dominance at the physiological activity of plants. Under the same conditions, the Km, Vmax and Kcat values of Ds TRI for tropinone were respectively 4.18 m M, 81.2 nkat.mg-1 and 2.40 S-1, the measurement results show that the Km values of Ba TRI for tropine less than Ds TRI, so Ba TRI has higher affinity to tropinone. In order to better illustrate Ba TRI reduction activity than Ds TRI, We also measured the Kcat of Ba TRI and Ds TRI to 5m M Tropinone at 6.4,6.8 and 7.0, without a doubt, the Kcat of Ba TRI higher than Ds TRI, the results show that catalytic efficiency of Ba TRI higher than Ds TRI.We analyze the tissue profiles of Da TRI by q PCR, Ba TRI was expressed in all examined organs but was most abundant in secondary roots, and the tissue profiles of Ba TRI differed from those in TAs-producing herbal plant species but it consistent with Previously reported Ba H6 H. Finally, we detected the TAs in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea and the leaves and stems of the content is higher than in other parts, which suggests that B. arborea is a scopolamine-rich plant species. The gene expression and TAs accumulation suggest that the biosynthesis of TAs occurred not only in the roots but also in the aerial parts of B. arborea.From the results for characterization of BaTRI, We can may determine that tropinone reductase I of B. arborea was a efficient enzyme, and it can provide for us a more efficient candidate genes to reconstruction TAs synthetic pathway on the TRI target, in order to obtain high yield scopolamine medicinal plants.