||Sasaki-Tabata K, Matsuo A, Fuchida H, Ojida A, Tanaka H and Morimoto S, Cloning, expression and characterization of polyketide synthase gene from Scopolia Japonica, The 9th CSP-KSP-JSP Joint Symposium on Pharmacognosy, 2016.05, BACKGROUND AND PURPOSE: Various important pharmacological compounds such as atropine and scopolamine are included in Scopolia japonica. These compounds are synthesized using ornithine as a starting material, however, it’s unclear which enzyme catalyzes the tropinone synthesis reaction. Hence, in the present study we aimed to identify function and structure of novel polyketide synthase (PKS) from S. japonica.
EXPERIMENTAL APPROACH: A total RNA was extracted from leaves of S. japonica and cDNA was synthesized using a reverse transcriptase. Then, degenerate PCR, 5' and 3' RACE was performed using cDNA as a template. Two novel genes were obtained and expressed in E. coli. The recombinant protein was purified with affinity column and ion exchange column. We analyzed the enzyme activity using HPLC. Moreover, we crystallized and performed crystallographic analysis of PKS.
KEY RESULTS: Two novel genes of PKS from S. japonica were obtained. The recombinant PKSs expressed in E. coli as a soluble fraction was highly purified using affinity and ion exchange column and the enzyme assay with HPLC showed that new compounds were synthesized using some substrates. Moreover, crystallization screening resulted in obtaining needle or plate crystals. We could get single crystals in an optimized condition. The crystal diffracted to 2.0 angstrom at KEK-PF and the structures of PKS were determined using molecular replacement method.
CONCLUSION AND IMPLICATIONS: These PKS genes seem to belong to PKS family because they showed high similarity to other CHS from various plants. These novel findings may contribute to clarifying the mechanism of tropane synthesis..
||Pongkitwitoon B, Kaori Tabata, Hiroyuki Tanaka, satoshi morimoto, Construction of a single-chain variable fragment antibody against daidzin and its potential use in an enzyme-linked immunosorbent assay, 61st International Congress and Annual Meeting of the Society for Medicinal Plant and Natural Product Research, 2013.09.
||Hiroyuki Tanaka, Chao Z, Kaori Tabata, satoshi morimoto, Preparation of antibodies to paclitaxel, 2013 International Congress on Natural Products Research, 2013.07.
||Pongkitwitoon B, Sakamoto S, Sasaki K, Tanaka H, Morimoto S, Molecular cloning and expression of O-methyltransferases from opium poppy, The XVI International Congress "PHYTOPHARM 2012, 2012.07.
||Tanaka H, Paudel MK, Takei A, Sakoda J, Juengwatanatrakul T, Sasaki-Tabata K, Putalun W, Shoyama Y, Morimoto S, Preparation of a recombinant Fab against the anti-malarial drugs, artemisinin and artesunate and their application, 2012 International Congress on Natural Products Research, 2012.07.