title: Functional characterisation of a tropine-forming reductase gene from Brugmansia arborea, a woody plant species producing tropane alkaloids.
abstract: Brugmansia arborea is a woody plant species that produces tropane alkaloids (TAs). The gene encoding tropine-forming reductase or tropinone reductase I (BaTRI) in this plant species was functionally characterised. The full-length cDNA of BaTRI encoded a 272-amino-acid polypeptide that was highly similar to tropinone reductase I from TAs-producing herbal plant species. The purified 29kDa recombinant BaTRI exhibited maximum reduction activity at pH 6.8-8.0 when tropinone was used as substrate; it also exhibited maximum oxidation activity at pH 9.6 when tropine was used as substrate. The Km, Vmax and Kcat values of BaTRI for tropinone were 2.65mM, 88.3nkatmg(-1) and 2.93S(-1), respectively, at pH 6.4; the Km, Vmax and Kcat values of TRI from Datura stramonium (DsTRI) for tropinone were respectively 4.18mM, 81.20nkatmg(-1) and 2.40S(-1) at pH 6.4. At pH 6.4, 6.8 and 7.0, BaTRI had a significantly higher activity than DsTRI. Analogues of tropinone, 4-methylcyclohexanone and 3-quinuclidinone hydrochloride, were also used to investigate the enzymatic kinetics of BaTRI. The Km, Vmax and Kcat values of BaTRI for tropine were 0.56mM, 171.62nkat.mg(-1) and 5.69S(-1), respectively, at pH 9.6; the Km, Vmax and Kcat values of DsTRI for tropine were 0.34mM, 111.90nkatmg(-1) and 3.30S(-1), respectively, at pH 9.6. The tissue profiles of BaTRI differed from those in TAs-producing herbal plant species. BaTRI was expressed in all examined organs but was most abundant in secondary roots. Finally, tropane alkaloids, including hyoscyamine, anisodamine and scopolamine, were detected in various organs of B. arborea by HPLC. Interestingly, scopolamine constituted most of the tropane alkaloids content in B. arborea, which suggests that B. arborea is a scopolamine-rich plant species. The scopolamine content was much higher in the leaves and stems than in other organs. The gene expression and TAs accumulation suggest that the biosynthesis of hyoscyamine, especially scopolamine, occurred not only in the roots but also in the aerial parts of B. arborea.
authors.affiliation: Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Qiang
authors.affiliation: Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China.
authors.forename: Ke
authors.initials: K
authors.lastname: Xia
authors.affiliation: Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China.
authors.forename: Qiaozhuo
authors.initials: Q
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China.
authors.forename: Junlan
authors.initials: J
authors.lastname: Zeng
authors.affiliation: College of Agronomy, Anshun University, Anshun 561000, China.
authors.forename: Yuanshe
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China.
authors.forename: Chunxian
authors.initials: C
authors.lastname: Yang
authors.affiliation: SWU-TAAHC Medicinal Plant Joint R&D Centre, College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China.
authors.forename: Min
authors.initials: M
authors.lastname: Chen
authors.affiliation: Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China.
authors.forename: Xiaoqiang
authors.initials: X
authors.lastname: Liu
authors.affiliation: TAAHC-SWU Medicinal Plant Joint R&D Centre, Agricultural and Animal Husbandry College, Tibet University, Nyingchi of Tibet 860000, China.
authors.forename: Xiaozhong
authors.initials: X
authors.lastname: Lan
authors.affiliation: Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), SWU-TAAHC Medicinal Plant Joint R&D Centre, School of Life Sciences, Southwest University, Chongqing 400715, China. Electronic address: zhliao@swu.edu.cn.
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Liao
mesh_terms: D000429:Alcohol Oxidoreductases / Q000378:metabolism*; D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004365:Drugs, Chinese Herbal / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D020539:Sequence Analysis, Protein; D019657:Solanaceae / Q000235:genetics / Q000378:metabolism; D014326:Tropanes / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004365:Drugs, Chinese Herbal; D014326:Tropanes; D000429:Alcohol Oxidoreductases; C091007:tropine dehydrogenase
keywords: Brugmansia arborea; Enzymatic kinetics; Solanaceae; Tissue profile; Tropane alkaloids; Tropinone reductase I