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Molecular Cloning, Expression and Characterization of Hyoscyamine

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Molecular Cloning, Expression and Characterization of Hyoscyamine ____________________________________________________________________________http://www.paper.edu.cn Tao Liu Ping Zhu Molecular Cloning, Expression and Characterization of Ke-di Cheng Hyoscyamine 6b-Hydroxylase from Hairy Roots of Chao Meng Hui-xia He Anisodus tanguticus Original Paper Abstract frame of 1035 bp encoding 344 amino acids with high homology to other known H6Hs. The equivalent amino acid sequence Anisodus tanguticus, one of the indigenous Chinese ethnological shows a typical motif of 2-oxoglutarate-dependent dioxygenase. medicinal plants of the Solanaceae, produces anticholinergic al- The A. tanguticus H6H was expressed in Escherichia coli and puri- kaloids such as hyoscyamine, 6b-hydroxyhyoscyamine and sco- fied for enzyme function analysis. This study characterized the polamine. Hyoscyamine 6b-hydroxylase (H6H), a key enzyme in recombinant AtH6H and showed it could generate scopolamine the biosynthetic pathway of scopolamine, catalyzes the hydroxy- from hyoscyamine. lation of hyoscyamine and epoxide formation from 6b-hydroxy- hyoscyamine to generate scopolamine. A full-length cDNA of Key words H6H has been isolated from A. tanguticus hairy roots. Nucleotide Anisodus tanguticus ´ Solanaceae ´ hyoscyamine 6b-hydroxylase sequence analysis of the cloned cDNA revealed an open reading (H6H) ´ molecular cloning ´ expression ´ characterization Introduction ground parts of the plant, and hairy roots but not suspension 249 cells are considered capable of producing more secondary meta- Tropane alkaloids constitute a group of secondary metabolites in bolites [1], [2], [3], [4]. Observations have revealed that the con- Solanaceae plants including four important medicinally used al- tent of scopolamine was much higher than that of hyoscyamine kaloids: hyoscyamine, 6b-hydroxyhyoscyamine, anisodine, and in hairy roots after 4 weeks cultivation [4]. This indicated that scopolamine. Anisodus, Atropa, Duboisia, Hyoscyamus, and the enzymes responsible for the conversion of hyoscyamine Datura are the major tropane alkaloid producing genera. Tropane should be very active and might have potential use in increasing alkaloids act mainly on the parasympathetic nervous system [1], the scopolamine production rate. Hyoscyamine 6b-hydroxylase [2]. 6-Hydroxyhyoscyamine may be used to improve the micro- (H6H, EC 1.14.11.11) is a key enzyme in the biosynthesis of scopo- circulation [1]. lamine. It belongs to the 2-oxoglutarate-dependent dioxygenase family and catalyzes two consecutive oxidation reactions: the Anisodus tanguticus (Maxim.) Pascher (Solanaceae) is one of the hydroxylation of hyoscyamine to 6b-hydroxyhyoscyamine and folk medicines used in Tibet and Qinghai Province in West China. the epoxidation of 6b-hydroxyhyoscyamine to scopolamine It is an important medicinal plant species yet its cultivation is (Fig.1) [5]. difficult. Tropane alkaloids are mainly present in the under- Affiliation Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, P. R. China Correspondence Prof. Ke-di Cheng ´ Institute of Materia Medica ´ Chinese Academy of Medical Sciences and Peking Union Medical College ´ # 1Xian Nong Tan Street ´ Beijing 100050 ´ P. R. China ´ Fax: +86-10-6301-7757 ´ E-mail: [email protected] Received May 27, 2004 ´ Accepted October 9, 2004 Bibliography Planta Med 2005; 71: 249±253 ´ Georg Thieme Verlag KG Stuttgart ´ New York DOI 10.1055/s-2005-837825 ISSN 0032-0943 中国科技论文在线___________________________________________________________________________http://www.paper.edu.cn and Atropa belladonna (AbH6H, GenBank Accession No. AB017153) as well as the enzymes of the 2-oxoglutarate depen- dent dioxygenase family. The primer pair of 5¢-AACTATTACCCAC- CATG-3¢ (sense primer) and 5¢-TCCCAAGTTGACCACAAAAGC-3¢ (antisense primer) was used to amplify the fragment from the first strand cDNA. PCR was performed in a total volume of 25 mL containing 1.5 mM MgCl2, 0.2 mM dNTP, 0.2 mM each primer, 200 ng first strand cDNA, and 2.5 U Platinum Taq DNA polymerase (Invitrogen, USA) in 1reaction buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl). After an initial 4 min denaturing step at 948C, 30 cy- cles of amplification were performed at 948C for 1min, 54 8C for 1min, and 72 8C for 1.5 min. Elongation was extendedÄ to 10 min at 728C after the last cycle. The amplified fragment (200 bp) was Original Paper ligated into pMD 18-T vector (TaKaRa, Japan) and sequenced. 3¢¢ and 5¢¢ rapid amplification of cDNA ends (RACE) To obtain 3¢ and 5¢-terminal sequences of the cDNA, 3¢ RACE and Fig. 1 Biosynthetic pathway from hyoscyamine to scopolamine. H6H 5¢ RACE were performed with RACE System Version 2.0 (Invitro- catalyzes the two reactions. gen, USA) as the manufacturer described. For 3¢ RACE, two gene-specific primers were designed: 5¢-CGAT- The aim of this study was to clone the H6H gene of A. tanguticus CAACAAGACTTGCCTGGCT-3¢ (3-GSP1), 5¢-CGAGCCTGGCTTG- (AtH6H) and analyze the function of the recombinant protein CAACAACTT-3¢ (3-GSP2). The 3¢ RACE Adapter Primer (AP) se- expressed by Escherichia coli. quence was 5¢-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT- 3¢ and Abridged Universal Amplification Primer (AUAP) sequence was 5¢-GGCCACGCGTCGACTAGTAC-3¢. Materials and Methods The first strand cDNA was synthesized from the total RNA (5 mg) Plant material with primer AP. The second strand cDNA was amplified with the Anisodus tanguticus (Maxim.) Pascher was identified by Profes- 3-GSP1forward and AUAP reverse primers (94 8C for 4 min, then sor Guangcheng Xia in the Institute of Materia Medica, Chinese 30 cycles at 948C for 4 min, 548C for 1min, and 72 8C for 1.5 min, Academy of Medical Sciences and Peking Union Medical College. with final extension at 728C for 10 min). The PCR product was di- 250 The voucher specimen (IH #673) was deposited in the institute. luted 100 times, and employed in a second PCR (same condi- tions) but substituting forward primer 3-GSP2 for 3-GSP1. The Hairy roots of A. tanguticus, established from aseptic seedlings by amplified fragment (~ 600 bp) was cloned into pMD 18-T vector Agrobacterium rhizogenes (ATCC No. 15834) on Linsmayer and and sequenced. Skoog (LS) medium without auxins as described [4], were trans- ferred from solid culture to 500-mL flasks containing 200 mL of For 5¢ RACE, three gene-specific primers were designed: 5¢- liquid LS medium without auxins on a rotary shaker at 80 rpm CAGGCAAGTCTTGTTGAAGC-3¢ (5-GSP1), 5¢-CGATTACCATCA- and 258C in the dark [6]. The six-day hairy roots were used for GAGTGTCCTCC-3¢ (5-GSP2), 5¢-CGACAGAGTGTCCTCCTGAACC-3¢ RNA preparation. The voucher specimen (NP #84) was deposited (5-GSP3). The 5¢ RACE Abridged Anchor Primer (AAP) sequence in the Institute of Materia Medica, Chinese Academy of Medical was 5¢-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3¢ and Sciences and Peking Union Medical College. Abridged Universal Amplification Primer (AUAP) sequence was 5¢-GGCCACGCGTCGACTAGTAC-3¢. Chemicals l-Hyoscyamine hydrobromide was purchased from Sigma, USA. The first strand cDNA, synthesized from total RNA (5 mg) with 6b-Hydroxyhyoscyamine and scopolamine were obtained from primer 5-GSP1, was purified by S.N.A.P. column (Invitrogen, Minsheng Pharmaceutical Co., Ltd. Hangzhou, China. USA) and then oligo-dC tailed using terminal deoxynucleotidyl transferase (Invitrogen, USA). The second strand cDNA was am- Reverse transcriptase-polymerase chain reaction (RT-PCR) plified with the AAP forward and 5-GSP2 reverse primers (948C Total RNA was isolated from A. tanguticus hairy roots by Concert for 4 min, then 30 cycles at 948C for 4 min, 548C for 1min, and Plant RNA Reagent (Invitrogen, USA) following the manufactur- 72 8C for 1.5 min, with final extension at 728C for 10 min). The er's instruction. Total RNA (5 mg) was reverse transcribed to syn- PCR product was diluted 100 times, and employed in a second thesize first strand cDNA with Oligo(dT)20 Primer (Invitrogen, PCR (same conditions) with AUAP forward and 5-GSP3 reverse USA) and SuperScript RT II Reverse Transcriptase (Invitrogen, primers. The amplified fragment (~ 750 bp) was cloned into USA). pMD 18-T vector and sequenced. Primers were designed from the homologous regions found in Heterologous expression of AtH6H in E. coli the nucleotide alignments of the hyoscyamine 6b-hydroxylase The open reading frame (ORF) of AtH6H was amplified using a of Hyoscyamus niger (HnH6H, GenBank Accession No.D26583) sense primer (5¢-CGCGGAATTCATGGCTACTCTTGTCTCCAAC-3¢) Liu T et al. Molecular Cloning, Expression ¼ Planta Med 2005; 71: 249±253 中国科技论文在线___________________________________________________________________________http://www.paper.edu.cn with EcoR I site (underlined) and an antisense primer (5¢- just the pH value of the mixture to 9.0, followed by the extraction CAGGGCGGCCGCGGCATTGATTTTATAAGGCT-3¢) with Not I site with an equal volume of EtOAc. The extracts were evaporated to (underlined). The PCR product was ligated into pMD 18-T vector dryness at 558C and the residue dissolved in 0.5 mL of MeOH. and sequenced. After sequence confirmation, the ORF fragment The quantitative analysis of alkaloids was performed by HPLC was digested with EcoR I and Not I (TaKaRa, Japan), and direc- on an LC-6A HPLC System (Shimadzu, Japan) with a ZORBAX tionally ligated into pET-32a (+) vector (Novagen, Germany) to ODS-C18 column (5 mm, 4.6200 mm, Agilent, USA), eluted iso- generate pETAtH6H which expressed as a histidine-tagged fu- cratically with a mobile phase (acetonitrile/0.05 M sodium hep- sion AtH6H protein in E. coli strain BL21trxB (DE3) (Novagen, tanesulfonate 35:65) at a flow rate of 1.5 mL/min and detected at Germany). 210 nm. The products yielded by the recombinant AtH6H were purified by preparative TLC (SiO2, toluene/acetone/EtOH/ For the enzyme preparation, E. coli cells transformed with NH4OH, 3:6:0.6 :0.4; Dragendorff reagent; Rf 6b-hydroxyhyos- pETAtH6H harboring AtH6H gene were grown at 378Cin50mL cyamine = 0.23, scopolamine = 0.68) [2] and identified by 1H- 13 of Luria-Bertani medium, supplemented with ampicillin (20 mg/ NMR (500 MHz, in CD3OD) and C-NMR (125 MHz, in CD3OD) mL). The cultures were induced by the addition of isopropyl b-D- spectra, which were recorded on an Inova-500 (Varian, USA).
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