Molecular cloning, expression and characterization of hyoscyamine 6β-hydroxylase from hairy roots of Anisodus tanguticus

Article abstract of DOI:10.1055/s-2005-837825

Anisodus tanguticus, one of the indigenous Chinese ethnological medicinal plants of the Solanaceae, produces anticholinergic alkaloids such as hyoscyamine, 6β-hydroxyhyoscyamine and scopolamine. Hyoscyamine 6β-hydroxylase (H6H), a key enzyme in the biosynthetic pathway of scopolamine, catalyzes the hydroxylation of hyoscyamine and epoxide formation from 6β-hydroxyhyoscyamine to generate scopolamine. A full-length cDNA of H6H has been isolated from A. tanguticus hairy roots. Nucleotide sequence analysis of the cloned cDNA revealed an open reading frame of 1035 bp encoding 344 amino acids with high homology to other known H6Hs. The equivalent amino acid sequence shows a typical motif of 2-oxoglutarate-dependent dioxygenase. The A. tanguticus H6H was expressed in Escherichia coli and purified for enzyme function analysis. This study characterized the recombinant AtH6H and showed it could generate scopolamine from hyoscyamine.

Full text of DOI:10.1055/s-2005-837825

Tao Liu
Ping Zhu
Ke-di Cheng
Chao Meng
Hui-xia He
Molecular Cloning, Expression and Characterization of
Hyoscyamine 6b-Hydroxylase from Hairy Roots of
Anisodus tanguticus
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
cells are considered capable of producing more secondary meta-
249
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: chengkd@imm.ac.cn
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

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 1”reaction 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 extended to 10 min
Ä
at 728C after the last cycle. The amplified fragment (200 bp) was
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
5¢ RACE were performed with RACE System Version 2.0 (Invitro-
gen, USA) as the manufacturer described.
Fig. 1 Biosynthetic pathway from hyoscyamine to scopolamine. H6H
catalyzes the two reactions.
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
Plant material
The first strand cDNA was synthesized from the total RNA (5 mg)
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-
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
250
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-
plified with the AAP forward and 5-GSP2 reverse primers (948C
Reverse transcriptase-polymerase chain reaction (RT-PCR)
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- 728C 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, Expression¼ Planta Med 2005; 71: 249±253

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, 4.6”200 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 378C in 50 mL cyamine = 0.23, scopolamine = 0.68) [2] and identified by 1H-
of Luria-Bertani medium, supplemented with ampicillin (20 mg/ NMR (500 MHz, in CD3OD) and 13C-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).
thiogalactoside to a final concentration of 0.6 mM until the OD600
reached 0.6±0.7. After incubation for another 8 h at 208C, the The inhibitory effects of EDTA (a metal chelating agent) and var-
cells were harvested by centrifugation at 8,000”g for 5 min at ious metal ions on the enzyme activity were investigated [7]. Ki-
48C to give cell pellets. The strain transformed with empty vector netic parameters were calculated by Lineweaver-Burk methods.
was used as the control.
Km values were determined at the substrate concentrations be-
tween 0.005 and 0.1mM. Each substrate concentration was
measured at least three times.
Purification of the recombinant AtH6H
All purification procedures were carried out at 0±48C. About
30 g of cell pellets were resuspended in 60 mL of buffer A (20 The protein concentrations were estimated according to the
mM sodium phosphate, pH 7.2) with 1mg/mL lysozyme and 10 method of Bradford using crystalline bovine serum albumin as
mM DTT, and placed on ice for 30 min and then freeze-thawed the standard [8].
for four times. This suspension was homogenized by sonication
and then centrifuged at 13,000”g for 10 min at 48C to pellet deb- Southern blotting analysis
ris. After determination by SDS-PAGE, the resulting supernatant Genomic DNA was extracted from A. tanguticus hairy roots with
was referred to as cell-free extract and stored at ±708C.
the DNeasy Plant Maxi Kit (Qiagen, USA). Genomic DNA (20 mg)
was digested with restriction enzyme EcoR I, EcoR V, Sac I, and
The cell-free extract was loaded on a HiTrap Chelating HP col- Xho I (TaKaRa, Japan), respectively. The fragments were fractio-
umn (Amersham Biosciences, Sweden) previously equilibrated nated on 0.7% agarose gel and then transferred onto a Hybond
with the binding buffer (buffer A containing 1M NaCl). The col-
umn was eluted stepwise at 1mL/min with a linear gradient of
N+ membrane (Amersham Biosciences, Sweden). The labeling,
hybridization and detection were performed following the in-
251
imidazole from 0 to 20 mM in 6 mL of elution buffer (buffer A struction of ECL Direct Nucleic Acid Labelling and Detection Sys-
containing 1M NH 4Cl). Each fraction was analyzed by SDS- tem (Amersham Biosciences, Sweden). The labeled AtH6H full-
PAGE. The fractions containing the recombinant AtH6H were length cDNA was used as the probe.
combined and dialyzed against buffer A.
The enzyme fraction obtained from the previous steps was load- Results and Discussion
ed on a DEAE Sepharose Fast Flow column (1.0”20 cm, Amer-
sham Biosciences, Sweden) previously equilibrated with buffer Cloning of AtH6H and sequence analysis
A. The column was washed with 100 mL of the same buffer. The Hairy roots of A. tanguticus were the original materials for the
enzyme was eluted with a linear gradient of NaCl from 0 to 600 cDNA preparation. RT-PCR was performed with primers that
mM in 150 mL of buffer A at a flow rate of 1 mL/min. Fractions were designed on the basis of the known H6H sequences. Se-
containing the recombinant AtH6H were combined and dialyzed quence analysis of the resulting 177-bp amplicon revealed a par-
against buffer A and stored at ±708C.
tial cDNA bearing 93% nucleotide-level homology to HnH6H and
AbH6H. Based on this sequence, several gene-specific primers
were designed to obtain the sequences of 3¢-end and 5¢-end by
Enzyme assay
AtH6H was assayed by measuring the formation of products RACE. Fragments about 600 bp of 3¢ RACE and 750 bp of 5¢ RACE
from different substrates supplemented. The complete reaction were amplified and sequenced, respectively. After a PCR with pri-
mixture contains 50 mM Tris-HCl buffer (pH 7.4), 0.4 mM FeSO4, mers based on the sequences of 3¢-end and 5¢-end, the full-
4 mM sodium ascorbate, 1mM 2-oxoglutarate, 0.2 mM l-hyos- length sequence of AtH6H cDNA was obtained.
cyamine hydrobromide or 0.2 mM 6b-hydroxyhyoscyamine,
2 mg/mL catalase (Worthington Biochemical Corp., USA), and The full-length cDNA was 1,305 bp in size containing 5¢-untrans-
the enzyme (0.2±0.5 mg of the protein) [6], [7]. The volume for lated region (5¢UTR), 3¢-untranslated region (3¢UTR), poly (A+)
the enzyme assay was 40 mL in total.
tail, and an ORF of 1,035 nucleotides encoding a deduced protein
of 344 amino acid residues (GenBank Accession No. AY356396).
The reactions were performed at 308C for 2 h. They were initi- The deduced amino acid sequence of AtH6H showed 83% identi-
ated by adding the enzyme and stopped by adding NaHCO3 to ad- ty with HnH6H and 84% identity with AbH6H. The alignment to
Liu T et al. Molecular Cloning, Expression¼ Planta Med 2005; 71: 249±253

Fig. 3 Silver-
HnH6H, AbH6H and other 2-oxoglutarate-ependent dioxygena-
ses is shown in Fig. 2.
stained SDS-PAGE
with extracts from
the bacteria expres-
sing AtH6H. Lanes:
M, molecular mass
marker; 1, pure re-
combinant AtH6H (7
mg protein); 2, frac-
tions after HiTrap
column (10 mg pro-
tein); 3,cell-free ex-
tract (15 mg pro-
tein); 4, control (15
mg protein, cell-free
extract of cells with
empty vector).
Additionally, AtH6H possesses the HXD¼H motif (residues
His217, Asp219, and His274, respectively) that has been found in
other 2-oxoglutarate dependent dioxygenases from plants, fungi
and bacteria. This sequence element has been considered to be
an iron-binding site [9], [10].
Recombinant expression and purification of AtH6H
The ORF fragment of AtH6H was sub-cloned into the expression
vector pET-32a (+) and expressed in E. coli strain BL21trxB (DE3)
cells. A protein of the appropriate size (57 kDa) was expressed in
the soluble form determined by SDS-PAGE. The resulting soluble
enzyme fraction was treated by HiTrap affinity chromatography
followed by DEAE Sepharose Fast Flow chromatography to give
the recombinant AtH6H (Fig. 3). The purified protein was used
for the enzyme assay and catalytic characterization analysis.
The biosynthetic products were further prepared and purified by
Biochemical characterization of the recombinant AtH6H
silica gel TLC and gave an NMR spectra identical to that of 6b-hy-
The catalytic functions and characters of the recombinant AtH6H droxyhyoscyamine and of scopolamine [11], [12], confirming
were investigated in this study. The recombinant AtH6H gener- that the enzyme was AtH6H which could convert hyoscyamine
ated different products depending on the substrates supplement- to scopolamine via 6b-hydroxyhyoscyamine [5]. The optical
ed. The enzyme reaction mixture containing hyoscyamine as the rotation values of the products: 6b-hydroxyhyoscyamine [a]D20
substrate yielded two reaction products with retention times of ±13.58 (c 1.9, MeOH) and scopolamine [a]D20: ±1 88 (c 1.5, EtOH).
2.33 min and 2.47 min on HPLC, which were corresponding ex-
:
actly to the standards of 6b-hydroxyhyoscyamine and scopola- The optimum temperature was 308C for enzymatic activity of
mine, respectively. When 6b-hydroxyhyoscyamine was used as the recombinant AtH6H. Lower temperatures (below 208C)
the substrate, only one reaction product was detected and were not suitable for the catalysis, whereas higher temperatures
showed retention time of 2.47 min equal to that of scopolamine. (above 508C) could denature the enzyme. The pH optimum for
No product was detected for the control when the same assay the recombinant AtH6H was found to be at pH of 7.4. The enzyme
was performed.
activity dropped to 20% at pH 5.0 and decreased rapidly when
252
Fig. 2 Deduced amino acid sequence comparison of AtH6H to other 2-oxoglutarate-dependent dioxygenases. AtH6H: hyoscyamine 6b-hydro-
xylase from Anisodus tanguticus (GenBank Accession No.AY356396). HnH6H: hyoscyamine 6b-hydroxylase from Hyoscyamus niger (D26583).
AbH6H: hyoscyamine 6b-hydroxylase from Atropa belladonna (AB017153). AtFLS1: flavonol synthase 1 from Arabidopsis thaliana (NM_120951).
HvIDS3: iron deficiency-specific protein 3 from Hordeum vulgare subsp. vulgare (AB024007). SmF3H: putative flavanone 3-hydroxylase from Saus-
surea medusa (AF509338).
Liu T et al. Molecular Cloning, Expression¼ Planta Med 2005; 71: 249±253

the pH reached 8.8. The effect of pH on enzyme activity of AtH6H roots, the best transgenic clone had a 100-fold increase of scopo-
was quite similar to that of the available HnH6H except for the lamine [14]. It also reported that H. niger hairy roots overexpres-
optimum pH value (pH 7.8 for HnH6H) [7].
sing putrescine N-methyltransferase (PMT) and H6H produced
significantly higher levels of scopolamine compared with the
Enzyme activity of the recombinant AtH6H was strongly inhib- wild-type [15]. With increased an understanding of the enzy-
ited by 5 mM Ca2+ (100% inhibition), Cu2+ (100%), Mn2+ (100%), mology, molecular genetics and regulation of the tropane alka-
Zn2+ (66%), Fe3+ (65%), Ni2+ (64%) and EDTA (61%), and slightly loids pathway, metabolic engineering could be another way to
by 5 mM Mg2+ (14%). In contrast with the reported data [6], Ca2+ provide a potential solution to produce compounds that are rare
had a strong inhibitory effect on the AtH6H enzyme activity, and not easily obtained.
while a slight inhibitory effect was observed on the HnH6H en-
zyme activity. The enzyme activity was decreased to 14% and
10% in the absence of catalase (2 mg/mL) and sodium ascorbate Acknowledgements
(4 mM), respectively. Without the addition of Fe2+, only 1.3% of
the enzyme activity was detected. It indicated that Fe2+ might This work was supported by the National 863 High Technology
be an essential cofactor for the enzyme activity [7].
Programs of China (project 2001AA234021 and project
2002AA2Z343B).
The Km values for hyoscyamine and 6b-hydroxyhyoscyamine at
pH 7.4 were determined to be 15.1  0.3 mM and 17.0  0.2 mM,
respectively. The Vmax value for 6-hydroxyhyoscyamine (4.2  References
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(Fig. 4). At least seven bands could be detected in Southern blot-
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Fig. 4 Genomic
Southern
blotting
analysis of AtH6H
gene. Xho I has one
recognition
site
within the cDNA
sequence, whereas
EcoR I, EcoR V and
14
15
Sac
I have none.
Weak signals are
marked with arrows.
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