Molecular cloning and heterologous expression of progesterone 5β-reductase from Digitalis lanata Ehrh.

Article abstract of DOI:10.1016/j.phytochem.2005.11.013

A full-length cDNA clone that encodes progesterone 5β-reductase (5β-POR) was isolated from Digitalis lanata leaves. The reading frame of the 5β-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph1/Sal1 5β-POR fragment was cloned into the pQE vector and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally expressed and the recombinant enzyme was characterized. The K_m and v_max values for the putative natural substrate progesterone were calculated to be 0.120 mM and 45 nkat mg^-1 protein, respectively. Only 5β-pregnane-3,20-dione but not its α-isomer was formed when progesterone was used as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also determined. The molecular organization of the 5β-POR gene in D. lanata was determined by Southern blot analysis. The 5β-POR is highly conserved within the genus Digitalis and the respective genes and proteins share considerable homology to putative progesterone reductases from other plant species.

Full text of DOI:10.1016/j.phytochem.2005.11.013

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PHYTOCHEMISTRY
Phytochemistry 67 (2006) 225–231
www.elsevier.com/locate/phytochem
Molecular cloning and heterologous expression of progesterone
q
5b-reductase from Digitalis lanata Ehrh.
*
Vanessa Herl , Gabriele Fischer, Frieder Muller-Uri, Wolfgang Kreis
¨
Lehrstuhl fu¨r Pharmazeutische Biologie, Friedrich-Alexander-Universita¨t Erlangen-Nu¨rnberg, Staudtstr. 5, D-91058 Erlangen, Germany
Received 5 September 2005; received in revised form 11 November 2005
Available online 28 December 2005
Dedicated to the memory of Prof. Dr. Ernst Reinhard.
Abstract
A full-length cDNA clone that encodes progesterone 5b-reductase (5b-POR) was isolated from Digitalis lanata leaves. The reading
frame of the 5b-POR gene is 1170 nucleotides corresponding to 389 amino acids. For expression, a Sph1/Sal1 5b-POR fragment was
cloned into the pQE vector and was transformed into Escherichia coli strain M15[pREP4]. The recombinant gene was functionally
expressed and the recombinant enzyme was characterized. The Km and vmax values for the putative natural substrate progesterone were
calculated to be 0.120 mM and 45 nkat mgꢀ1 protein, respectively. Only 5b-pregnane-3,20-dione but not its a-isomer was formed when
progesterone was used as the substrate. Kinetic constants for cortisol, cortexone, 4-androstene-3,17-dione and NADPH were also deter-
mined. The molecular organization of the 5b-POR gene in D. lanata was determined by Southern blot analysis. The 5b-POR is highly
conserved within the genus Digitalis and the respective genes and proteins share considerable homology to putative progesterone reduc-
tases from other plant species.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Digitalis lanata; Plantaginaceae; Veronicaceae; Progesterone 5b-reductase (5b-POR); Cardenolide biosynthesis; Over-expression
1. Introduction
chains of variable length are attached at position C-3 of the
cardenolide genins. Through studies using radiolabelled
precursors, the putative biosynthetic pathway was basically
deduced, but it is not yet fully understood on a biochemical
level (Kreis et al., 1998).
Taking cholesterol as the starting point, about 20 enzymes
which probably affect the formation of cardenolides have
been identified and characterized in Digitalis (Lindemann
and Luckner, 1997; Kreis et al., 1998). But only some of them
have been purified, including the progesterone 5b-reductase
(5b-POR), a key enzyme of cardenolide biosynthesis catalys-
ing the conversion of progesterone to 5b-pregnane-3,20-
dione. The enzyme has been partially sequenced (Ga¨rtner
et al., 1994). To find a possible route for manipulating car-
denolide biosynthesis in plants, a more detailed knowledge
of the enzymes and genes involved in cardenolide formation
is necessary for studying the regulation and engineering of
Leaves of Digitalis plants are still the major source for
the isolation of cardenolides that are used to treat cardiac
insufficiency in humans. Cardenolides are characterized
by a steroid nucleus with its four rings connected cis–
trans–cis, having a 14b-hydroxy group and an unsaturated
five-membered lactone ring at C-17b. Typically, sugar side
Abbreviations: 3b-HSD, D5-3b-hydroxysteroid dehydrogenase/D5-D4-
ketosteroid isomerase; 5b-POR, progesterone 5b-reductase; RT-PCR, re-
verse transcriptase polymerase chain reaction; tR, relative retention time.
q
The nucleotide sequences reported in this paper have been submitted to
GenBanke Data Base with the corresponding Accession Numbers
AY574950/AY585867.
*
Corresponding author. Tel.: +49 9131 8528251/50; fax: +49 9131
8528243.
E-mail address: vherl@biologie.uni-erlangen.de (V. Herl).
0031-9422/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2005.11.013

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V. Herl et al. / Phytochemistry 67 (2006) 225–231
the cardenolide pathway (Kreis et al., 1998; Eisenbeiß et al.,
1999; Luckner and Wichtl, 2000).
So far, molecular data from Digitalis are available only
for a few house keeping genes (tRNA-Leu, 18S ribosomal
RNA) and several enzymes like aldo-keto reductase (Gavi-
dia et al., 2002), acyl-CoA-binding protein (Metzner et al.,
account (www.kazusa.or.jp). The resulting fragments
showed high sequence homology to the genomic clone of
Dop5br gene of D. obscura and to the progesterone 5b-
reductase (p5br) from D. purpurea. After submission of
the sequence for p5br gene (AJ310673; Roca-Perez et al.,
2004) the results were confirmed by PCR amplification with
distinct primers. The 5b-POR was amplified by RT-PCR
from cDNA prepared from D. lanata, D. purpurea and D.
obscura. DNA fragments of nearly identical length were
also obtained when genomic DNA of D. purpurea was used
as template. After subcloning of the PCR fragments into the
TOPO cloning vector system the nucleotide sequence was
elucidated by MWGꢁ Biotech AG (Ebersberg, Germany).
We here isolated a full-length cDNA clone that encodes
a progesterone 5b-POR from leaves of D. lanata. An iden-
tical match was observed between the deduced and directly
determined amino acid sequence of the progesterone 5b-
reductase peptides from D. purpurea (Ga¨rtner et al.,
1994). RT-PCR using RNA and/or mRNA from mature
leaves resulted in one single DNA fragment of the appro-
priate size. The DNA fragments and the nucleotide
sequences obtained from D. lanata, D. purpurea and D. obs-
cura did not differ in size (Fig. 2A). PCR amplification with
genomic DNA as a template resulted in a fragment of 1247
bp, slightly different in size from the cDNA fragments
(Fig. 2A, Lane 4). Actually, the sequence of the genomic
clone contained a small intron as obtained after sequencing
data analysis.
2000), cyclophilins (Scholze et al., 1999; Kullertz et al.,
¨
1999), for review see Luckner and Wichtl (2000). Cardeno-
lide specific genes are described for: cardenolide-160-O-
glucohydrolase (Scho¨ninger et al., 1998; Framm et al.,
2000), lanatoside-150-O-acetylesterase (Kandzia et al.,
1998), and D5-3b-hydroxysteroid dehydrogenase (Fins-
terbusch et al., 1999; Lindemann et al., 2000). The gene
for progesterone 5b-reductase of D. obscura (Dop5br;
AJ555127) was reported by Roca-Perez et al. (2004). The
p5br gene from D. purpurea was cloned and a partial geno-
mic clone from D. obscura has been used to analyse the car-
denolide production in 10 natural populations under
seasonal aspects. We here report for the first time the clon-
ing and heterologous functional expression of 5b-POR
from leaves of Digitalis lanata Ehrh. and the biochemical
characterization of the recombinant enzyme.
2. Results and discussion
2.1. PCR amplification and cloning
The early steps of cardenolide biosynthesis are usually
described as outlined in Fig. 1 (for review, see Kreis
et al., 1998). We here focussed on the crucial step leading
to 5b-configured pregnanes supposed to be the direct pre-
cursors of Digitalis cardenolides.
Initial experiments were carried out using degenerated
oligo nucleotide primers derived from the peptide sequences
of progesterone 5b-reductase from D. purpurea (Ga¨rtner
et al., 1990, 1994) taking KazusaÕs codon usage system into
2.2. Alignments
Several authors proposed that 5b-POR has a key func-
tion in cardenolide biosynthesis (Ga¨rtner et al., 1990;
Lindemann and Luckner, 1997) producing the required
5b-configured pregnane intermediates leading to the vari-
ous cardenolide genins. Fig. 3 shows the alignment of
deduced 5b-POR protein sequences for D. lanata
O
O
3 -HSD
HO
HO
O
cholesterol
pregnenolone
progesterone
isoprogesterone
O
5 -POR
5 -POR
O
O
O
H
5 -pregnane-3,20-dione
5 -pregnane-3,20-dione
5 -cardenolide
Fig. 1. Early steps in cardenolide biosynthesis.

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V. Herl et al. / Phytochemistry 67 (2006) 225–231
227
5a-POR (Kreis et al., 1998), several conserved functional
protein domains (LEGFXAF, LIHX, IPFV) were described
by Thigpen and Russell (1992) and Bhattacharyya et al.
(1999). None of these domains could be found in the 5b-
PORs isolated so far. On the other hand, the two peptide
fragments sequenced by Ga¨rtner et al. (1994) were identified
in all 5b-POR products sequenced so far (Roca-Perez et al.,
2004; AY585865-68, AY750898, AY574950, AY738710-12).
2.3. Southern blot analysis
The molecular organization of the 5b-POR from D.
lanata was determined by Southern blot analysis of geno-
mic DNA (Sambrook et al., 1989), digested with the
restriction endonucleases EcoR1, BamH1 and HindIII
(Fig. 2B). The full-length cDNA clone of 1170 bp was
32P-dGTP labelled and used as a probe for hybridization.
Under high stringency conditions two bands were detected
for cuts by EcoR1 and BamH1 restrictases, while three
bands were found for HindIII, as outlined in Fig. 2B. These
results indicate that 5b-POR from D. lanata consists of
almost 1–2 genes, as the size of the genomic clone of the
5b-POR is known (1250 bp, AY585867).
2.4. Over-expression
Over-expression of 5b-POR was achieved in Escherichia
coli after IPTG (0.1 mM) induction at low temperature
(4 ꢂC). The enzyme was purified by Ni-NTA batch fraction-
ation under native conditions and analysed by SDS–PAGE
(Fig. 4). As a control, the bacterial host cells were trans-
formed with an empty pQEX vector. The Ni-NTA-purified
r5b-POR was analysed by SDS–PAGE where a strong band
at 40 kDa indicated that the recombinant protein has the
same size as the 5b-POR isolated from D. purpurea leaves
by Ga¨rtner et al. (1994). Traces of other proteins (less than
3% of overall protein) could also be detected in the SDS–
PAGE but these were also found in the control extracts of
the bacteria containing the pQEX vector only. Since Jan-
knecht et al. (1991) stated that proteins containing neigh-
bouring histidins are not common in bacteria we suggest
that these ‘‘contaminating’’ proteins remained bound to
the column material due to the low stringency condition
(20 mM imidazole) chosen in the washing step.
Fig. 2. (A) PCR amplification of 5b-POR from D. lanata; (Lane 1) DNA
fragment of genomic DNA from D. lanata; (Lane 2) cDNA fragment from
RNA of D. obscura after RT-PCR; (Lane 3) cDNA fragment from RNA
of D. purpurea after RT-PCR; (Lane 4) cDNA fragment from RNA of D.
lanata after RT-PCR; (M) DNA-Marker SmartLadder (200–10,000 bp).
(B) Southern blot of D. lanata DNA digested with EcoR1 (E), HindIII (H)
and BamH1 (B) hybridized with 32P-dGTP labelled 5b-POR cDNA.
(AY574950), D. purpurea (AY585868) and the Dop5br gene
of D. obscura (AJ555127). The sequences of the deduced 5b-
POR gene products are 95–99% identical. A high degree of
homology was also seen when the nucleotide sequence of
the cDNA was analysed in silico and compared with the
reports for Dop5br of D. obscura (Roca-Perez et al., 2004)
and Dop5br of D. purpurea (AJ310673) (Fig. 3). Hence, it
seems as if the 5b-POR genes are highly conserved within
the genus Digitalis. Recently, Bra¨uchler et al. (2004) pro-
posed a phylogenetic cladogramme of the genus Digitalis
on the basis of ITS- and trnL-F sequences and found D.
lanata and D. obscura more closely related to each other
than to D. purpurea which is supported by our data.
2.5. Function of r5b-POR
The 5b-POR gene over-expressed in E. coli yielded an
enzymatically active protein. The Ni-NTA-purified protein
was checked for 5b-POR activity using the assay described
earlier (Stuhlemmer and Kreis, 1996). A typical experiment
is shown in Fig. 5A. TLC analysis revealed the formation of
one single product, namely 5b-pregnane-3,20-dione, when
progesterone was used as the substrate (Fig. 5, Lane 3).
5a-Pregnane-3,20-dione, the 5a-isomer of the pregnane-
3,20-dione was not formed. This fact is supported by the
different Rf-values and the different colours of the two
The deduced 5b-POR protein sequences were found
similar to those of Oryza sativa (about 58%), Populus tremu-
loides (about 64%) and to the Arabidopsis thaliana wound-
induced gene AWI (Yang et al., 1997). For the competing

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V. Herl et al. / Phytochemistry 67 (2006) 225–231
Fig. 3. Alignment of deduced 5b-POR proteins from different sources: AY574950 – D. lanata; AJ555127 – D. obscura; AJ310673 – D. purpurea.
Differences are bold.
isomers on TLC (Fig. 5A, Lane 4–5). The results have been
further confirmed by GC analyses which proofed that only
the 5b-isomer but not the 5a-isomer of pregnane-3,20-dione
was produced (Fig. 5B). The minor compounds seen in
Fig. 5B/III at tR = 28.30 and tR = 27.94 do not represent
pregnane isomers as could be deduced from the MS spectra.
Interestingly, 5b-pregnane-3,20-dione, the product of the
enzyme reaction was not converted to other products as it
was seen in partially purified enzyme preparations from
D. lanata leaves (Kreis et al., 1998), indicating that those
extracts contained other pregnane-modifying enzymes and
that r5b-POR catalyses only the 5b-reduction of progester-
one. It should be stressed that 5b-POR or homologous
genes have not been found in bacteria as yet. Actually,
5b-POR activity could not be detected in protein extracts
from bacteria containing the pQEX vector only.
2.6. Substrate preferences
The substrate preferences and kinetic properties of r5b-
POR were investigated using an HPLC method for product
identification and quantification. Besides the putative nat-
ural substrate progesterone other steroid substrates were
tested. Relative activities as well as Km- and vmax-values
were calculated and are shown in Table 1 and Fig. 6. The
r5b-POR did not only accept progesterone but also testos-
terone, 4-androstene-3,17-dione, cortisol and cortisone.
Fig. 4. Expression of recombinant 5b-POR in E. coli analyzed on SDS–
PAGE (12%). M – molecular weight marker BioRad (Munchen); (1)
¨
bacterial homogenate not induced by IPTG; (2) bacterial homogenate
induced by 0.1 mM IPTG and cultivated at 4 ꢂC for 96 h; (3) cell lysate
before Ni-NTA column; (4) flow through fraction from Ni-NTA column;
(5) wash fraction; (6) imidazole eluate from Ni-NTA column (recombinant
protein arrowed).