Δ5-3β-hydroxysteroid dehydrogenase (3βHSD) from Digitalis lanata. Heterologous expression and characterisation of the recombinant enzyme

Article abstract of DOI:10.1055/s-2007-981537

During the biosynthesis of cardiac glycosides, Δ⁵-3β- hydroxysteroid dehydrogenase (3βHSD, EC 1.1.1.51) converts pregnenolone (5-pregnen-3β-ol-20-one) to isoprogesterone (5-pregnene-3,20-dione). A 3βHSD gene was isolated from leaves of Digitalis lanata. It consisted of 870 nucleotides containing a 90 nucleotide long intron. A full-length cDNA clone that encodes 3βHSD was isolated by RT-PCR from the same source. A Sph I/Kpn I 3βHSD cDNA was cloned into the pQE30 vector and then transferred into E. coli strain M15[pREP4]. 3βHSD cDNA was functionally expressed as a His-tagged fusion protein (pQ3βHSD) composed of 273 amino acids (calculated molecular mass 28,561 Da). PQ3βHSD was purified by metal chelate affinity chromatography on Ni-NTA. Pregnenolone and other 3β-hydroxypregnanes but not cholesterol were 3β-oxidised by pQ3βHSD when NAD was used as the co-substrate. Testosterone (4-androsten-17β-ol-3-one) was converted to 4-androstene-3,17-dione indicating that the pQ3βHSD has also 17β-dehydrogenase activity. pQ3βHSD was able to reduce 3-keto steroids to their corresponding 3β-hydroxy derivatives when NADH was used as the co-substrate. For comparison, 3βHSD genes were isolated and sequenced from another 6 species of the genus Digitalis. Gene structure and the deduced 3βHSD proteins share a high degree of similarity. Georg Thieme Verlag KG Stuttgart.

Full text of DOI:10.1055/s-2007-981537

Vanessa Herl
D5-3b-Hydroxysteroid Dehydrogenase (3bHSD) from
Digitalis lanata. Heterologous Expression and
Characterisation of the Recombinant Enzyme*
Jördis Frankenstein
Nadine Meitinger
Frieder Müller-Uri
Wolfgang Kreis
Abstract
one) was converted to 4-androstene-3,17-dione indicating that
the pQ3bHSD has also 17b-dehydrogenase activity. pQ3bHSD
During the biosynthesis of cardiac glycosides, D5-3b-hydroxy- was able to reduce 3-keto steroids to their corresponding 3b-hy-
steroid dehydrogenase (3bHSD, EC 1.1.1.51) converts pregneno- droxy derivatives when NADH was used as the co-substrate. For
lone (5-pregnen-3b-ol-20-one) to isoprogesterone (5-pregnene- comparison, 3bHSD genes were isolated and sequenced from an-
3,20-dione). A 3bHSD gene was isolated from leaves of Digitalis other 6 species of the genus Digitalis. Gene structure and the de-
lanata. It consisted of 870 nucleotides containing a 90 nucleotide duced 3bHSD proteins share a high degree of similarity.
long intron. A full-length cDNA clone that encodes 3bHSD was
isolated by RT-PCR from the same source. A Sph I/Kpn I 3bHSD Key words
cDNA was cloned into the pQE30 vector and then transferred D5-3b-Hydroxysteroid dehydrogenase ´ D5-3-ketosteroid isomer-
into E. coli strain M15[pREP4]. 3bHSD cDNA was functionally ase ´ Digitalis lanata ´ cardenolide biosynthesis ´ gene expression ´
expressed as a His-tagged fusion protein (pQ3bHSD) composed Plantaginaceae ´ pregnenolone ´ progesterone
of 273 amino acids (calculated molecular mass 28,561 Da).
pQ3bHSD was purified by metal chelate affinity chromatography Supporting information available online at
on Ni-NTA. Pregnenolone and other 3b-hydroxypregnanes but http://www.thieme-connect.de/ejournals/toc/plantamedica
not cholesterol were 3b-oxidised by pQ3bHSD when NAD was
704
used as the co-substrate. Testosterone (4-androsten-17b-ol-3-
Introduction
lone (5-pregnen-3b-ol-20-one) in a two-step reaction (see Fig. 4
below) [1], [2]. 3b-Hydroxysteroid dehydrogenase (3bHSD; EC
Cardenolides, used for treatment of cardiac insufficiency in hu- 1.1.1.51) converts pregnenolone into isoprogesterone (5-preg-
mans, are still isolated from plants, the most important source nene-3,20-dione) which is then isomerised to progesterone.
being Digitalis lanata leaves. Cardenolides are supposed to be 3bHSD has been demonstrated in Digitalis cell cultures and
formed from cholesterol or other phytosterols (see [1] for a com-
prehensive review). The first step in the putative biosynthetic
route being studied extensively is the one facilitating the forma-
tion of progesterone (4-pregnene-3,20-dione) from pregneno-
* The nucleotide sequences reported in this paper have been submitted to
GenBankꢁ Data Base with the corresponding accession numbers:
DQ466890; AY844960; AY789449-453; AY844959.
Affiliation
Lehrstuhl für Pharmazeutische Biologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen,
Germany
Dedication
Dedicated to Professor A. Wilhelm Alfermann on the occasion of his 65th birthday
Correspondence
Prof. Dr. Wolfgang Kreis ´ Lehrstuhl Pharmazeutische Biologie ´ Friedrich-Alexander-Universität Erlangen-
Nürnberg ´ Staudtstr. 5 ´ 91058 Erlangen ´ Germany ´ Phone: +49-9131-852-8241 ´ Fax: +49-9131-852-8243 ´
E-mail: wkreis@biologie.uni-erlangen.de
Received March 7, 2007 ´ Revised April 24, 2007 ´ Accepted May 4, 2007
Bibliography
Planta Med 2007; 73: 704±710 ꢀ Georg Thieme Verlag KG Stuttgart ´ New York
DOI 10.1055/s-2007-981537 ´ Published online June 12, 2007
ISSN 0032-0943

plants [3] and was isolated and purified from a D. lanata cell cul- PCR DNA amplification
ture [4]. Lindemann et al. [5] amplified and sequenced a 700 bp Polymerase chain reaction amplifications were performed ac-
PCR fragment coding for 233 amino acids and the same group cording to [12]. Gene-specific primers for PCR amplification
published the sequence for a full-length 3bHSD cDNA in Gen- were generated based on the submitted sequence for 3bHSD
Bank (Acc.-Nr. AJ345026).
from D. lanata (Acc.-Nr. AJ345026). The full-length clones were
amplified using the two synthetic oligonucleotide primers
3bHSDs are involved in steroid biosynthesis in animal tissues (Eurogentec GmbH; Köln, Germany) 5¢-ATG TCG TCA AAG CCA
and steroid transformation in microorganisms. In animals, AGG T-3¢ (HSDdir) and 3¢-CTA ACG CAC GAC GGT GAA G-5¢
3bHSD and ketosteroid isomerase activities reside in one single (HSDrev).
protein. Several membrane-bound isoenzymes have been re-
ported from mammals, all being products of the 3bHSD gene Each reaction vessel (50 mL total volume) contained 2.5 units SA-
family [6]. On the other hand, 3b/17b-hydroxysteroid dehy- WADY Taq-DNA-Polymerase (Peqlab Biotechnologie GmbH), 1x
drogenase (3b/17bHSD), a soluble protein in Commamonas reaction buffer S, 0.5 mM MgCl2, 0.5 mM of each dNTP, 2 mM of
testosteroni, catalyses the reversible reduction/dehydrogenation primers and 0.2 mg of genomic DNA. A Personal Cycler 20 (Bio-
of the oxo/b-hydroxy groups at positions C-3 and C-17 of steroid metra GmbH; Göttingen, Germany) was used for amplification
compounds but does not exhibit D5-3-ketosteroid isomerase (3- according to the supplier's recommendation. 31 cycles of 3 min
oxosteroid D5-D4-isomerase, KSI; EC 5.3.3.1) activity (see [7] for denaturation at 95 8C each, followed by 1 min annealing at 56 8C
review). The 3bHSD from D. lanata shares high homology with and 2 min extension at 72 8C. An additional 5 min extension at
prokaryotic hydroxysteroid dehydrogenases lacking isomerase 72 8C was added to complete the amplification. PCR products
activity.
were analysed using 1% agarose gel electrophoresis in a TAE buf-
fer system. DNA was stained with ethidium bromide and made
We here cloned 3bHSD genes from several Digitalis species, func- visible under UV365nm light. SmartLadder (Eurogentec GmbH)
tionally expressed a 3bHSD cDNA isolated from D. lanata and was used for size determination, producing a pattern of 14 equal-
characterised the recombinant protein.
ly spaced bands ranging from 200 up to 10 000 bp. Usually three
clones each were sequenced from both sites for complete con-
sensus sequence (MWG Biotech AG; Martinsried, Germany).
Material and Methods
Chemicals
RT-PCR was performed with a Titan One Tube RT-PCR System
(Roche Diagnostics GmbH; Mannheim, Germany) using RNA
Most of the steroids used here were obtained from commercial and mRNA for the preparation of templates.
sources (e.g., Steraloids Inc.; Newport, RI, USA). Isoprogesterone
(5-pregnene-3,20-dione) was synthesised following the proce- In silico analysis
dure advised by Djerassi et al. [8] with minor modifications and After sequencing, all data have been analysed by different soft-
705
purified by preparative TLC.
ware packages provided by the European Bioinformatics Insti-
tute (http://www.ebi.ac.uk). Searching and sequence analysis
were performed with BLAST software in the GenBank Data Base.
Plant material
Plant seeds were obtained from the Genbank of the Institute for The nucleic acid as well as the translated (TRANSEQ) amino acid
Plant Genetics and Research on Cultivated Plants in Gatersleben, sequences were aligned by ClustalW (http://www.ebi.ac.uk/
Germany and were grown under standard greenhouse condi- clustalw/index.html).
tions. Leaves from D. lanata Ehrh. and several other species,
namely D. purpurea, D. ferruginea, D. grandiflora, D. parviflora, D. Heterologous expression
mariana and D. thapsi were included in this study.
The open reading frame of the full-length 3bHSD cDNA was am-
plified by PCR using the primers 5¢-TATAGCATGCATGC ATG TCG
Suspension cultures of the D. lanata cell line K3OHD [9] were TCA AAG CCA AGG T-3¢ (HSDsphdir) and 5¢-TATAGGTAC CTA
grown in 300-mL Erlenmeyer flasks kept in the dark at 24 8C on ACG CAC GAC GGT GAA G-3¢ (HSDkpnrev) to introduce SphI and
gyratory shakers (110 rpm). Cells were sub-cultured every 7 d by KpnI restriction sites, respectively. The amplified fragment was
inoculating 10 g cells (wet weight) into 100 mL of fresh growth cloned into the pQE30 vector system (Qiagen) for over-expres-
medium [10].
sion in E. coli strain M15[pREP4]. 1.0 mM IPTG (isopropyl-b-D-
thiogalactoside) was added to the medium to induce 3bHSD for-
mation. Cultivation continued at 37 8C for 5 h. 3bHSD cDNA was
DNA/RNA extraction
Fresh plant tissues were ground to a fine powder in liquid nitro- expressed as a His-tagged fusion protein containing the follow-
gen using a mortar and pestle. Genomic DNA as well as total RNA ing peptide at its N-terminus: MRSGSHHHHHHGSAC. The re-
extraction was carried out with E.Z.N.A. Plant DNA or RNA Mini combinant 3bHSD (termed pQ3bHSD) was isolated following
Kits, respectively (Peqlab Biotechnologie GmbH; Erlangen, Ger- the supplier's protocol (QIAexpressionist; Qiagen). The Ni-NTA-
many). Messenger RNA was isolated by Oligotexdirect mRNA
matrix(nickel-nitrilotriacetic acid coupled to Superflow resin)
Kit (Qiagen GmbH; Hilden, Germany). All molecular methods was washed extensively with 20 mM imidazole buffer before
were performed according to [11].
elution with 250 mM imidazole buffer. Protein analysis on SDS-
PAGE was performed as reported earlier [13], [14]. Protein was
quantified using the method described by Bradford [15].
Herl V et al. D5-3b-Hydroxysteroid Dehydrogenase¼ Planta Med 2007; 73: 704±710

D
5±3b-Hydroxysteroid dehydrogenase (3bHSD) assay
Dehydrogenase reaction (NAD as co-substrate): Substrate concen-
The solution containing pQ3bHSD was passed through PD-10 tration range 0.04±1.00 mM; concentrations for determination
columns (Amersham Biosciences Europe GmbH; Freiburg, Ger- of kinetic constants for co-substrate: 0.1±2.0 mM NAD.
many) for buffer exchange (20 mM NaPi buffer, pH 7.8) and the
protein eluate was diluted if necessary. The standard assay con- Reductase reaction (NADH as co-substrate): Substrate concentra-
tained in a final volume of 1 mL: 0.2 mg protein, 0.3 mmol preg- tion range 0.2±2.5 mM; concentrations for determination of ki-
nane and 1.0 mmol NAD in 20 mM NaPi buffer, pH 7.8. To check netic constants of co-substrate: 0.5±5.0 mM NADH.
the reverse reaction the same buffer was used. The assay then
contained in a final volume of 1 mL: 0.2 mg protein, 2 mM NAD, Isoelectric point (pI) of pQ3bHSD: This was determined by isoelec-
2 mmol glucose 6-phosphate and 6.4 nkat glucose 6-phosphate tric focusing on Criterionꢁ Gels IEF 3±9/Criterionꢁ Cell (BioRad;
dehydrogenase. The mixture was pre-incubated for 10 min at München, Germany).
30 8C prior to the addition of 0.3 mmol pregnane. After the addi-
tion of the respective substrate, samples were incubated at 50 8C Supporting information
and 550 rpm for 1 h (Eppendorf Thermomixer 5437, Eppendorf; PCR amplification of 3bHSD genes from genomic DNA and align-
Hamburg, Germany). Heat-inactivated samples served as con- ment of deduced amino acid sequences for 3bHSD and other relat-
trols. The reaction was terminated by adding 1 mL dichlorome- ed enzymes of the dehydrogenase family from different plant spe-
thane. After vortexing for 30 s, the organic phase containing the cies are available online as Supporting Information and at http://
pregnanes was removed, evaporated and the residue was dis- www.biologie.uni-erlangen.de/pharmbiol/Herletal3bHSD.pdf.
solved in 50 mL ethanol. This solution was qualitatively analysed
using thin layer chromatography (TLC). The samples and the re-
spective standards were spotted on TLC plates (silica gel 60, Results and Discussion
Merck; Darmstadt, Germany), and subsequently developed with
dichloromethane: ethyl acetate (8:2). Pregnane spots were vi- Oligonucleotide primers from peptide fragments of the 3bHSD
sualised with anisaldehyde reagent [16].
isolated from D. lanata leaves [4] have been deduced by Linde-
mann et al. [5]. The same authors amplified and sequenced a
In addition, pregnanes were analysed by HPLC according to [3] 700-nucleotide cDNA fragment for a putative 3bHSD. Based on
using a 1525 Binary HPLC Pump equipped with a 2487 Dual l Ab- their reports and the submitted GenBank entry for a full length
sorbance Detector (Waters; Eschborn, Germany). 20 mL were ap- 3bHSD cDNA (AJ345026, 780 nucleotides, 259 amino acids), we
plied on a Symmetry column (C18, 5 mM, 4.6”150 mm) and the generated primers for PCR amplification and RT-PCR. Leaves of
pregnanes eluted with a step gradient of H2O (solvent A) and Digitalis lanata were used for genomic DNA and cDNA prepara-
acetonitrile (solvent B): start (25% B), 10 min (65% B), 18 min tion. The genomic clone for D. lanata (DQ466890) was obtained
(100% B), 21 min (100% B), 23 min (25% B), 25 min (25% B). Preg- using two 19 nucleotide synthetic primers (HSDdir and HSDrev).
nanes were detected at 205 nm and 240 nm and identified by The sequenced 3bHSD gene consisted of 870 nucleotides contain-
their retention times by comparison with authentic pregnanes. ing a 90 nucleotides long intron (18±107). Genomic sequences for
The retention time was for progesterone 15.8 min, for pregneno- 3bHSD were isolated from additional six Digitalis species, namely
lone 16.4 min and for isoprogesterone 16.8 min. For quantitative D. purpurea, D. ferruginea, D. grandiflora, D. parviflora, D. mariana,
analyses an internal standard, 3-keto-D4-19-O-acetylstrophan- D. thapsi, and analysed. All PCR products were found to be of a
thidol, was added to the assay prior to dichloromethane extrac- similar size; they did not differ significantly from each other
706
tion followed by HPLC analysis.
(Fig.1S, Supporting information). All genomic 3bHSD gene se-
quences contained a 90±96 bp intron at the 5¢ end of the gene.
The deduced protein sequences coded by these genes are aligned
Enzyme properties
Determination of pH optimum: The effect of the buffer pH on the in Fig.1. The sequence identity within the genus is very high, about
enzyme activity was examined with the following buffers: 20 94% on the nucleotide level and 94±98% on the amino acid level.
mM NaPi (Buffer I) at pH 6.0 to 7.5, Tris-HCl (pH 7.1 to 8.9) and
20 mM glycine-NaOH (pH 8.6 to 10.5). The enzyme was dissolved The putative 3bHSD genes sequenced here have all been submitted
in the respective buffer after passing through PD-10 columns to GenBank (Acc.-Nr. DQ466890, AY789449-453, AY844959-960).
(Amersham Biosciences Europe GmbH) for buffer exchange.
Aligned with genes of other plant species available in public da-
tabases, Digitalis putative 3bHSD genes display considerable si-
Determination of temperature optimum: Incubations were carried milarities with putative alcohol dehydrogenase genes obtained
out at temperatures ranging from 20 to 70 8C. The protein ex- from Pisum sativum, Arabidopsis thaliana and Oryza sativa. They
tracts (Buffer I, pH 7.8) were pre-incubated at the respective in- are also very similar to isopiperitenol dehydrogenase derived
cubation temperature for 5 min.
from Mentha x piperita [17] and secoisolariciresinol dehydrogen-
ase from Forsythia x intermedia [18], both genes involved in the
Kinetic constants: These were determined for all substrates in formation of plant secondary metabolites. Deduced Digitalis
both reaction directions at pH 7.8 and at 50 8C using the standard lanata 3bHSD shares 47% and 49% identity with the proteins of
enzyme assay containing 0.1 mmol of NAD or 0.2 mmol NAD and Mentha piperita and Forsythia intermedia, respectively (Fig. 2S,
an NADH-regenerating system to check forward or reverse reac- Supporting information).
tion, respectively.
Since the genomic sequences of all Digitalis 3bHSD genes ana-
lysed here contained an intron, RT-PCR amplification was used
Herl V et al. D5-3b-Hydroxysteroid Dehydrogenase¼ Planta Med 2007; 73: 704±710

Fig. 1 Alignment of deduced 3bHSD amino
acid sequences from different Digitalis spe-
cies. D. pur-Digitalis purpurea; D. fer-D. ferru-
ginea; D. tha-D. thapsi; D. lan-D. lanata; D.
par-D. parviflora; D. gra-D. grandiflora; D.
mar-D. mariana. Symbols: ª*º means that
the residues in that column are identical in
all sequences in the alignment,ª :º means
that conserved substitutions have been ob-
served and ª.º means that semi-conserved
substitutions are observed.
to synthesise 3bHSD cDNA for subsequent cloning into the ex- presence of NAD. Under standard incubation conditions pregne-
pression vector pQE. The 3bHSD cDNA isolated from D. lanata nolone was mainly transformed into isoprogesterone together
contained an open reading frame of 780 nucleotides correspond- with variable amounts of progesterone. As a result of non-enzy-
ing to 259 amino acids (calculated molecular mass 26,857 Da). matic isomerisation, prolonged exposition yielded increasing
The primers HSDsphdir and HSDkpnrev (see Material and Meth- amounts of progesterone even if the enzyme reaction was termi-
ods) were used to amplify 3bHSD cDNA and integrate it into the nated by boiling.
707
expression vector. Finally, 3bHSD cDNA was expressed as a His-
tagged fusion protein composed of 273 amino acids (calculated In addition to pregnenolone, assumedly the biosynthetic precur-
molecular mass 28,561 Da).
sor of cardenolides, several other steroids with a 3b-hydroxy
group were tested as substrates for the pQ3bHSD. 5b-Pregnan-
After IPTG induction the pQ3bHSD gene was expressed in E. coli. 3b-ol-20-one, 5a-pregnan-3b-ol-20-one, 21-hydroxypregneno-
The protein was purified by metal chelate affinity chromatogra- lone (C21-steroids) and 5-androsten-3b-ol-17-one (a C17-steroid)
phy on Ni-NTA. After purification the recombinant 3bHSD ap- were all well accepted, whereas cholesterol was not. For many
peared as a homogenous band of 30 kDa (Fig. 2). As a control microorganisms, the enzyme responsible for the modification of
M15[pREP4] cells were transformed with an empty pQE30 vec- 3b-hydroxy-5-ene to 3-keto-4-ene belongs to the oxidases (ªcho-
tor. The protein extracts prepared from bacteria containing only lesterol oxidaseº, ChO; EC 1.1.3.6) which accept C17, C21 as well as
the vector did not display an induced 30 kDa protein band al- C27 3b-steroids. Since cholesterol was not accepted by the
though they contained traces of other proteins (Fig. 2, lanes 6 pQ3bHSD it is assumed to be unrelated to these oxidases.
and 7).
Testosterone (4-androsten-17b-ol-3-one), a C17-steroid with a 3-
The pQ3bHSD was most active at a pH of 8.5 (20 mM Tris-HCl) carbonyl group and a 17b-hydroxy group, was converted to 4-an-
and a temperature of 55 8C. The isoelectric point (pI) of recombi- drostene-3,17-dione. This indicates that pQ3bHSD also possesses
nant His-tagged 3bHSD, as determined by isoelectric focusing 17b-dehydrogenase activity.
(IEF), was 6.5; the theoretical value was calculated to be 6.86
(http://www.expasy.ch/tools/pi_tool.html).
Moreover, pQ3bHSD was able to catalyse the reduction of 3-ke-
tosteroids when NADH was used as co-substrate. Pregnane-
With a view to further characterise recombinant His-tagged 3,20-diones without a D4- or D5-double bond like 5b-pregnane-
3bHSD, we tested several steroids as substrates. It was found 3,20-dione and 5a-pregnane-3,20-dione were accepted. The 5b-
that pQ3bHSD converts pregnenolone to isoprogesterone in the configurated dione yielded two products, namely 5b-pregnan-
Herl V et al. D5-3b-Hydroxysteroid Dehydrogenase¼ Planta Med 2007; 73: 704±710

Fig. 2 SDS-PAGE analysis of the heterologously expressed pQ3bHSD. Marker proteins (lane M); lanes 1±6: protein from cells with pQ3bHSD,
lanes 7±12: protein from control cells containing the empty pQE expression vector only. No IPTG added (lane 1); IPTG-induced cells cultivated
for 5 h at 37 8C (lane 2); Cell lysate before Ni-NTA column (lane 3); column flow-through (lane 4); column wash fraction (lane 5); imidazole (200
mM) eluate with pQ3b-HSD band at ca. 29 kDa (lane 6); Imidazole eluate of control (lane 7); wash fraction of the Ni-NTA column (lane 8); column
flow-through (lane 9); bacterial lysate (lane 10); IPTG-induced cells cultivated at 37 8C for 5 hours (lane 11); No IPTG added (lane 12).
with those of the partially purified plant 3bHSD reported by Fin-
sterbusch et al. [4] (Table 1). The Vmax values for substrates con-
taining D4- or D5-double bonds differ considerably whereas other
values appear to be very similar. Especially the values for pregne-
nolone must be regarded as ªapparentº since part of the isopro-
gesterone formed is isomerised non-enzymatically to progester-
one.
The hydroxysteroid dehydrogenases described so far belong to
one of two protein families, namely aldo-keto reductases (AKR)
or short-chain dehydrogenases/reductases (SDR) [19], [20]. Both
families contain prokaryotic and eukaryotic members. The
Digitalis 3bHSD is a soluble enzyme and it shares this property
with members of the SDR family [21], [22]. In contrast, mamma-
lian 3bHSDs are localised in the membranes of the smooth ER
and mitochondrial cristae [23] (see [6] for a recent review). SDR
type proteins may occur as homo-oligomers of subunits with
molecular masses of about 30 kDa whereas 3bHSDs belonging
to the AKR family have higher molecular masses. As already
pointed out by Finsterbusch et al. [4] the Digitalis 3bHSDs prob-
ably belong to the SDR enzyme superfamily (group of hydroxy-
steroid dehydrogenases).
708
Fig. 3 pQ3bHSD activity measured by HPLC analysis: 3-keto-D4-19-O-
acetyl-strophanthidol (IS,internal standard); progesterone ( 1); preg-
nenolone (2); isoprogesterone (3).
3a-ol-20-one and 5b-pregnan-3b-ol-20-one whereas the 5a-
configurated dione yielded only one product, namely 5a-preg- The pQ3bHSD described here exhibits 3a-, 3b- and 17b-dehydro-
nan-3b-ol-20-one. 4-Androstene-3,17-dione was also accepted genase and 3/17-keto reductase activities. A 3b/17bHSD was also
as a substrate, but the 17-keto instead of the 3-keto function reported to occur in Commomonas testosteroni, whereas other
was reduced.
HSDs display stricter substrate specificities (see [24] for more
details).
A clear preference for NAD (NADH for reduction) as co-sub-
strate(s) was observed. NADP and NADPH were also accepted, The results obtained with pQ3bHSD have to be compared with
but were about 3 to 5 times less effective.
data reported for putative hydroxysteroid oxidoreductases sup-
posed to be involved in cardenolide metabolism [1], [4]. For ex-
HPLC was used to study the kinetic properties of pQ3bHSD and to ample, the 3a-hydroxysteroid-5b-reductase described by Stuh-
quantify enzyme activity. A typical chromatogram of an experi- lemmer et al. [25] did not accept 5a-steroids or D4/D5-unsaturat-
ment using pregnenolone as the substrate is shown in Fig. 3. Ki- ed pregnenes, whereas 5b-steroids including digitoxigenone
netic constants for pQ3bHSD were determined with several sub- were converted to their respective 3a-derivatives. From inhibi-
strates. The kinetic data calculated for pQ3bHSD were compared tion studies with crude enzyme preparations it was concluded
Herl V et al. D5-3b-Hydroxysteroid Dehydrogenase¼ Planta Med 2007; 73: 704±710

Table 1 Substrates and products of pQ3bHSD. Kinetic constants determined here were compared with published data [4]
Substrate(s)
Product(s)
Km pQ3bHSD (mM)a
Vmax pQ3bHSD (mkat/kg)a
Vmax/KM
NAD
260 (32.0)
90  9 (20)
19.5 (n.d)
0.08
2.02
1.55
1.78
0.84
7.40
5.26
4.04
+ Pregnenolone
Isoprogesterone + Progesteroneb
5b-Pregnane-3,20-dione
5a-Pregnane-3,20-dione
5-Androstene-3,17-dione
182  43 (770)c
488  111 (610)c
206  20 (690)c
539 (680)c
+ 5b-Pregnan-3b-ol-20-one
+ 5a-Pregnan-3b-ol-20-one
+ 5-Androsten-3b-ol-17-one
NADH
314  66 (110)
116  23 (120)
642 (130)
642 (n.d.)
4750 (n.d.)
+ 5b-Pregnane-3,20-dione
+ 5a-Pregnane-3,20-dione
5b-Pregnan-3b-ol-20-one + 5b-Pregnan-3a-ol-20-one
5a-Pregnan-3b-ol-20-one
1725 (n.d.)
106  1 (n.d.)
9071 (n.d.)
428  30 (n.d.)c
a Data of Finsterbusch et al. [4] in brackets; n.d. = not determined.
b Isoprogesterone was the main product (about 90%),see Fig. 3.
c Means  SD of at least two independent experiments.
Fig. 4 Putative early steps
and enzymes in cardenolide
biosynthesis (3bHSD: D5-3b-hy-
droxysteroid dehydrogenase,
KSI: D5-3-ketosteroid isomer-
ase,5 bPOR: progesterone 5b-
reductase.
709
that the formation of 3a- and 3b-hydroxy-5b-pregnanes are cat- sterone are still fundamental problems to solve this question.
alysed by two different stereospecific oxidoreductases [26]. In KSI is one of the most efficient enzymes known, with kcat/KM ap-
many aspects the r3b-HSD described here behaves like these en- proaching the diffusion control limit [27]. Therefore, even trace
zymes which have never been isolated and characterized in pure amounts of KSI present in a 3bHSD enzyme preparation might si-
form. Therefore, our results support the hypothesis of Finster- mulate KSI side reaction. On the other hand, the non-enzymatic
busch et al. [4] who discussed that 3bHSD might be a multifunc- isomerisation of b,g-unsaturated ketones, such as isoprogester-
tional SDR type enzyme in steroid metabolism. As far as the one or 5-androstene-3,17-dione, has been studied extensively
cardenolide pathway is concerned, we presume that 3bHSD cat- [28]. The isomerisation itself is a multi-step reaction which
alyses two steps in cardenolide biosynthesis, namely the dehy- might be influenced by many parameters, such as the presence
drogenation of pregnenolone and the reduction of 5b-pregnane- of acetate or carboxylate residues and, of course, biocatalysts.
3,20-dione (Fig. 4). The presence of 3bHSD in other species not
accumulating cardenolides [3], [4] indicates that the enzyme Lindemann et al. [5] already stated that 3bHSD from D. lanata
may also be involved in other metabolic pathways. RNAi experi- shows sequence similarities with microbial hydroxysteroid de-
ments may help to elucidate this issue further.
hydrogenases but not with animal enzymes and contains a con-
served putative short chain dehydrogenase (SDR) [29] domain.
One of the crucial questions still remains, namely whether Hence, it may be speculated that 3bHSD and KSI activities do
3bHSD of D. lanata actually possesses KSI activity. This question not reside in one single protein in D. lanata. The molecular struc-
has already been addressed by Finsterbusch et al. [4] but could ture of the Digitalis 3bHSD supports the biochemical evidence
not be answered conclusively. The authors therefore advised since no KSI-like domains can be found in the protein sequences
studies employing a recombinant form of 3bHSD. However, deduced from the published nucleotide sequences. However, al-
even when studying the recombinant enzyme, the catalytic though preliminary experiments indicate that isoprogesterone
properties of KSI and non-enzymatic isomerisation of isoproge- was isomerised only non-enzymatically and that thus progester-
Herl V et al. D5-3b-Hydroxysteroid Dehydrogenase¼ Planta Med 2007; 73: 704±710

12
13
Williams B, Tsang A. A maize gene expressed during embryogenesis is
ABA-inducible and highly conserved. Plant Mol Biol 1991; 16: 919±23.
Müller-Uri F, Reva VA. Overexpression and catalytic function of cyclo-
philin 18 from Digitalis lanata Ehrh. Pharm Pharmacol Lett 2000; 10:
5±7.
Herl V, Fischer G, Müller-Uri F, Kreis W. Molecular cloning and hetero-
logous expression of progesterone 5b-reductase from Digitalis lanata
Ehrh. Phytochemistry 2006; 67: 25±31.
Bradford MM. A rapid and sensitive method for the quantitation of mi-
crogram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 1976; 72: 248±54.
Jork H. Thin Layer Chromatography: Reagents and Detection Methods,
Vol. 1. In: Jork H., Funk W, Fischer W, editors. Thin-Layer-Chromatog-
raphy. New York: VCH Publishers; 2004: 195±8.
Ringer KL, Davis EM, Croteau R. Monoterpene metabolism. Cloning,
expression, and characterization of (±)-isopiperitenol/(±)-carveol de-
hydrogenase of peppermint and spearmint. Plant Physiol 2005; 137:
863±72.
Xia Z-Q, Costa MA, PØlissier HC, Davin LB, Lewis NG. Secoisolariciresi-
nol dehydrogenase purification, cloning, and functional expression. J
Biol Chem 2001; 276: 12614±23.
Jörnvall H, Persson B, Krook M, Atrian S, Gonzalez-Duarte R, Jeffery J et
al. Short-chain dehydrogenases/reductases (SDR). Biochemistry 1995;
34: 6003±13.
one was an artefact rather than a product of pQ3bHSD catalysis,
the question is still open and we intend to give experimental evi-
dence for the assumption that KSI is not associated with HSD in
the Digitalis 3b-HSD using the recombinant enzyme described
here.
14
15
16
17
Acknowledgements
The authors thank G. Fischer for excellent technical assistance.
Plants, seeds and herbal material were kindly provided by Dr.
W. Welû, Botanical Garden FAU Erlangen, Prof. Dr. A. Graner,
Genbank Gatersleben, Prof. G. Heubl, LMU München and Prof. V.
Melzheimer, Marburg. We thank Barbara White for linguistic ad-
vice.
18
19
References
1
Luckner M, Wichtl M. In: Luckner M, Wichtl M, editors. Digitalis.
20 Jez JM, Bennett MJ, Schlegel BP, Lewis M, Penning TM. Comparative
anatomy of the aldo-keto reductase superfamily. Biochem J 1997;
326: 625±36.
Stuttgart: WVGmbH; 2000: 1±352.
2 Kreis W, Hensel A, Stuhlemmer U. Cardenolide biosynthesis in fox-
glove. Planta Med 1998; 64: 491±9.
21
Oppermann UCT, Maser E. Characterization of a 3a-hydroxysteroid
3 Seidel S, Kreis W, Reinhard E. D5-3b-Hydroysteroid dehydrogenase/D5-
D4-ketosteroid isomerase (3b-HSD), a possible enzyme of cardiac gly-
coside biosynthesis, in cell cultures and plants of Digitalis lanata
EHRH. Plant Cell Rep 1990; 8: 621±4.
dehydrogenase/carbonyl reductase from the gram-negative bacter-
ium Commamonas testosteroni. Eur J Biochem 1996; 209: 459±66.
22 Oppermann UCT, Filling C, Berndt KD, Persson B, Benach J, Ladenstein
R et al. Active site directed mutagenesis of 3b/17b-hydroxysteroid de-
hydrogenase establishes differential effects on short-chain dehydro-
genase/reductase reactions. Biochemistry 1997; 36: 34±40.
23 Thomas JL, Berko EA, Faustino A, Myers RP, Strickler RC. Human pla-
cental 3b-hydroxy-5-ene-steroid dehydrogenase and steroid 5-4-
ene-isomerase: purification from microsomes, substrate kinetics, and
inhibition by product steroids. J Steroid Biochem 1988; 31: 785±93.
24 Benach J, Filling C, Oppermann UC, Roversi P, Bricogne G, Berndt KD et
al. Structure of bacterial 3b/17b-hydroxysteroid dehydrogenase at 1.2
A resolution: a model for multiple steroid recognition. Biochemistry
2002; 41: 4659±68.
4 Finsterbusch A, Lindemann P, Grimm R, Eckerskorn C, Luckner M. D5-
3b-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh ± a mul-
tifunctional enzyme in steroid metabolism?.Planta 1999; 209: 479±
86.
5 Lindemann P, Finsterbusch A, Pangert A, Luckner M. Partial cloning of
a D5±3b-hydroxysteroid dehydrogenase from Digitalis lanata. In: Oka-
moto M, Ishimura Y, Nawata, H, editors. Molecular steroidogenesis,
Proceedings of the Yamada Conference LII Tokyo: Universal Academy
Press, Frontiers Science Series 29 ± XXIV; 2000: 333±4.
6 Simard J, Ricketts M-L, Gingras S, Soucy P, Feltus FA, Melner MH. Mo-
lecular biology of the 3b-hydroxysteroid dehydrogenase/D5-D4 iso-
merase gene family. Endocrine Rev 2005; 26: 525±82.
7 Persson B, Krook M, Jörnvall H. Characteristics of short-chain alcohol
dehydrogenases and related enzymes. Eur J Biochem 1991; 200:
537±43.
710
25 Stuhlemmer U, Kreis W. Cardenolide formation and activity of preg-
nane-modifying enzymes in cell suspension cultures, shoot cultures
and leaves of Digitalis lanata. Plant Physiol Biochem 1996; 34: 85±91
26 Seitz HU, Gärtner DE. Enzymes is cardenolide-accumulating shoot
cultures of Digitalis purpurea L. Plant Cell Tissue Organ Cult 1994; 38:
337±44.
8 Djerassi C, Engle RR, Bowers A. The direct conversion of steroidal D5-
3b-alcohols to D5- and D4-3-ketones. J Org Chem 1956; 21: 547±9.
9 Kreis W, Zhu W, Reinhard E. 12b-hydroxylation of digitoxin by
Digitalis lanata cells. Production of deacetyllanatoside C in a 20-L air-
lift bioreactor. Biotechnol Lett 1989; 11: 25±30.
27 Pollack RM. Enzymatic mechanisms for catalysis of enolization: keto-
steroid isomerase. Bioorg Chem 2004; 32: 341±53.
28 Houck WJ, Pollack RM. Activation enthalpies and entropies for the mi-
croscopic rate constants of acetate-catalyzed isomerization of 5-an-
drostene-3,17-dione. J Am Chem Soc 2003; 125: 10206±12.
29 Krozowski Z. The short-chain alcohol dehydrogenase superfamily:
variations on a common theme. J. Steroid Biochem Mol Biol 1994; 51:
125±30.
10
Kreis W, Reinhard E. 12b-hydroxylation of digitoxin by suspension-
cultured Digitalis lanata cells. Production of deacetyllanatoside C
using a two-stage culture method. Planta Med 1988; 54: 95±100.
Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. A laboratory
11
manual, 2nd edition. New York: Cold Spring Harbor Press; 1989.
Herl V et al. D5-3b-Hydroxysteroid Dehydrogenase¼ Planta Med 2007; 73: 704±710