Insights into the Substrate Promiscuity of Novel Hydroxysteroid Dehydrogenases

Article abstract of DOI:10.1002/adsc.202000120

Hydroxysteroid dehydrogenases (HSDHs) are valuable biocatalysts for the regio- and stereoselective modification of steroids, bile acids and other steroid derivatives. In this work, we investigated the substrate promiscuity of this highly selective class of enzymes. In order to reach this goal, a preliminary search of HSDH homologues in in-house or public available (meta)genomes was carried out. Eight novel NAD(H)-dependent HSDHs, showing either 7α-, 7β-, or 12α-HSDH activity, and including, for the first time, enzymes from extremophilic microorganisms, were identified, recombinantly produced, and characterized. Among the novel HSDHs, four highly active (up to 92 U mg^?1) NAD(H)-dependent 7β-HSDHs showing negligible similarity towards previously described 7β-HSDHs, were discovered. These enzymes, along with previously characterized HSDHs, were tested as biocatalysts for the stereoselective reduction of a panel of substrates including two α-ketoesters of pharmaceutical interest and selected ketones that partially resemble the structural features of steroids. All the reactions were coupled with a suitable cofactor regeneration system. Regarding the α-ketoesters, nearly all of the tested HSDHs showed a good activity toward the selected substrates, yielding the reduced α-hydroxyester with up to 99% conversions and enantiomeric excesses. On the other hand, only the 7β-HSDHs from Collinsella aerofaciens and Clostridium absonum showed appreciable activity toward more complex ketones, i. e., (±)-trans-1-decalone, but with interesting as well as different selectivity. (Figure presented.).

Full text of DOI:10.1002/adsc.202000120

Title: Insights into the Substrate Promiscuity of Novel Hydroxysteroid
Dehydrogenases
Authors: Susanna Bertuletti, Erica Elisa Ferrandi, Stefano Marzorati,
Marta Vanoni, Sergio Riva, and Daniela Monti
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To be cited as: Adv. Synth. Catal. 10.1002/adsc.202000120
Link to VoR: https://doi.org/10.1002/adsc.202000120

10.1002/adsc.202000120
Advanced Synthesis & Catalysis
FULL PAPER
DOI: 10.1002/adsc.202000120
Insights into the Substrate Promiscuity of Novel
Hydroxysteroid Dehydrogenases
Susanna Bertuletti,+a,b Erica Elisa Ferrandi,+a Stefano Marzorati,a Marta Vanoni,a Sergio
Riva,a,* and Daniela Monti a,*
a
Istituto di Scienze e Tecnologie Chimiche “G. Natta” (SCITEC), CNR, Via Mario Bianco 9, 20131 Milano, Italy
E-mail: sergio.riva@scitec.cnr.it; daniela.monti@scitec.cnr.it
Università degli Studi di Milano, Via Giuseppe Colombo 60, 20133 Milano, Italy
Equal author contribution
b
+
Received:((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.
Abstract. Hydroxysteroid dehydrogenase (HSDHs) are These enzymes, along with previously characterized
valuable biocatalysts for the regio- and stereoselective HSDHs, were tested as biocatalysts for the stereoselective
modification of steroids, bile acids and other steroid reduction of a panel of substrates including two α-
derivatives. In this work, we investigated the substrate ketoesters of pharmaceutical interest and selected ketones
promiscuity of this highly selective class of enzymes. In that partially resemble the structural features of steroids. All
order to reach this goal, a preliminary search of HSDH the reactions were coupled with a suitable cofactor
homologues in in-house or public available (meta)genomes regeneration system. Regarding the α-ketoesters, nearly all
was carried out. Eight novel NAD(H)-dependent HSDHs, of the tested HSDHs showed a good activity toward the
showing either 7α-, 7β-, or 12α-HSDH activity, and selected substrates, yielding the reduced α-hydroxyester
including, for the first time, enzymes from extremophilic with up to 99% conversions and enantiomeric excesses. On
microorganisms, were identified, recombinantly produced, the other hand, only the 7β-HSDHs from Collinsella
and characterized. Among the novel HSDHs, four highly aerofaciens and Clostridium absonum showed appreciable
active (up to 92 U mg-1) NAD(H)-dependent 7β-HSDHs activity toward more complex ketones, i.e., (±)-trans-1-
showing negligible similarity towards previously described decalone, but with interesting as well as different
7β-HSDHs, were discovered.
selectivity.
Keywords: Hydroxysteroid dehydrogenase; Bile acids;
Substrate promiscuity; Enzyme discovery; Stereoselectivity
Introduction
Hydroxysteroid dehydrogenases (HSDHs) are a
group of NAD(P)(H)-dependent oxidoreductases that
are characterized by their ability to catalyze the
oxidation/reduction of the hydroxyl/keto groups of
bile acids and steroids.[1–3] Most of the HSDHs
described so far show an exceptionally high
selectivity that enables their application in the
straightforward modification of their natural complex
substrates, which is instead quite challenging by
conventional chemical synthesis.
Specifically, HSDHs have been shown to oxidize
the hydroxyl groups at different positions, e.g., at C-3,
C-7, and C-12 of bile acids in a very high
regiospecific manner (Scheme 1). Moreover, for each
one of these positions, HSDHs usually show a
practically absolute stereoselectivity by oxidizing
only either the hydroxyl group above (β
configuration) or below (α configuration) the plane of
the steroid molecule.[4,5] The reactions catalyzed by
HSDHs are reversible, thus these enzymes can be
applied in the regioselective reduction of the
corresponding keto derivatives as well.[4,6,7]
Scheme 1. Chemical structure of the most common bile
acids, natural substrates of hydroxysteroid dehydrogenases.
Thanks to these interesting features, HSDHs have
been widely studied during the last years for their
1
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10.1002/adsc.202000120
Advanced Synthesis & Catalysis
exploitation in the biocatalyzed synthesis of key
intermediates of the drug ursodeoxycholic acid
(UDCA), a largely applied therapeutic agent used for
the dissolution of cholesterol gallstones and for the
treatment of different hepatic diseases. These efforts
have led to the characterization of different enzymes
showing either 7α-, 7β- or 12α-HSDH activity[2,3], as
well as to the development of biocatalyzed processes
for the preparation of UDCA intermediates.[8–11]
dehydrogenases/reductases” (SDR) superfamily, a
very large and highly divergent protein family with
generally low sequence identity (20-30%), thus
making quite challenging their functional annotation
if a biochemical characterization is not performed.[21]
On the other side, most of the known HSDHs are
produced by gut or soil bacteria belonging to the
phyla of Proteobacteria, e.g., Escherichia[22] and
Stenotrophomonas[5] strains, Actinobacteria, e.g.,
Collinsella[23] and Eggerthella[8,24,25] strains, and
The ability of enzymes to act on substrates
possessing chemical structures significantly different
from their natural ones is a quite common feature.[12–
Firmicutes,
e.g.,
Clostridium,[26–30]
Ruminococcus,[31,32] and Lactobacillus[5] strains, thus
resulting in an overall limited diversity.
14]
This “substrate promiscuity” was also observed
with HSDHs a long time ago by Davies and
To improve the diversity of the HSDHs library
already available in our lab, a bioinformatic screening
was performed in search of HSDH homologues in in-
house or public available (meta)genomes, with a
special focus for those from extreme environments.
Indeed, no HSDHs have been characterized so far
coworkers who reported the activity of
a
3SDH from Mortierella ramanniana toward
3-hydroxybicyclo-heptan-6-ones.[15]
However, to date only few additional examples of
HSDH promiscuous activities towards non-steroidal
substrates have been described. For example,
from
extremophilic
microorganisms,
e.g.,
Comamonas
11HSDH[17] were found able to reduce p-
nitrobenzaldehyde, p-nitroacetophenone and
testosteroni
3α-SDH[16]
and
thermophiles and halophiles. However, enzymes from
these sources may have practical interest exceeding
the scope of the present work, since, thanks to their
higher robustness, they could be more suitable than
mesophilic enzymes to industrial applications.[33–36]
The search of novel HSDH sequences was carried
metyrapone, while 17-HSDH from the filamentous
fungus Cochliobolus lunatus[18] was active toward
quinones,
Moreover,
menadione,
3,17-HSDH
p-nitrobenzaldehyde.
from Comamonas
out
by
using
either
the
Blastp
tool
testosteroni and 3-HSDH from Streptomyces
hydrogenans showed activity toward muscarine
precursors.[19] More recently, the catalytic
promiscuous property of the 7α-HSDH from
Bacteroides fragilis has been investigated by
studying the enzyme substrate specificity and
stereoselectivity in the reduction of different aromatic
and aliphatic α-ketoesters to the corresponding α-
hydroxyesters.[20] To the best of our knowledge, only
these six papers have been published so far,
describing, as just summarized, the results obtained
with unrelated HSDHs (3SDH, 3α-SDH,
11HSDH, 17-HSDH, 3,17-HSDH, 3-
HSDH and 7α-HSDH) on very different substrates
(3-hydroxybicyclo-heptan-6-ones, metyrapone, p-
nitroacetophenone, p-nitrobenzaldehyde, quinones,
menadione, muscarine precursors, aromatic and
aliphatic α-ketoesters).
In this work, we aimed at filling this gap by
performing a more systematic investigation on
HSDHs substrate promiscuity. This result was
achieved by i) increasing the diversity of the
available HSDHs by discovering novel enzyme
homologues, ii) establishing their regio- and
stereoselectivity in the respect of their natural
substrates, i.e., bile acids, and iii) evaluating their
possible substrate promiscuity toward a set of
different non-steroidal substrates.
(https://blast.ncbi.nlm.nih.gov/) for mining into the
NCBI database (https://www.ncbi.nlm.nih.gov/) or
the program LAST (http://last.cbrc.jp/) for the
analysis of metagenomes of samples collected in hot
terrestrial environments.[33,37,38] In both cases, only
sequences of functionally characterized HSDHs
(Table S1 in the Supporting Information) were used
as queries during the multiple sequence alignments.
At the end of these analyses, ten SDR sequences
were selected for cloning and characterization (Table
1).
Two sequences (Dm7α-HSDH and Hh7α-HSDH,
entries 1-2 in Table 1) showing about 75% similarity
with known 7α-HSDHs (see also Fig. S1 in the
Supporting Information) were discovered for the first
time in genomes from extremophilic bacteria. Dm7α-
HSDH was annotated as a putative glucose 1-
dehydrogenase in the genome of Deinococcus
marmoris strain PAMC 26562, a radiation-resistant
and psychro- and draught-tolerant bacterium isolated
from an Antarctic rock sample.[39,40] Instead, Hh7α-
HSDH was already annotated as a 7α-HSDH in the
genome of the halophilic bacterium Halomonas
halodenitrificans, but not yet characterized.
Since genes coding for HSDHs are often clustered
in bacterial genomes, e.g., the gene coding for
Clostridium absonum 7β-HSDH was found upstream
that coding for a co-expressed 7α-HSDH,[29] the
genome regions contiguous to Dm7α-HSDH and
Hh7α-HSDH genes were checked. No putative
dehydrogenase was found in proximity of the gene
coding for Dm7α-HSDH.
Results and Discussion
Instead, a gene annotated as a SDR family
oxidoreductase (Genbank: WP_027961749.1) was
found upstream the Hh7α-HSDH gene. Interestingly,
Discovery of novel HSDHs
To the best of our knowledge, all the HSDHs
characterized so far belong to the “short-chain
2
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10.1002/adsc.202000120
Advanced Synthesis & Catalysis
this sequence showed only a modest similarity to 7α-
HSDHs (Table 1, entry 3, 28-38% identity at the
deduced amino acid level), and no significative
similarity to other HSDSs, including known 7β-
HSDHs.
Even more curiously, when searching for
homologues of this new sequence into the NCBI
database, we found several uncharacterized SDRs
from diverse sources with up to 79% identity. Three
SDRs, i.e., one from plasmid material of the
Recombinant production and characterization of
novel HSDHs
The codon-optimized synthetic genes coding for the
selected putative HSDHs were cloned into the pETite
expression vector (Lucigen) in frame with a C-term
His-Tag sequence. Protein over-expression in the
corresponding
recombinant
Escherichia
coli
BL21(DE3) strains was achieved by induction with
isopropyl-β-D-thiogalactopyranoside (IPTG) (see
Experimental section for details).
photosynthetic
cyanobacterium
PCC 7437
Stanieria
As shown in Table 1, all the target enzymes could
be produced in recombinant form in E. coli and
homogeneous protein samples were obtained from
the cell extracts with up to >300 mg L-1 yields by
nickel-nitriloacetic acid (Ni-NTA) chromatography,
as confirmed by SDS-PAGE analysis (Fig. S3,
Supporting Information).
The assessment of the catalytic activity of these
novel SDRs toward different bile acids (Scheme 1)
was first carried out by spectrophometric assays in
oxido-reduction reactions in the presence of
NAD(P)(H) cofactors. Subsequently, the respective
HSDH activity was confirmed by setting up small-
scale oxido-reduction reactions with suitable bile
acids (see Experimental section, Fig. S4 and Scheme
S1 in the Supporting Information for details).
As expected from the previously reported sequence
analysis, Dm7α-HSDH (Table 1, entry 1), Hh7α-
HSDH (entry 2), and Ngi1_7α-HSDH (entry 7)
showed NAD(H)-dependent 7α-HSDH activity, thus
demonstrating for the first time the occurrence of
HSDH activities in extremophilic microorganisms.
Interestingly, Dm7α-HSDH (entry 1) showed a
thermophilic behavior by displaying an optimum
temperature at 60°C in the regioselective oxidation of
cholic acid (see Fig. S5 in the Supporting
Information), which is, to our knowledge, the highest
value reported so far for HSDHs. It is also
noteworthy the broad temperature range at which this
enzyme showed activity, since about 15% of the
maximal activity recorded at 60°C was retained at
both 20°C and 80°C. The finding of a thermostable
enzyme in a cold adapted microorganism looks
contradictory at a first glance. However, despite their
psychrophilic origin, the occurrence of thermophilic
and thermostable enzymes in Antarctic microbes has
been previously reported.[42–45] To a lower extent, also
Ngi1_7α-HSDH (entry 7), whose origin, as
previously mentioned, is likely close to psychrophilic
bacteria, showed a moderate thermophilic behavior,
with an optimum temperature at 40°C (see Fig. S5 in
the Supporting Information). Remarkably, Dm7α-
HSDH and Ngi1_7α-HSDH are phylogenetically
related (Fig. 1), and close also to the recently
discovered 7α-HSDH from Stenotrophomonas
maltophilia (Sm7α-HSDH), which showed a marked
thermophilicity as well,[5] thus constituting a distinct
clade from the other (mesophilic) 7α-HSDHs.
cyanosphaera
(Genbank:
WP_015212061.1), one from a Brucella strain
(Genbank: WP_004684107.1), and one from a
Rhodobacter
sphaeroides
strain
(Genbank:
WP_011911126.1) were then selected for further
studies (Table 1, entries 4-6, see also Fig. S2 in the
Supporting Information).
As far as the metagenomes mining concerns, three
sequences were found, two of them from the
metagenomes of samples collected at drilling wells in
the Norwegian continental shelf (Ngi1_7α-HSDH
and Ngi7-SDR, entries 7 and 9, Table 1) and one
from the metagenome obtained from Icelandic hot
spring sediments (Is2-SDR, entry 8, Table 1).
Ngi1_7α-HSDH (entry 7) showed about 70%
similarity with known 7α-HSDHs. By performing a
Blast analysis in the NCBI database, a putative 7α-
HSDH with 99.7% identity in the respect of
Ngi1_7α-HSDH was found in the genome of a
Psychrobacter sp. strain (Genbank: HAR74729.1).
It was more difficult to make hypotheses about the
possible catalytic activity of the other two selected
metagenomic sequences. In fact, Is2-SDR showed a
quite low similarity with respect to both known
HSDHs and sequences in the NCBI database [38%
and 53% identity with the 7α-HSDH from Bosea sp.
and with a putative D-threitol dehydrogenase from an
Aerophobetes strain (Genbank: TKJ47585.1),
respectively]. In the case of Ngi7-SDR, it showed a
35% identity with the 12α-HSDH from Clostridium
sp., and was identical at the amino acidic level to an
uncharacterized SDR family oxidoreductase from a
Pseudomonas pelagia strain (Genbank: QFY56536).
Finally, a sequence from Lysinibacillus (formerly
Bacillus) sphaericus showing about 47% identity
with known 12α-HSDH was discovered and included
in further studies (Ls12α-HSDH, entry 10, Table 1).
In fact, from the same source, a commercially
available NAD(H)-dependent 12α-HSDH (in the past
from Genzyme Biochemicals Ltd., today from
Creative Enzymes®) was widely used in previous
studies,[11,41] but never cloned and produced in
recombinant form.
3
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10.1002/adsc.202000120
Advanced Synthesis & Catalysis
Table 1. Expression yields and activity of the SDRs selected in this work.
Entry Enzyme
Source
Identity with
known HSDHsa)
(%)
HSDH
Cofactor
Yield
Yield Specific
activityb)
activity
(U)b) c) (mg)c)
(U mg-1)
1
Dm7α-HSDH
Deinococcus
marmoris
Halomonas
halodenitrificans (Sh7α-HSDH)
Halomonas 38.89
halodenitrificans (Bs7α-HSDH)
Stanieria
cyanosphaera
Brucella sp.
75.40
(Sm7α-HSDH)
74.51
7α-HSDH
7α-HSDH
7β-HSDH
7β-HSDH
7β-HSDH
7β-HSDH
7α-HSDH
n.a.d)
NAD(H)
NAD(H)
NAD(H)
NAD(H)
NAD(H)
NAD(H)
NAD(H)
NADP(H)
NAD(H)
788
14
64
95
7
56
31
10
38
92
29
142
-
2
Hh7α-HSDH
Hh7β-HSDH
Sc7β-HSDH
Bsp7β-HSDH
Rs7β-HSDH
Ngi1_7α-HSDH
Is2-SDR
1986
955
3
4
38.40
(Gri7α-HSDH)
36.00
(Pa7α-HSDH)
39.68
(Bs7α-HSDH)
69.05
(Sm7α-HSDH)
38.49
(Bsp7α-HSDH)
35.50
(Csp12α-HSDH)
47.39
(El12α-HSDH)
261
5
8400
2348
91
80
6
Rhodobacter
sphaeroides
metagenome
7
24780 174
8
metagenome
metagenome
-
107
326
135
9
Ngi7-SDR
n.a.d)
-
-
10
Ls12α-HSDH
Lysinibacillus
sphaericus
12α-HSDH NAD(H)
348
2.6
a)
For details about known HSDHs (source and Genbank accession numbers), see Table S1 in the Supporting
Information. b) Activity assays were performed in the presence of NAD+ as cofactor and respective substrates: 7α-HSDH,
cholic acid; 12α-HSDH, deoxycholic acid; 7β-HSDH, ursodeoxycholic acid (see Experimental section for details).
c) Yields refer to 1 L-scale enzyme production under optimized conditions (see Experimental section for details). d) n.a.:
not active towards any of the tested bile acids (see Experimental section for details).
The third novel 7α-HSDH, Hh7α-HSDH (entry 2),
showed, in agreement with the phylogenetic analysis
(Fig. 1), a mesophilic behavior (optimal temperature
at 30°C). Moreover, consistently with its origin, it
showed a slight halophilic character. In fact, an about
40% decrease of activity was observed when
removing by dialysis the salts contained in the elution
buffer of the Ni-NTA chromatography.
Coming to the next four sequences (entries 3-6,
Table 1), these enzymes showed unequivocally
NAD(H)-dependent 7β-HSDH activity and were
consequently named Hh7β-HSDH, Sc7β-HSDH,
Bsp7β-HSDH, and Rs7β-HSDH, respectively. As
shown in Fig. 1, these 7β-HSDHs constitute a
separate clade from that including other known 7β-
HSDHs, which are all NADP(H)-dependent enzymes
with the only exclusion of the recently discovered
NAD(H)-dependent 7β-HSDH from Lactobacillus
spicheri (Ls-7β-HSDH).[5] Moreover, the novel 7β-
HSDHs showed a significantly higher specific
activity (up to 92 U mg-1) than Ls-7β-HSDH (3.1 U
mg-1),[5] thus suggesting a highly interesting potential
for further biocatalytic applications in bile acid
modifications.
U mg-1) than that observed after the affinity
chromatography step (10 U mg-1). Interestingly,
Hh7β-HSDH fully recovered its starting activity by
addition of NaCl (1 M final concentration) to the
dialyzed solution.
Sc7β-HSDH (entry 4) showed a rather limited
solubility during the recombinant production with a
detrimental effect on the expression yields, even
under optimized conditions (Table S3, Supporting
Information). However, this enzyme is quite
thermophilic, showing an optimum of temperature at
around 50°C (Figure S4, Supporting Information).
Excellent yields and specific activity were obtained
instead with Bsp7β-HSDH (entry 5) and Rs7β-HSDH
(entry 6), both enzymes showing a mesophilic
character.
As far as the metagenomic sequences Is2-SDR and
Ngi7-SDR concern, both proteins were obtained with
very good yields in soluble form (entries 8 and 9,
Table 1). However, neither of the two showed
catalytic activity toward any of the tested bile acids
(see Experimental section for details). Indeed, both
enzymes
demonstrated
to
be
functional
dehydrogenases in the presence of NADPH (Is2-
SDR) or NADH (Ngi7-SDR) as cofactors and
different ketone substrates (see in the following).
Subsequent characterization of Hh7β-HSDH (entry
3) showed that the presence of salts had a stronger
influence on its activity than that observed with its
clustered enzyme Hh7α-HSDH (entry 2). In fact,
dialyzed samples of Hh7β-HSDH showed about 2
orders of magnitude lower specific activity (0.07-0.16
4
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10.1002/adsc.202000120
Advanced Synthesis & Catalysis
B. fragilis 7α-HSDH, and three bicyclic ketones (3-5)
partially resembling the structural features of steroids.
To allow the use of catalytic amounts of the
cofactors and to provide the necessary driving force
to product formation, the reactions were coupled with
a suitable cofactor regeneration protocol exploiting a
formate/formate dehydrogenase (FDH) system (see
Experimental section and Scheme S2 in Supporting
information).
Reaction conversions were estimated after 24-48 h
by GC-MS (1, 3-5) or by HPLC (2) analysis and
enantiomeric excesses (ees) were evaluated by chiral
GC (1 and 5) or chiral HPLC analysis (2).
For the sake of comparison, compounds 1-5 were
also submitted to the action of the two new identified
SDRs not showing HSDH activity (Is2-SDR and
Ngi7-SDR, lines 8 and 9 of Table 1) and the results
are reported in the Supplementary Materials (Table
S4).
Figure 1. Unrooted phylogenetic tree of HSDHs. 7α-
HSDHs are indicated in red, 7β-HSDHs in blue, 12α-
HSDHs in green. Novel enzymes discovered in this work
are in bold. Thermophilic 7α-HSDHs and the novel
NAD(H)-dependent 7β-HSDHs are in light red and light
blue background, respectively. The tree was generated with
Clustal Omega[46] and visualized with iTOL.[47]
Finally, Ls12α-HSDH (entry 10, Table 1) was
obtained with excellent yield from recombinant
production and confirmed as a NAD(H)-dependent
12α-HSDH, thus included, together with the other
seven novel HSDHs, in the following study of the
substrate promiscuity of this group of enzymes.
Substrate scope of HSDHs
The demonstration of the catalytic activity of the
eight novel HSDHs in oxidoreduction reactions of
different bile acids paves the way to their exploitation
as biocatalysts for the stereo- and regioselective
preparation of steroid derivatives. Our group is
currently performing specific investigations on this
topic and the results will be reported in due time.
Additionally, we considered of synthetic interest a
more detailed evaluation of the substrate promiscuity
of this group of enzymes. Accordingly, the newly
discovered 7α-, 7β-, and 12α-HSDHs were tested,
along with our in-house collection of previously
characterized HSDHs (Table 2, for details see also
Table S1, Supporting Information), in the
stereoselective reduction of a panel of substrates (1-5)
(Scheme 2).
Scheme 2. Substrates 1-5 and their possible reduction
products.
Regarding the α-ketoesters 1-2, nearly all the
tested HSDHs showed a good activity towards these
substrates, yielding the corresponding reduced α-
hydroxyesters, with high conversions and high
enantiomeric excesses (Table 2).
Specifically, quantitative conversions of 1 were
obtained with Ca7α-HSDH, Dm7α-HSDH, Ca7β-
HSDH, Cae7β-HSDH and Bsp7β-HSDH (Table 2,
Specifically, the tested substrates included two α-
ketoesters (1-2) of pharmaceutical interest,[48] chosen
among the panel of ketoesters that, as previously
discussed,[20] were selected by Zhu and co-workers to
investigate the catalytic promiscuous properties of the
5
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10.1002/adsc.202000120
Advanced Synthesis & Catalysis
entries 2, 3, 6, 7, and 10), while complete conversions
of 2 were obtained with Dm7α-HSDH, Ca7β-HSDH,
Cae7β-HSDH, Bsp7β-HSDH, Rs7β-HSDH and
Ls12α-HSDH (Table 2, entries 3, 6, 7, 10, 11 and 13).
On the other hand, the most enantioselective
HSDHs in the reduction of 1 were Ngi1_7α-HSDH,
Rs7β-HSDH and Csp12α-HSDH (Table 2, entries 5,
11, and 12), while Ec7α-HSDH and Hh7α-HSDH
(Table 2, entries 1 and 4) showed the best
enantioselectivity in the reduction of 2.
Interestingly, the stereoselectivity in the reduction
of 1 was significantly lower than that observed with 2
when testing the NADP(H)-dependent Ca7β-HSDH
and Cae7β-HSDH (entries 6 and 7, respectively), and,
to a minor extent, the novel Ls12α-HSDH enzyme
(entry 13). These results suggest that in these selected
HSDHs the aromatic substituent in 1 may promote
alternative substrate binding modes in either pro-R or
pro-S fashion, whereas in the case of the aliphatic α-
keto ester 2 only the pro-R binding mode is
productive.
It is also worth of mentioning that reactions
catalyzed by Hh7α-HSDH and Hh7β-HSDH (Table 2,
entries 4 and 9) were carried out in parallel in the
presence and in the absence of NaCl (0.4 M). As
expected, conversions were very low in the absence
of NaCl (<5%, data not shown), while they were up
to about 98% in the presence of salts, thus confirming
the halophile behavior of these novel HSDHs.
The thermophilic Dm7α-HSDH (Table 2, entry 3)
demonstrated to perform well also at sub-optimal
temperatures, giving complete conversions of 1 and 2
at 25°C.
being consistent with the value reported in literature
for 2a.[49] Thus, also in the case of the reduction of 2,
all the HSDHs demonstrated to be (R)-selective.
Table 2. Conversions and enantioselectivity of HSDH-
catalyzed reduction of substrates 1 and 2.
Entry Enzymea)
Compound 1 Compound 2
b)
b)
cb)
(%)
eeR
(%)
cb)
(%)
eeR
(%)
1
Ec7α-HSDH
89.7 90.2 81.6 >99
2
3
4
5
Ca7α-HSDH
Dm7α-HSDH
Hh7α-HSDH
>99
>99
88.3 42.3 91.1
82.9 >99 93.9
95.9 97.9 81.0 >99
Ngi1_7αHSDH 26.1 >99 64.9 92.4
6
7
8
9
10
11
12
13
Ca7β-HSDH
Cae7β-HSDH
Sc7β-HSDH
Hh7β-HSDH
Bsp7β-HSDH
Rs7β-HSDH
>99
>99
45.2 >99
23.3 >99
89.3
97.2
61.7 92.5 92.0 94.6
97.4 95.2 65.8 64.9
>99
89.6 >99
>99
96.0
95.5
75.4 >99
Csp12α-HSDH 94.5 >99
Ls12α-HSDH
93.9 85.7
93.6
94.1 64.7 >99
a)
Sources of previously characterized HSDHs: Ec7α-
HSDH (entry 1): Escherichia coli; Ca7α-HSDH (entry 2)
and Ca7β-HSDH (entry 6): Clostridium absonum; Cae7β-
HSDH (entry 7): Collinsella aerofaciens; Csp12α-HSDH
(entry 12): Clostridium sp. (see Table S1, Supporting
b)
Information, for details).
Conversions and ees were
assigned via chiral HPLC for reduction of 1, while chiral
GC was used for reduction of 2 (see Experimental section
for details).
The stereochemistry of the products obtained from
the enzymatic reduction of 1 (the enantiomers 1a or
1b) was determined via analytical comparison with
standards. All the HSDHs showed to be (R)-selective,
compound 1a being the preferred product. Instead, in
order to assign the absolute configurations of the
products obtained by the reduction of 2 (the
enantiomers 2a or 2b), the reaction catalyzed by 7β-
HSDH from Collinsella aerofaciens (Cae7β-HSDH,
Table 2, entry 7), which combined quantitative
conversion of 2 and high enantioselectivity, was
scaled up (100 mg). Optical rotation measurements
were carried out on the purified product (63%
Concerning the reduction of the bicyclic substrates
3-5, none of the tested enzymes showed activity
towards the tetralones 3 and 4, whereas only Ca7β-
HSDH and Cae7β-HSDH were found able to catalyze
the reduction of (±)-trans-1-decalone ((±)-5). These
two enzymes, working under unoptimized reaction
conditions, gave moderate conversions (18% and
35% after 48 h for Ca7β-HSDH and Cae7β-HSDH,
respectively), but accompanied by interesting
stereoselectivity (Scheme 3).
22
isolated yield), the resulting [α]D value (-2.70°)
6
This article is protected by copyright. All rights reserved.

10.1002/adsc.202000120
Advanced Synthesis & Catalysis
Considering that commercially available 5 is a
isolated and analyzed by NMR spectroscopy. In this
way, it was possible to assign the relative
stereochemistry of the diastereomers 5a and 5b (see
Supporting Information for further details).Moreover,
the four isomeric products (5a1, 5a2, 5b1, 5b2) could
be separated by chiral GC.
racemate of the enantiomers (+)-5 (4aS,8aR) and (-)-5
(4aR,8aS), its chemical reduction can yield four
different stereoisomers, i.e., 5a1 (1S,4aS,8aR), 5a2
(1R,4aR,8aS), 5b1
(1R,4aS,8aR),
and 5b2
(1S,4aR,8aS) (Scheme 2). The four isomers were
prepared by chemical reduction of 5 with NaBH4,
Scheme 3. Enzymatic reductions of substrate (±)-5 with A) Ca7β-HSDH and B) Cae7β-HSDH.
The same analysis of the samples obtained from
Table 3.Conversion and enantioselectivity of reduction of
the two enzymatic reductions indicated that Ca7β-
HSDH catalyzed the formation of one enantiomer of
each diastereomer (Scheme 3A, see also Scheme S3
in the Supporting Information for details). On the
substrate (±)-5 catalyzed by Cae7β-HSDH and Ca7β-
HSDH.
Enzyme
ca)
eea)
contrary, Cae7β-HSDH performed
a
kinetic
residual
product product
resolution of the starting ketone. The unreacted
substrate 5 showed an ee of 72%, whereas the
product 5b showed an ee of 87% (Scheme 3B).
Therefore, Ca7β-HSDH and Cae7β-HSDH showed a
different stereopreference for the enantiomers of (±)-
5.
substrate
5b
5a
(%) (%)
(%)
(%)
Cae7β-HSDH 35
72 (4aS,8aR) 87b)
9 (4aR,8aS)
-
Ca7β-HSDH
18
> 99b)
> 99c)
a) Conversions and ees were assigned via chiral GC (see
b)
To assign the absolute configurations of the
residual substrate and of the formed products, the
reduction of (±)-5 catalyzed by Cae7β-HSDH was
scaled up to 100 mg.
Experimental section for details).
stereochemistry: (1S,4aR,8aS).
stereochemistry: (1S,4aS,8aR).
Major product
Major product
c)
The remaining substrate and the product (5b, 15%
isolated yield; product 5a was present in traces and
therefore it was not isolated) were purified via flash
chromatography, then submitted to NMR analysis in
order to identify the relative stereochemistry and to
optical rotation measurements to assess the absolute
configurations (see Supporting Information for
further details). The resulting [α]D25 values (+44.45°
and +2,75° for produced 5b and residual 5,
respectively) were compared with the data reported in
literature,[50] indicating that the residual ketone was
enriched in the enantiomer (+)-5 and the product was
enriched in the enantiomer 5b2 (1S,4aR,8aS). These
data are summarized in Table 3.
Our previous investigations on the 3D structure of
Cae7β-HSDH[51] showed that its substrate binding
site, including
a
mobile substrate loop, is
substantially different from that of other HSDHs, e.g.,
E. coli 7α-HSDH. Although the overall active site
architecture results quite similar in this family of
oxidoreductases, it is likely that substrate recognition
and, consequently, stereoselectivity, are ruled by a
network of very subtle interactions between the
substrate and the active site residues. Further
investigations in this respect are currently under
considerations in our lab.
7
This article is protected by copyright. All rights reserved.

10.1002/adsc.202000120
Advanced Synthesis & Catalysis
H2SO4(50% v/v in H2O, 40 mL), MeOH (400 mL)]. The
isolation of pure products was allowed via extraction with
EtOAc and subsequent flash chromatography on silica gel
60 (70-320 mesh, Merck, eluent CH2Cl2).
Conclusion
As far as the mining of (meta)genomes for novel
HSDHs is concerned, the results obtained in the first
part of this work show that the occurrence of redox
enzymes acting on steroids largely exceeds the range
of microbes usually considered as possible HSDHs
sources so far, i.e., gut and soil bacteria. In fact,
functionally active HSDHs have been found for the
first time in extremophilic bacteria as well as in
photosynthetic cyanobacteria. This finding poses
several interesting questions that were clearly out of
the scope of this investigation, e.g., about the
physiological role of these HSDHs in these microbes,
as well as about how these enzymatic functions were
acquired or evolved from common ancestral genes.
However, since extremophilic enzymes are very often
more robust than their mesophilic counterparts,[52] the
discovery of these novel HSDHs could also be useful
in the development of industrial biocatalytic
processes for the stereo- and regioselective
modification of steroid derivatives. We are currently
working on this specific topic, as well as on the
synthetic exploitation of the novel and highly active
NAD(H)-dependent 7β-HSDHs for the preparation of
bile acid derivatives.
Coming to the study of the activity of HSDHs
toward non-steroidal substrates, we have confirmed
that enzymes evolved to accommodate structurally
complex and bulky substrates in their active sites,
maintain their selectivity towards much simpler
molecules, as observed with compounds 1, 2 and 5.
These results corroborate the few scant previous
literature reports, point out the importance of having
in hands libraries of enzymes and encourage us to
continue these studies in order both to acquire more
information on the general substrate scope of HSDHs
and to exploit molecular modeling approaches[53] to
find suitable rationales for the observed selectivity.
Environmental sample collection, DNA extraction from
samples, DNA sequencing, and generation of databases of
metagenomic sequences were carried out as previously
described.[54] The metagenomic sample “ngi” was collected
in drilling wells in Svalbard island and DNA extraction
and sequencing from this sample was performed as
described previously for sample It3.[54]
Bacterial strains.
E. coli BL21(DE3) and E. coli HI-Control 10G were from
Lucigen (Wisconsin, USA).
E. coli BL21(DE3)/pET24b-Ec7αHSDH producing the 7α-
HSDH from E. coli, E. coli BL21(DE3)/pET24b-
Cae7βHSDH producing the 7β-HSDH from Collinsella
aerofaciens, E. coli BL21(DE3)/pETite-Ca7βHSDH
producing the 7β-HSDH from Clostridium absonum, E.
coli TOP10/pBAD-Ca7αHSDH producing the 7α-HSDH
from Clostridium absonum were part of our in-house
collection and were expressed as fusion proteins with a
(6x)His-tag at the C-terminus.
Analytical methods
At scheduled times, reaction samples (50 µl) were
extracted with EtOAc and dried over Na2SO4 to obtain a 10
mM final concentration sample suitable to chiral GC
(and/or GC-MS) analysis, or evaporated, resuspended in
CH3CN to 10 mM final concentration and analyzed by
chiral RP-HPLC.
GC-MS analyses were performed using an Agilent HP-
5MS column (30 m × 0.25 mm × 0.25 μm) on a Finnigan
TRACE DSQ GC/MS instrument (ThermoQuest, San Jose,
CA). Elution conditions: 60°C, 1 min; +10°C min-1 until
300°C; hold 1 min; flow rate: 1.0 mL min-1; inlet
temperature: 250°C; ion source temperature: 250°C; MS
transfer line temperature: 250°C. Retention times: (1): 9.53
min; (1a, 1b): 9.38 min; ((-)-5 and(+)-5): 9.57 min; (5b1,
5b2): 9.25 min.
Chiral GC analyses were carried out on an Agilent
Technologies 6850 Network GC system gas
chromatograph equipped with split/splitless injector, FID
detector and MEGA-DEX DAC Beta chiral capillary
column (25 m x 0.25 mm x 0.25 μm). Elution conditions
for 2 and 5: 80°C, 1 min; + 1°C min-1 until 90°C; +10°C
min-1 until 180°C; hold 5 min. Retention times: (2): 9.1
min; (2a, 2b): 7.9 min, 8.8 min ((-)-5 and(+)-5,
respectively): 17.5 min, 17.6 min; (5b1, 5b2): 17.7 min,
18.6 min. Elution conditions for 3 and 4: 120°C, 1 min; +
5°C min-1 until 180°C; +10°C min-1 until 200°C; hold 5
min. Retention times: (3) 8.8 min; (3a, 3b): 9.1 min, 9.3
min; (4) 10.5 min; (4a, 4b): 10.0 min. Flow rate: 1.5 mL
min-1; detector temperature 200°C; inlet temperature
250°C.
Experimental Section
General
Methyl benzoylformate (purity 98%), ethyl 3-methyl-2-
oxobutyrate (purity 97%), 1-tetralone (purity 97%), 2-
tetralone (purity 98%) and (±)-trans-1-decalone (purity
98%) were purchased from Merck (Darmstadt, Germany)
(catalog numbers M30507, 218456, T19003, T19208 and
156655, respectively) and used with no further purification.
The analytical standards methyl D-mandelate (purity≥99%,
[α]D20=144°±2, c=2 in MeOH) and methyl L-mandelate
(purity>99%) were from Fluka Chemika (catalog numbers
63456 and 63466, respectively). Formate dehydrogenase
(FDH) from Candida boidinii and lactate dehydrogenase
(LDH) from rabbit muscle were from Merck. The
NADP(H) dependent FDH from methylotrophic bacterium
Pseudomonas sp. 101 was a kind gift from Prof. Tishkov
(M.V. Lomonosov Moscow State University). Unless
otherwise stated, all other chemicals were of analytical
grade and were purchased from Merck.
Chiral HPLC analyses were performed on a Shimadzu LC-
20AD high performance liquid chromatography system
equipped with a Shimadzu SPD-20A UV detector and a
Phenomenex Lux Cellulose-1 5µ chiral column. HPLC
conditions: injection volume 10 µL; mobile phase H2O +
0.1% trifluoroacetic acid : CH3CN = 75 : 25 (isocratic
elution); flow rate: 1.0 mL min-1; detection 254 nm;
temperature 30°C. Retention times: (1): 4.0 min; (1b): 5.5
min; (1a): 7.0 min. Prior to performing HPLC analyses, the
molar extinction coefficients of 1 and 1a, 1b were
determined (see Supporting information for details), to be
able to assess conversions while considering the different
molar absorbivity of substrate and products.
Reactions were monitored via TLC (thin-layer
chromatography) on pre-coated glass plates silica gel 60
with fluorescent indicator UV254 and treated with an
oxidizing solution [4-hydroxybenzaldehyde (6.3 g),
8
This article is protected by copyright. All rights reserved.

10.1002/adsc.202000120
Advanced Synthesis & Catalysis
The NMR spectra (1H and 13C) were acquired in CDCl3 or
in DMSO-d6 at rt on a Bruker AV 400 MHz spectrometer
Csp12α-HSDH cloning into the pETite N-His Kan vector
in frame with N-term His-Tag sequence was carried out by
gene amplification with primers F10/R10 (Table S2,
Supporting Information), using the PCR protocol described
above. The amplified sequence was subsequently cloned in
the pETite N-His Kan vector using the same kit described
for the cloning in the pETite C-His Kan plasmid.
1
with a z gradient at 400 MHz for H-NMR analysis and
101 MHz for 13C-NMR.
ESI-MS spectra were recorded on a Bruker Esquire 3000
PLUS instrument (ESI Ion Trap LC/MSn System),
equipped with an ESI source and a quadrupole ion trap
detector (QIT). The samples were dissolved in methanol to
10-2 g L-1 and then directly syringed in the ESI-MS at 4 μL
min-1 rate. The analyses were performed in positive mode.
The acquisition parameters were optimized as such: 4.5 kV
needle voltage, 10 L h-1 N2 flow rate, 40 V cone voltage,
trap drive set to 46, 115.8 V capillary exit, 13000 (m/z) s-1
scan resolution over the 35-900 m/z mass/charge range,
source temperature 250°C.
The resulting plasmids were purified from E. coli HI-
Control 10G cells using the E.Z.N.A. Plasmid Mini kit II
(Omega/VWR) and plasmid inserts were sequenced on
both strands by Biofab Research (Rome, Italy) using
primers T7 promoter and pETite reverse (Table S2,
Supporting Information). Recombinant pETite plasmids
were finally transformed in E. coli BL21(DE3) chemically
competent cells (Lucigen) for the expression of the
corresponding genes.
Optical rotations were measured on a Jasco P-2000
polarimeter. The specific rotation was calculated as the
HSDH/SDR and FDH expression and purification
T
[α]λ =α100/cd, where α represents the recorded optical
rotation, c the analyte concentration (mg mL-1), d the
cuvette length (dm). As such, the specific rotation is
expressed as (10-1 deg cm-2 g-1). λ is reported in nm and T
in °C. λ usually corresponds to sodium D line (589 nm),
thus the optical rotation is referred to as [α]D. T, c and the
solvent were chosen according to references reported in
literature: c = 1 in CHCl3 at 22°C for 2; c = 0.5 in EtOH at
25°C for 5; c = 0.75 in CHCl3 at 25°C for 5a and 5b.[49,50]
Recombinant E. coli strains from our in-house collection
(see bacterial strain section) and recombinant E. coli
BL21(DE3) strains prepared as described above, were
inoculated overnight in LB medium supplemented with the
opportune antibiotic (Table S3, Supporting Information)
(100 mL) and grown at 37°C, 220 rpm. 25 mL of
precultures were subsequently inoculated in 500 mL of LB
medium containing the corresponding antibiotic and
incubated at 37°C, 220 rpm till the OD600 cell density
reached 0.5–1. Gene expression was induced by the
addition of IPTG (for IPTG final concentrations, see Table
S3, Supporting Information) and the culture was
transferred to 17-37°C (see Table S3, Supporting
Information) with shaking at 220 rpm and grown for 4-72h
(see Table S3, Supporting Information). In case of E. coli
TOP10/pBAD-Ca7αHSDH, precultures were inoculated in
TB medium containing rhamnose as protein expression
inducer.
In silico screening for novel HSDHs and bioinformatic
analysis
Bioinformatic search for new HSDHs was performed by
aligning query sequences (entry 1, 2, 4, 14-21, 33 Table S1
Supporting Information) with database metagenomic
sequences using the program LAST (http://last.cbrc.jp/)
with default settings[55] or with GenBank database
sequences
using
standard
protein
Blast
tool
(https://blast.ncbi.nlm.nih.gov/).
After recovery by centrifugation (5000 rpm, 30 min, 4°C),
cell pellet was resuspended in 20 mL wash buffer (20 mM
potassium phosphate (KP) buffer, pH 7.0, 500 mM NaCl,
20 mM imidazole) and cells were disrupted by sonication.
Soluble protein fraction was separated from the cell debris
by centrifugation (11,000 rpm, 30 min) and clear lysates
were assessed for the presence of soluble protein by SDS-
PAGE (12% T, 2.6% C).
Phylogenetic trees were created using the Clustal Omega
tool (http://www.ebi.ac.uk/Tools/msa/clustalo/)[46] and
visualized with iTOL webserver (http://itol.embl.de/).[47]
Gene cloning and generation of recombinant bacterial
strains
The codon-optimized genes coding for putative HSDHs
(Table S1, Supporting Information, entries 11-13, 26-29,
31, 43, and 44,) and for Csp12α-HSDH (entry 33) were
synthesized and cloned into the pUC57 vector by
BaseClear (Leiden, The Netherlands).
For protein purification, Ni Sepharose 6 Fast Flow agarose
resin (Ni-NTA) (GE Healthcare, Italy) was incubated with
clear cell lysates containing soluble protein for 90 min at
4 °C under mild shaking. The mixture was then loaded
onto a glass column (10 × 110 mm) and the resin was
washed with 20 mL of wash buffer (20 mM imidazole, 500
mM NaCl, 20 mM KP buffer). His tagged proteins were
eluted using a 3-step gradient (10 ml washing buffer with
100, 200, and 300 mM imidazole, respectively) and, if not
stated otherwise, dialyzed against 5 L of a suitable buffer
(Table S3, Supporting Information), at 4°C for 16 h and
stored at −80°C. Protein content was measured using the
Bio-Rad Protein Assay according to the method of
Bradford and protein purity was verified by SDS-PAGE
analysis (12% T, 2.6% C). The molecular weight protein
standard mixture from Bio-Rad (Karlsruhe, Germany) was
used as reference. Gels were stained for protein detection
with Coomassie Brilliant Blue.
Putative HSDH genes were amplified by PCR using
primers reported in Table S2 (Supporting Information), for
the subsequent cloning in the pETite C-His Kan vector in
frame with C-term His Tag sequence. PCR amplifications
were carried out in 50 µl reaction mixtures containing 20
ng of pUC57 vector including the desired gene, primers (1
µM each), dNTPs (0.2 mM each), 2 U of Xtra Taq
polymerase and 5 µl of buffer containing MgCl2. All PCR
reagents were from Genespin (Milan, Italy). PCR
conditions were as follows: 95 °C for 2 min, followed by
35 cycles at 94 °C for 30 s, primers Tm -5°C for 30 s, 72 °C
for 1 min, and then 72 °C for 10 min. PCR products were
then purified from agarose gel (0.7% (w/v)) using the
Wizard® SV Gel and PCR Clean-Up System (Promega,
Wisconsin, USA) before cloning. Purified sequences were
subsequently cloned in the pETite C-His Kan plasmid
using the Expresso T7 Cloning and Expression kit from
Lucigen (Wisconsin, USA). Specifically, purified
sequences were mixed with the pETite C-his linear
plasmid and transformed in chemically competent E. coli
HI-Control 10G cells following the manufacturer's
instructions.
Activity assays
Dehydrogenase activity of HSDHs and FDHs was
determined spectrophotometrically by measuring the
reduction of NAD(P)+ at 340 nm (ε: 6.22 mM-1cm-1) in the
presence of the opportune substrate. If not stated otherwise,
cholic acid was used for 7α- and 12α-HSDHs,
ursodeoxycholic acid was used for 7β-HSDHs, and
ammonium formate was used for FDHs.
Specifically, assays were carried out in polyethylene
cuvettes at 25°C by adding the opportune purified
9
This article is protected by copyright. All rights reserved.

10.1002/adsc.202000120
Advanced Synthesis & Catalysis
dehydrogenase (1-20 µL) to the following assay mixtures
(1 mL final volume):
4a+4b: 1H NMR (400 MHz, CDCl3) δ 7.24 – 7.02 (m, 4H),
4.30 – 3.98 (m, 1H), 3.12 (dd, J = 15.9, 4.5 Hz, 1H), 3.07 –
3.06 (m, 1H), 2.99 (dt, J = 17.0, 5.8 Hz, 1H), 2.92 – 2.84
(m, 1H), 2.80 (dd, J = 16.3, 7.9 Hz, 1H), 2.15 – 2.04 (m,
1H), 1.92 – 1.79 (m, 1H).
HSDH assay: 2.5 mM substrate; 50 mM potassium
phosphate buffer at pH 9.0; 0.20 mM NAD(P)+.
FDH assay: 20 mM substrate; 50 mM potassium phosphate
buffer at pH 7.0; 0.20 mM NAD(P)+.
5a1+5a2: 1H NMR (400 MHz, CDCl3) δ 3.76 (q, J = 2.5
Hz, 1H), 1.92 – 1.74 (m, 2H), 1.74 – 1.55 (m, 4H), 1.56 –
1.43 (m, 3H), 1.44 – 1.16 (m, 5H), 1.15 – 0.85 (m, 3H).
One unit of activity is defined as the enzyme activity that
reduces 1 µmol of NAD(P)+ per min under the assay
conditions described above.
HSDHs optimal temperatures were determined by heating
the assay solution in cuvettes in a water bath in the range
20–90 °C for 10 min before adding the enzyme. Results
were compared to blanks.
1
5b1+5b2: H NMR (400 MHz, CDCl3) δ 3.20 (ddd, J =
10.2, 9.1, 4.3 Hz, 1H), 2.18 – 2.07 (m, J = 6.1, 3.0 Hz, 1H),
2.04 – 1.91 (m, 1H), 1.87 – 1.59 (m, 4H), 1.61 – 1.17 (m,
7H), 1.12 – 0.81 (m, 5H).
Small-scale biotransformations of bile acids
Enzymatic reduction of substrates 1-5 catalyzed by
HSDHs
Oxidation of cholic Acid to 3α,12α-dihydroxy-7-oxo-5β-
cholanoic acid or to 3α,7α-dihydroxy-12-oxo-5β-cholanoic
acid. Cholic acid oxidation reactions were coupled with a
pyruvate/LDH system to regenerate NAD+. Specifically,
reactions were carried out in a 1 mL solution containing 50
mM potassium phosphate buffer, pH 8.0, 10 mM cholic
acid, 50 mM sodium pyruvate, 0.4 mM NAD+, 0.5 U LDH
from rabbit muscle, 2 U of opportune 7α-HSDH (entry 11-
13, Table S1, Supporting Information) to obtain 3α,12α-
dihydroxy-7-oxo-5β-cholanoic acid (Scheme S1, A) or
Ls12α-HSDH to obtain 3α,7α-dihydroxy-12-oxo-5β-
cholanoic acid (Scheme S1, B) at 25°C for 24 h.
Reactions catalyzed by HSDHs were coupled with a
formate/FDH system to regenerate NAD(P)H. For the
initial activity screening the general reaction protocol was
as follows: 76 mM NH4HCO2; 0.2 U mL-1 FDH; 0.4 mM
NAD(P)+; HSDH, 3.4 U mL-1; 12.5 mM substrate; 5% v/v
DMSO; 50 mM potassium phosphate buffer, pH 7.0 (total
volume: 1 mL). Reaction catalyzed by Hh7α-HSDH and
Hh7β-HSDH were performed in the presence and in the
absence of 0.4M NaCl. The mixtures were shaken at 25°C
and 100 rpm for 24 to 48 h and monitored over time via
TLC (eluent CH2Cl2). Reaction conversions and
enantiomeric excesses were evaluated by GC-MS, chiral
GC or chiral HPLC analyses. The absolute configurations
were assigned via analytical comparison with standards
(products 1a and 1b) or via optical rotation measurements
and confrontation with values reported in literature
(products 2a and 2b, (+)-5 and (-)-5, 5b1 and 5b2).[49,50]
Reaction progress was monitored by TLC using
chloroform-methanol-acetic acid, 10:1:0.08, as eluting
system.
Reduction of 3α,12α-dihydroxy-7-oxo-5β-cholanoic acid to
ursocholic acid. 3α,12α-diHydroxy-7-oxo-5β-cholanoic
acid reduction was coupled with a formate/FDH system to
regenerate NADH. Specifically, reactions were performed
in a 1 mL solution containing 50 mM potassium phosphate
buffer, pH 8.0, 10 mM cholic acid, 50 mM NH4HCO2, 0.4
mM NADH, 0.5 U FDH from Candida boidinii, 2 U of
opportune 7β-HSDH (entry 26-29, Table S1, Supporting
Information) (Scheme S1, C) at 25°C for 24 h.
Reaction progress was monitored by TLC using the same
eluting system described for cholic acid oxidation
(chloroform-methanol-acetic acid, 10:1:0.08), as eluting
system.
Reactions on substrates 2 and 5 catalyzed by Cae7β-HSDH
were subsequently scaled up to semi-preparative scale (100
mg, 0.7 mmol in 50 mL total volume) following the
protocol described above. The isolated products were
1
characterized via H- and 13C-NMR, ESI-MS, chiral GC
25
and [α]D (see Supporting information, and analytical
methods).
2a+2b: 1H NMR (400 MHz, DMSO) δ 5.05 (d, J = 5.9 Hz,
1H), 4.04 – 3.89 (m, 2H), 3.64 (t, J = 5.4 Hz, 1H), 1.83 –
1.70 (m, 1H), 1.05 (t, J = 7.1 Hz, 3H), 0.74 (d, J = 6.9 Hz,
3H), 0.69 (d, J = 6.8 Hz, 3H); 13C NMR (101 MHz,
CDCl3) δ 174.90, 77.35, 77.04, 76.72, 74.99, 61.48, 41.00,
32.14, 18.74, 15.97, 14.23; [M+Na]+: 167.1; conversion:
99.9%; isolated yield: 63.2%; ee (chiral GC): 97.1%;
[α]D22: -2.70°.
Preparation of standard racemate mixtures
The reductions of substrates 1-5 were performed following
a standard protocol of reduction with NaBH4.[56] To a
stirred solution of 0.13 M substrate (1 eq, 100 mg) in
MeOH (5 mL) at 0°C, NaBH4 (1 eq, 13 mg) was added.
When the reaction mixture became clear, it was brought to
rt and stirred for 2-4 h. The reaction was quenched with a
saturated aqueous solution of NH4Cl, then it was extracted
with EtOAc (3x). The organic phase was dried over
Na2SO4, filtrated and the solvent was evaporated under
reduced pressure to yield the desired products (1a+1b,
2a+2b, 3a+3b, 4a+4b, 5a1+5a2+5b1+5b2) in quantitative
yields. The products were characterized via 1H-NMR
analysis and chiral GC or HPLC analyses (see Supporting
information, and analytical methods).
5: 1H NMR (400 MHz, CDCl3) δ 2.41 – 2.25 (m, J = 13.5,
5.0, 1.8 Hz, 2H), 2.10 – 2.00 (m, J = 16.3, 5.9, 2.8 Hz, 1H),
2.00 – 1.85 (m, 2H), 1.85 – 1.59 (m, 5H), 1.49 – 1.30 (m,
2H), 1.30 – 1.09 (m, 4H); 13C NMR (101 MHz, CDCl3) δ
212.72, 77.35, 77.03, 76.71, 55.08, 44.96, 41.80, 34.38,
33.04, 26.48, 25.75, 25.43, 25.10; [M+Na]+: 175.1; ee
(chiral GC): 62.2%; [α]D25: +2,75°.
1
5b1+5b2: H NMR (400 MHz, CDCl3) δ 3.29 – 3.11 (m,
1H), 2.23 – 2.06 (m, J = 6.0, 3.0 Hz, 1H), 2.02 – 1.91 (m,
1H), 1.84 – 1.58 (m, 4H), 1.58 – 1.43 (m, 2H), 1.41 – 1.13
1a+1b: 1H NMR (400 MHz, CDCl3) δ 7.49 – 7.30 (m, 1H), (m, 4H), 1.11 – 0.77 (m, 5H); 13C NMR (101 MHz,
5.20 (s, 1H), 3.79 (s, 1H).
CDCl3) δ 77.33, 77.01, 76.70, 75.04, 50.46, 41.13, 35.82,
33.59, 33.43, 29.00, 26.34, 26.15, 24.03; [M+Na]+: 177.1;
conversion (GC-MS): 35.8%; isolated yield: 15%; ee
(chiral GC): 89.1%; [α]D25: +44.45°.
2a+2b: 1H NMR (400 MHz, CDCl3) δ 4.42 – 4.15 (m, 1H),
4.04 (s, 1H), 2.72 (s, 1H), 2.19 – 2.00 (m, J = 13.7, 6.9, 3.5
Hz, 1H), 1.33 (t, J = 7.1 Hz, 1H), 1.05 (d, J = 6.9 Hz, 1H),
0.89 (d, J = 6.9 Hz, 1H).
Acknowledgements
3a+3b: 1H NMR (400 MHz, CDCl3) δ 7.48 – 7.41 (m, 1H),
7.26 – 7.18 (m, 2H), 7.15 – 7.08 (m, 1H), 4.88 – 4.72 (m,
1H), 3.04 – 2.52 (m, 2H), 2.21 – 1.74 (m, 4H).
We would like to kindly acknowledge Prof. Vladimir I. Tishkov
and Dr. Anastasia A. Pometun (M.V. Lomonosov Moscow State
University) for their support in the preparation of the
10
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10.1002/adsc.202000120
Advanced Synthesis & Catalysis
Pseudomonas sp. FDH. We warmly thank also Laura Cucinotta
for valuable technical assistance in enzyme production and
characterization and Walter Panzeri (SCITEC-CNR) for
analytical support.
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Insights into the Substrate Promiscuity of Novel
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Adv. Synth. Catal. Year, Volume, Page – Page
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