Biotechnol Lett (2021) 43:1063–1073

https://doi.org/10.1007/s10529-021-03097-y (0123456789().,-volV)
( 01234567
89().,-volV)

ORIGINAL RESEARCH PAPER

Characterization of substrate specificity and inhibitory

mechanism of bile salt hydrolase from Lactobacillus reuteri
CRL 1098 using molecular docking analysis
Ana Estela Ledesma . Marı́a Pı́a Taranto . Ana Yanina Bustos

Received: 25 April 2020 / Accepted: 3 February 2021 / Published online: 16 February 2021
Ó The Author(s), under exclusive licence to Springer Nature B.V. part of Springer Nature 2021

Abstract with glyco- and tauro-conjugated BAs, respectively.

Objectives To elucidate the molecular mechanisms
involved in the substrate interaction of the bile salt
hydrolase of Lactobacillus reuteri CRL 1098 (LrBSH)
with bile acids (BAs) and to evaluate potential enzyme
inhibitors based on computer and in vitro modeling
assays.
Results Asp19, Asn79, and Asn171 participated in
the LrBSH interaction with all BAs tested while Leu56
and Glu 222 played an important role in the interaction
Supplementary Information The online version contains
supplementary material available at https://doi.org/10.1007/
s10529-021-03097-y.
A great percentage of hydrophobic and polar interac-
tions were responsible for the binding of LrBSH with
glyco- and tauro-conjugated BAs, respectively.
Remarkably, the four binding pocket loops partici-
pated in the substrate binding site of LrBSH unlike
most of the reported BSHs. Inhibition assays showed
that ascorbic acid, citric acid, penicillin G, and
ciprofloxacin decreased LrBSH activity by 47.1%,
40.14%, 28.8%, and 9%, respectively. Docking anal-
ysis revealed that tetracycline and caffeic acid
phenethyl ester had the low binding energy (-7.32
and -7.19 kcal/mol, respectively) and resembled the

A. E. Ledesma
Departamento Académico de Quı́mica, Facultad de
Ciencias Exactas y Tecnologı́as, Centro de Investigación
en Biofı́sica Aplicada y Alimentos (CIBAAL-UNSE-
CONICET), Universidad Nacional de Santiago del Estero,
Av. Belgrano Sur 1912, 4200 Santiago del Estero,
Argentina
M. P. Taranto
Centro de Referencia para Lactobacilos (CERELA-
CONICET), Chacabuco 145, 4000 San Miguel de
Tucumán, Argentina
A. Y. Bustos
Facultad de Agronomı́a y Agroindustrias (FAyA),
Universidad Nacional de Santiago del Estero, Av.
Belgrano Sur 1912, 4200 Santiago del Estero, Argentina
A. Y. Bustos
Facultad de Humanidades, Ciencias Sociales y de la Salud
(FHU), Universidad Nacional de Santiago del Estero, Av.
Belgrano Sur 1912, 4200 Santiago del Estero, Argentina

A. Y. Bustos (&)
Centro de Investigación en Biofı́sica Aplicada y
Alimentos (CIBAAL-UNSE-CONICET), RN 9- Km
1125, 4206 Santiago del Estero, Argentina
e-mail: abustos@uspt.edu.ar

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interaction pattern of GDCA (-6.88 kcal/mol) while
penicillin (-6.25 kcal/mol) and ascorbic acid
(-5.98 kcal/mol) interacted at a longer distance.
Conclusion This study helps to delve into the
molecular mechanisms involved in the recognition
of substrates and potential inhibitors of LrBSH.
Keyword Bile salt hydrolase
BSH inhibitors

Homology modeling
Lactobacillus reuteri
CRL1098
Molecular docking analysis
Introduction
The metabolism of bile acids (BAs), the major
compounds of bile, is an excellent example of a close
symbiotic relationship with the microbiota since it
depends on both the host and the microbial enzymes
(Bustos et al. 2018). BAs deconjugation by bile salt
hydrolase enzymes (BSH, EC 3.5.1.24) that are
present in microbiota members is one of the most
relevant reaction of this microsystems (Chand et al.
2017; Song et al. 2019). BSH activity is also consid-
ered a functional probiotic biomarker since it may lead
to serum cholesterol lowering and alterations in
energy homeostasis (Dong and Lee 2018; Adebola
et al. 2020) and even play a role in the anti-giardial
effect of some lactobacilli since deconjugated BAs are
toxic to the parasite (Allain et al. 2018). Furthermore,
BSH has also been proposed as a potential microbial
mechanism of resistance to bile (Bustos et al. 2018).
On the other hand, BSH enzyme inhibitors have
been proposed as an alternative to antibiotic growth
promoters (AGP) due to their frequent side effects.
The purpose is to decrease the BSH activity which
may increase fat deposition in farm animals (Rani
et al. 2017; Geng et al. 2020). To successful devel-
opment of effective, safe, and low cost BSH inhibitors,
a detailed understanding of the molecular basis of
BSH substrate recognition in different microorgan-
isms, is strongly needed. Hence, computational strate-
gies such as homology modeling and molecular
docking studies can be used to identify protein–ligand
interactions and the effective binding patterns (Rani
et al. 2017).
Previously, we cloned and described the BSH
enzyme of Lactobacillus (L.) reuteri CRL 1098
(LrBSH), a valuable probiotic bacterium with a proven
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Biotechnol Lett (2021) 43:1063–1073
hypocholesterolemic effect (Bustos et al. 2016). In this
study, structural homology modeling was used for the
LrBSH and the differences in electronic properties by
six glyco- and tauro-conjugated that explain the BSH
interaction with BAs were evaluated. Molecular
docking analysis was performed for a better under-
standing of the substrate specificity molecular fea-
tures, and the role of specific residues play in the
interaction of LrBSH with BAs. The interaction of
LrBSH with potential enzyme inhibitors was also
studied by in vitro and molecular docking assays.
Materials and methods
Bile salt hydrolase structure modeling
The amino acid sequence of the BSH from L. reuteri
CRL 1098 (Bustos et al. 2016) was submitted to
Swiss-model, a protein structure homology-modeling
server (https://swissmodel.expasy.org/) (Bordoli et al.
2009; Bienert et al. 2017). The program provides a
graphical representation as well as numerical calcu-
lations for the structure alignments. A three-dimen-
sional structure of BSH protein was generated and the
positions of the amino acid residues were analyzed
with PyMol program.
Ligands structures
The structures of six conjugated BAs molecules,
glycocholic acid (GCA), taurocholic acid (TCA),
glycodeoxycholic acid (GDCA), taurodeoxycholic
acid (TDCA), glycochenodeoxycholic acid
(GCDCA), and taurochenodeoxycholic acid
(TCDCA) (Supplementary Fig.1) and the potential
inhibitors penicillin, ascorbic acid, tetracycline, and
caffeic acid phenethyl ester (CAPE) were initially
built with the GaussView program and then optimized
in aqueous solution phases at B3LYP/6-31G* level of
theory using Gaussian Program (Frisch et al. 2009).
Docking procedure
The binding site between the generated BSH protein
and ligands was explored by molecular docking with
the AutoDock 4.2 program using the Lamarckian
genetic algorithm (LGA). Ligands including BAs,
penicillin V, ascorbic acid, tetracycline, and CAPE,
Biotechnol Lett (2021) 43:1063–1073
obtained as described above, were used in the docking
analysis.
The LrBSH enzyme was considered as rigid and all
the rotatable torsional bonds of each BA were allowed
to relax during docking calculations. Default Auto-
Dock parameters were used as well as a grid volume
(60 9 60 9 60 with 0.375 Å grid spacing) for a more
precise evaluation of thee the atomic interactions at
the binding sites. To identify the best docked com-
plexes, the root mean square deviation (RMSD)
tolerance of 1.0 Å was used. The ligand conformation
with the lowest free energy of binding (DG), chosen
from the most favored conformers, was selected to
perform NBO calculations using the DFT method.
To better understand the role of key residues in the
substrate preference of LrBSH, two in silico site-
directed mutants using the PyMOL program were
generated. In this study, the amino acids Leu 56 and
Glu222 were selected as mutation points with glyco-
and tauro-conjugated BAs, respectively. Selected
amino acids were replaced as follows: hydrophobic
Leu56 residue was substituted by electrically charged
Glu56 residue, and Glu222 by Leu222 to obtain
mutant A and B, respectively. Mutant A was tested
against GCA, GDCA, and GCDCA while mutant B
against TCA, TDCA, and TCDCA using the procedure
described above.
Potential inhibitors of L. reuteri CRL 1098 bile salt
hydrolase activity
L. reuteri CRL 1098 was grown in MRS broth at 37 °C
for 16 h prior to use. Cells were washed, suspended
(volume 20-fold concentrated respect to the broth
culture) in 50 mM sodium acetate buffer (pH 5.2) and
disrupted with zirconium beads and vortex. The
suspension was centrifuged and the cell free extract
was used to determine the efficiency of LrBSH activity
inhibitors. Protein concentration of the cell free extract
(Bradford technique) was 500 lg/mL.
The reaction mixture (1 ml) containing 50 ll of cell
free extract and 50 mM acetate buffer (pH 5.2) was
pre-incubated separately, with penicillin G, ciproflox-
acin, ascorbic acid or citric acid at a concentration of
5 mM for 30 min at 37 °C (Rani et al. 2017). The
enzymatic reaction was started by the addition of
5 mM GDCA and was stopped after 10-min incuba-
tion at 37 °C by the addition of 1 ml trichloroacetic
acid (20%, w/v). Samples were centrifuged to remove
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precipitated proteins, and the amino acids released by
LrBSH activity were determined by o-phthalaldehyde
(OPA) method, as previously described (Bustos et al.
2016). The inhibition efficiency of LrBSH inhibitors
was calculated by dividing the activity of the control
(without inhibitor) by that of the treatment group (with
inhibitor). The experiments were performed in
triplicate.
Results and discussion
Comparison of the bile acid structure
As shown in Table 1, the lowest stabilization energy
and higher dipolar moment were predicted by B3LYP/
6-31G* method for tauro-derivatives, especially for
TCA. This fact can be explained by the greater
stability granted by the presence of taurine in the
molecule. The highest stabilization energy corre-
sponded to GDCA and GCDCA acids by the presence
of two hydroxyl groups if compared with cholic acid
derivatives. The Mulliken charges showed slight
changes in the charge values of all BAs atoms with
the highest negative values for the O atom of glycine
group of glyco-conjugated BAs and the O atom of the
hydroxyl group nearest the amino acid in the structure.
These results showed that the presence of both the
third OH group in cholic acid and the glycine residue
in the structure notably increased the negative Mul-
liken charges on the O atoms of BAs (Supplementary
Fig. 2).
It is known that the gap energy, defined as the
difference between the highest occupied molecular
orbital (HOMO) and the lowest unoccupied molecular
orbital (LUMO) determines the chemical reactivity
and kinetic stability of a molecule. Thus, a low
difference means that the molecule has high chemical
reactivity and low kinetic stability. The HOMO
energies were more negative for TCA and TDCA
compounds while the highest chemistry reactivity was
predicted for the GCA compounds.
Figure 1 shows the electronic distribution of the
frontier orbitals for GDCA and TCA. The HOMO
orbitals are localized on the carbonyl bound of the
glycine group in GCA, GDCA, and GCDCA, while
these orbitals are localized on the hydroxyl group in
opposite position to the taurine group in TCA, TDCA,
and TCDCA. This electronic distribution
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Table 1 Stabilization Bile acid Energy (Hartree) Dipolar moment (Debye) HOMO LUMO GAP
energies, dipolar moment,
frontier orbital energies GCA -1522.3393 48.89 1.9982 0.0037 -1.99
(HOMO and LUMO), and
GDCA -1447.1218 47.18 -0.2266 0.0331 0.26
energy difference between
them (GAP) GCDCA -1447.1222 46.95 -0.2264 0.0332 0.26
TCA -1996.9192 55.12 -0.2410 0.0288 0.27
TDCA -1921.7021 53.14 -0.2410 0.0286 0.27
TCDCA -1921.7017 53.84 1.9942 0.0038 -1.99

Fig. 1 Energy potential surface, HOMO and LUMO frontier orbitals for GDCA and TCA compounds. The red area indicates a negative
region, and the green area a positive region

(Supplementary Fig. 3) may justify the different
reactivity between two kinds of compounds suggest-
ing that the glycine and taurine residues give the BAs
different electronic properties.
Structural homology modeling: LrBSH 3D
structure
Lactobacillus reuteri CRL 1098 is a probiotic bac-
terium with a proven hypocholesterolemic effect
associated to the presence of the BSH enzyme. In a
previous work, we characterized the bsh operon of
CRL 1098 strain as a single open reading frame of 978
nucleotides that encodes a predicted protein of 325
amino acids with a calculated mass of 36,098.1 Da
(Bustos et al. 2016). The sequence of bsh gene was
submitted to Swiss Model program. The BSH struc-
ture of Enterococcus (E.) faecalis T2 obtained by
X-ray diffraction with 2.01 Å resolution (PDB acces-
sion code 4WL3) was used as a template for modeling.
The chosen sequence showed an identity of 56.44%
with LrBSH.
Based on Swiss model prediction, the secondary
structure of LrBSH would have 32.2% strand, 17.4%
alpha helix, 8% 3–10 helix, and 47.7% loops and turns.
The final structure was modeled with PyMOL program
using 321 residues obtained from structural homology
modeling and with the addition of Asn322, Gln323,
Asn324, and Gln325 residues. The catalytic residues,
Cys2, Asp19, and Asn171 in the generated structure
were observed as shown in Fig. 2. This molecule is
consistent with other BSH enzymes reported in PDB
database and was used for docking studies with
different substrates.
Substrate specificity and binding site
characterization of LrBSH by molecular docking
studies
In the present work, molecular docking analysis was
performed to explore the molecular features of the

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Fig. 2 Three-dimensional structure of monomer LrBSH
protein. The catalytic site of the enzyme shows the conserved
amino acids, Cys2. This figure was created by PyMOL program
(PyMOL Executable Build is CopyrightÓ 2006 Delano Scien-
tific LLC, South San Francisco, California, USA)
enzyme substrate specificity. As shown in Fig. 3,
different binding modes for glyco- or tauro-conjugated
BAs were predicted. The GCA, GDCA, and GCDCA
interact with Cys2 residue of the LrBSH in the binding
cavity with the steroidal hydroxyl group directed
Fig. 3 Glyco- (top) and tauro- (bottom) conjugated BAs at the LrBSH binding site. The residue in blue ball and sticks is Cys2. In red,
the nucleophile site (negative region), in blue, the electrophile site (positive region), and in green, the neutral region
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towards the inside and the glycine group directed
towards the outside. In contrast, TCA, TDCA, and
TCDCA interact in a longitudinal manner with the
taurine group directed towards the inside cavity. The
electronic distribution showed that the oxygen atom in
C = O group of glycine derivative is a nucleophilic
region, which strongly interacts with the N atom of N–
H group of Cys2 of LrBSH with a shorter interaction
length than tauro-derived BAs. The electronic density
effects seem to be of importance for the enzyme-BAs
interaction.
Tables 2 and 3 show the binding energy for all BAs
and the residues involved in the binding site of LrBSH
observed with AutoDock. According to docking
analysis, GDCA had the higher binding energy
(-6.88 kcal/mol) followed by TCDCA, GCDCA,
and GCA. The increase in binding energies for TDCA
and TCA may indicate a weak interaction with the
enzyme (Supplementary Fig. 4). These results agree
with our previous experimental assays (included in
Table 2 for comparison purpose) since LrBSH showed
substrate preference toward glyco-conjugated BAs
(GDCA and GCA) compared to tauro-conjugated
(Taranto et al. 1999; Bustos et al. 2016). Data
available in the literature indicate that BSHs predom-
inantly recognize the amino acid moieties in the
substrates and most of BSH enzymes prefer glyco-
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Table 2 Binding energy (kcal/mol), inhibition constant (lM) of LrBSH-bile acid complex predicted by molecular docking, and
reported enzyme activities
Bile acid Binding energy (kcal/mol) Inhibition constant Hydrogen bonds and Hydrogen bond length Enzyme
(lM) (Å) activitya

GCA -5.88 32.91 O–H–O-Asp220 (1.89) 29

GDCA -6.88 9.00 65
GCDCA -6.29 24.36 N–H–O–Glu270 (1.92) ND
TCA -5.46 85.47 14
TDCA -5.63 74.08 26
TCDCA -6.32 23.35 ND
ND not determined
a -1 -1
lmol amino acids released min (mg proteins) , taken from Taranto et al. (1999)

Table 3 Residues involved Bile acids

in the binding site of LrBSH
with glyco- (GCA, GDCA, Residues GCA GDCA GCDCA TCA TDCA TCDCA
and GCDCA) and tauro-
conjugated BAs (TCA, CYS2 X X X X X X
TDCA, TCDCA) ARG16 X X X X X
LEU18 X X X X X
ASP19 X X X X X X
LEU20 X X
LEU24 X X X
LEU56 X X X
PHE65 X X X X
GLY77 X
ASN79 X X X X X X
ASP81 X
GLN134 X X
ALA135 X X X
ALA136 X X
LEU138 X X X X X
ASN171 X X X X X X
ASN172 X X X
ASP220 X X
SER221 X X X
GLU222 X X X
ARG224 X X
GLU270 X X X X X

over tauro-conjugated BAs, even when the substrate
preference is strain dependent (Dong and Lee 2018).
This can be attributed mainly to the steric hindrance
caused by the sulfur atom in tauro-conjugated com-
pounds, as it was observed in our results, and also to an
abundance of glyco-conjugated bile salts in nature
(Chand et al. 2017). In addition, the LrBSH enzyme
had higher binding affinity for deoxycholic and
chenodeoxycholic acids derivatives compared to those
from cholic acid, in agreement with the suggestion that
BSHs evolved to recognize BAs in both the amino acid
groups and steroid nucleus (Dong and Lee 2018).

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As shown in Fig. 4, the docking analysis revealed
that the Cys2, Asp19, Asn79, Asn171, and Glu270
residues participated in the binding site of LrBSH,
which formed hydrogen bonds only with GCA and
GCDCA compounds with bond lengths from 1.89 Å to
1.92 Å. Table 3 shows other residues involved in the
catalytic reaction aside from Cys2 such as Arg16,
Leu24 which are also strictly conserved in other BSHs
reported (Rani et al. 2017; Öztürk and Önal 2019).
Interestingly, Asp19, Asn79, and Asn171 participated
in the interaction of LrBSH with all BAs tested.
A great percentage of hydrophobic interactions
with Leu20, Leu24, Leu56, and Ala135 and Ala136
residues took place at the binding site of LrBSH with
glyco-conjugated BAs while polar and ionic interac-
tions with Ser221, Asn172, and Glu222 residues for
tauro-conjugated BAs were detected. The interaction
of LrBSH with GCA, GDCA, and GCDCA revealed
that the hydrophobic residues Leu20, Leu24, Leu56,
and Phe65 are near to the catalytic center and within
the cavity of enzyme substrates. For TCA, TDCA, and
TCDCA, the polar residues Asn172, Ser221, and
Glu222 are involved in the active site and substrate
binding of the enzyme.
Despite the remarkable progress in the identifica-
tion and characterization of new BSH enzymes, the
structural and molecular basis of the substrate recog-
nition by different BSHs remains not fully clarified. In
most of the BSHs studied, the substrate-binding
residues are not strongly conserved with some excep-
tions such as Leu141 (Leu138 in LrBSH) (Dong and
Lee 2018). Our findings are in line with previously
Fig. 4 Interaction distance (in Å) of the N–H Cys2 residue of LrBSH with GDCA and TDCA bile acids
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reported crystallographic and molecular docking
studies for other BSH enzymes. Most of the residues
described in the binding pocket of LrBSH seem to be
conserved in E. faecalis,. In fact, it was reported that
GCA interacted with the amino acids Cys2, Arg16,
Asp19, Tyr65 (Phe 65 in LrBSH), Asn79, Asp170
(Asp171 in LrBSH), and Arg223 (Arg224 in LrBSH)
of the BSH suggesting the importance of these
residues (Dong and Lee 2018). Moreover, Leu20
was important in the interaction between BSH and
glyco-conjugated BAs in Bifidobacterium (B.) longum
(Kumar et al. 2006) while Ile56 also appeared in L.
salivarius (Leu 56 in LrBSH) (Xu et al. 2016). In L.
gasseri, TCA formed hydrogen bonds with Gln135
(Gln134 in LrBSH) (Rani et al. 2017) and with Met20
in Clostridium (C.) perfringes (Leu20 in LrBSH)
(Dong and Lee 2018).
Our results showed a variation in the substrate
binding pocket of glyco-conjugated BAs in compar-
ison with tauro-conjugated ones. As it is shown in
Fig. 5, of the four binding pocket loops (Chand et al.
2017), loop I (Leu20-Leu24), loop II (Leu56-Phe65),
and loop III (Gln134-Ala136) constitute the binding
pocket for glyco-conjugated BAs while only loop III
and loop IV constitute the binding pocket for tauro-
conjugated Bas. In this sense, the four loops for most
of the BSHs characterized are predominantly
hydrophobic. In an exhaustive review, Dong and Lee
(2018) reported that the residues involved in substrate
binding were located in loops I, II and III but no key
residues were reported in loop IV, except for E.
faecalis where all binding loops (loop I–IV) constitute
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Fig. 5 Substrate binding studies on the surface of BSH from Lactobacillus reuteri with tauro- and glyco-conjugated acids after
stabilizing the protein
the binding pocket for GCA. This fact represents an
important difference of LrBSH respect to most of the
BSH enzymes described.
Since molecular docking results agreed with our
previous experimental findings and enabled us to
identify the residues involved in the enzyme–substrate
interaction, the role of two important amino acids
using this computational approach was evaluated. To
determine the role of loop I residues in the interaction
with glyco-conjugated BAs, in silico mutant A was
generated by replacing hydrophobic Leu56 residue by
electrically charged Glu56 residue since it is a key
amino acid for the binding site of LrBSH. Binding
pocket analysis of Glu56 mutant demonstrated that
loop II of the enzyme provided a higher interaction for
GCA, GDCA and GCDCA substrates at a different
binding site than the native LrBSH. New residues
participated in the interaction of the last two substrates
with the LrBSH-E56 mutant including hydrophobic
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Biotechnol Lett (2021) 43:1063–1073
residues (Leu133, Val102, Tyr64, Phe99) and posi-
tively charged ones (Arg58, Lys132) (Supplementary
Fig. 5).
When Glu222 of loop IV was in silico replaced by
Leu222, the interaction for all tauro-conjugated BAs
drastically improved and took place at another binding
site where the Arg58, Phe99, and Leu133 residues
have a significant role. The selected residues seem to
play an important role in the interaction of the enzyme
with the substrates since its replacement may affect the
enzyme activity. These findings provide evidences to
deepen the understanding on this valuable enzyme.
Interaction of LrBSH with potential inhibitors:
in vitro assays and docking analysis
In this work, some commonly used feed additives and
AGPs were experimentally tested as potential LrBSH
enzyme inhibitors. Ascorbic and citric acids,
Biotechnol Lett (2021) 43:1063–1073
commonly used as preservatives and antioxidants in
the food industry, and penicillin and ciprofloxacin,
widely used as antibiotic growth promoters (AGP)
were chosen for the study.
Among the tested compounds, 47.11 ± 3.4% inhi-
bition with ascorbic acid followed by citric acid
(40.14 ± 1.7%) and penicillin G (28.8 ± 3.5%) was
obtained, corresponding the lowest (9 ± 0.5%) inhi-
bition percentage to ciprofloxacin. In this sense,
different compounds such as Hg2?, Cu2?, and Cd2?,
Mg2?, Ca2?, Zn2?, Mn2?, EDTA, p-mercuribenzoate,
and iodoacetamide displayed different degrees of
inhibition on LrBSH activity as reported by Taranto
et al. (1999).
Recently, some authors claimed the high correla-
tion existing between the growth-promoting effects of
AGP and a decreased BSH activity in the gut. Based
on these findings, they proposed the use of BSH
enzyme inhibitors as an alternative to replace AGP due
to numerous side effects. It is expected that BSH
inhibitors modify the lipid and energy metabolism of
the host and consequently lead to body weight gain in
farm animals (Geng et al. 2020). Among the BSH
inhibitory compounds identified through screens,
CAPE seem to be one of the most effective (Rani
et al. 2017; Geng et al. 2020). CAPE is an emerging
natural food additive that attracted extensive attention
for human and animal application because of its
multiple potential bioactivities such as anti-carcino-
genic, anti-oxidation, and anti-inflammatory effects
(Geng et al. 2020). The interaction of LrBSH with the
AGP penicillin and tetracycline was evaluated by
molecular docking. Ascorbic acid and CAPE mole-
cules were also included in this study.
Results obtained revealed that tetracycline and
CAPE resembled the interaction pattern of GDCA
with LrBSH close to Cys2 residue, as depicted in
Fig. 6, while penicillin and ascorbic acid interacted at
a longer distance. Table 4 shows the binding energies
and the residues involved in the binding site of
LrBSH-inhibitors. Docking analysis revealed that
tetracycline and CAPE had lower binding energy
(-7.32 kcal/mol and -7.19 kcal/mol, respectively)
than GDCA (-6.88 kcal/mol) and they interacted
with residues such as Asn79, Asp81, Cys2 and Leu18
of the LrBSH similarly to GDCA. These results
indicate that both compounds could competitively
inhibit GDCA binding to LrBSH and are consistent
with previous reports where tetracycline and CAPE
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Fig. 6 Docking analysis of GDCA (cyan), CAPE compound
(red), penicillin (Pen, magenta), tetracycline (TC, white) and
ascorbic acid (Asc, yellow) with LrBSH to determine inhibitory
effects
showed more than 95% inhibition of BSH activity
(Smith et al. 2014; Rani et al. 2017). On the other
hand, penicillin and ascorbic acid reacted with LsBSH
with lower binding energies than GDCA
(-6.25 kcal/mol and -5.98 kcal/mol, respectively)
and other kind of residues are involved in this
interaction.
Our results showed that molecular docking may be
a useful tool for rapid screening of BSH inhibitors and
also provide new insights respect to the interaction of
the LrBSH enzyme with potential inhibitors.
Conclusion
In this work, the BSH enzyme of L. reuteri CRL 1098
was modeled by using structural homology modeling.
Molecular docking analysis was performed to have a
deeper understanding of the substrate specificity and
interaction of potential inhibitors with LrBSH as well.
The participation of Asp19, Asn79, and Asn171 in the
LrBSH interaction with all BAs tested was evidenced
together with the fact that N–H group of Cys2
interacted with C = O of all BAs, being greater for
glyco-conjugated BAs. Besides, Leu56 (located in
loop I) and Glu 222 (in loop IV) played an important
role in the interaction with glyco- and tauro-conju-
gated BAs, respectively. Remarkably, it was found
that the four binding pocket loops (loop I–IV)
identified participated in the substrate binding site of
LrBSH unlike most of the reported BSHs. Regarding
inhibitors, docking analysis revealed that tetracycline
and CAPE interacted with residues such as Asn79,
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Table 4 Binding energy (kcal/mol) and residues involved in the binding site of LrBSH with inhibitors
Inhibitor Binding energy Residues of the binding site
(kcal/mol)

CAPE -7.19 CYS2-LEU18-ASP19-LEU20-PHE65-ASN79-PHE99-PHE129-LEU133-ALA135-ALA136

Tetracycline -7.32 GLU21-ASN79-ASP81-ASN171-ASN172-MET219-ASP220-SER221-ARG224-GLN257-
ASP262-GLU270
Penicillin G -6.2 LEU179-LEU182-SER183-TYR185-ARG186-VAL188-LEU215-VAL226-PHE230
Ascorbic -5.98 TYR64-PHE65-GLY77-SER98-PHE99-LEU101-VAL102-LEU138-TRP140
acid

Asp81, Cys2 and Leu18 of the LrBSH in a similar way
than GDCA while penicillin and ascorbic acid inter-
acted at a longer distance and different residues are
involved.
These results contribute to a greater knowledge of
the molecular mechanisms involved in the recognition
of substrates by BSH enzymes, which may be
extrapolated to other fields related to enzyme engi-
neering. This work provides some new insights into
the inhibitory effect of potential BSH inhibitors.
Acknowledgements This work was supported with grants
from UNSE Project No. 23/C147 (Consejo de Investigaciones,
Universidad Nacional de Santiago de Estero).
Data availability The authors declare that all the material and
data of this work are available.
Code availability Not applicable.
Compliance with ethical standards
Conflict of interest The authors declare that they have no
conflict of interests.
Research involving human and animal rights This article
does not contain any studies with human participants or animals
performed by any of the authors.
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