A highly active bile salt hydrolase from Enterococcus faecalis shows positive cooperative kinetics

Process Biochemistry 51 (2016) 263–269

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A highly active bile salt hydrolase from Enterococcus faecalis shows positive cooperative kinetics Deepak Chand, Sureshkumar Ramasamy ∗ , C.G. Suresh ∗ Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India

a r t i c l e

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Article history: Received 18 September 2015 Received in revised form 8 December 2015 Accepted 11 December 2015 Available online 19 December 2015 Keywords: Bile salt hydrolase Gut organism Bile acids Allosteric regulation Penicillin V acylase Enzyme kinetics

a b s t r a c t Bile salt hydrolase (BSH; cholylglycine hydrolase, EC 3.5.1.24) is an important enzyme that catalyses the deconjugation of bile acids conjugated with glycine or taurine and assists in the reduction of blood cholesterol levels. In the present study, we report the cloning, overexpression and characterisation of BSH gene from a gut-associated microbe Enterococcus faecalis (EfBSH). The overexpressed protein in Escherichia coli was purified to homogeneity. Optimum pH and temperature for activity were found to be 5.0 and 50 ◦ C, respectively. The enzyme was considerably stable in the pH range of 5.0–7.0 and at a temperature of up to 50 ◦ C. It showed high specific activity of 1390 U mg−1 and 1289 U mg−1 for substrates such as glycocholic acid (GCA) and glycodeoxycholic acid (GDCA), respectively. The effect of additives on enzyme activity was assessed, and the detergent Triton X showed a marginally enhanced activity. The enzyme demonstrated unique enzyme kinetics of non-linear regression, thereby displaying positive cooperativity. In addition, the modulating effect of the non-substrate ligand Pen V on the hydrolysing ability of EfBSH towards bile acid such as GDCA was measured. It was observed that Pen V significantly enhanced the BSH activity. This is markedly different from the previously reported competitive inhibition of BSH activity by Pen V. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Bile salt hydrolase (BSH; cholylglycine hydrolase, E.C.3.5.1.24) is an enzyme involved in the crucial metabolic pathway of mammalian bile acid metabolism. Although distributed among bacteria (gram positive and gram negative) and the archaea kingdom, the enzyme is more widely found in gut microbes [1]. Several microbial species, including Bifidobacterium, [2,3–8] Lactobacillus [9–16], Clostridium perfringens [17,18], Brevibacillus [19], Bacteroides [20] and Xanthomonas maltophilia [21], are reported to show BSH activity. In addition, some pathogenic bacteria such as Listeria monocytogen [22,23] and Brucella abortus [24] also possess BSH activity. The BSH enzyme belongs to the Ntn (N-terminal nucleophile) hydrolase structural superfamily and the cholylglycine hydrolase subfamily (Pfam: PF02275). Evolutionarily closely related BSH and penicillin V acylase (PVA) share all conserved active-site residues. Bile salts are produced by liver hepatocytes and they assist in dietary absorption of fat in the small intestine. Bile acids help in the

solubilisation and transport of cholesterol and fat across the intestinal epithelium [25,4,26] these are the key signalling molecules that regulate host lipid metabolism and mucosal defence in the intestine [1,27]. BSHs are present in most intestinal microbes, conferring resistance against the antimicrobial activity of bile. BSHs from Lactobacillus have been studied in rats [28,29] and humans [12,30]. BSH significantly lowers the serum cholesterol levels [31] as well as maintains bile acid pool homeostasis in the gastrointestinal (GI) tract, which consequentially reduces further complications related to hypercholesterol. Therefore, BSH activity is of great concern in the study of probiotic strains. In this study, we describe the cloning, overexpression and biophysical and detailed biochemical characterisation of EfBSH. This enzyme has demonstrated many-fold higher specific activity and distinctive enzyme kinetics as compared to other reported BSH enzymes. In addition, the non-substrate ligand Pen V exerted a positive modulating effect on EfBSH activity. 2. Material and methods 2.1. Materials

∗ Corresponding authors. E-mail addresses: [email protected] (S. Ramasamy), [email protected] (C.G. Suresh). http://dx.doi.org/10.1016/j.procbio.2015.12.006 1359-5113/© 2015 Elsevier Ltd. All rights reserved.

Genomic DNA was isolated from E. faecalis strain NCIM 2403 obtained from NCIM of NCL. All enzymes used for DNA manipu-

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lation were procured from New England Biolabs (NEB). pET22b+ vector was used for cloning and E. coli DH5␣ cells were used as the maintenance host. The clone was transformed in the BL21* (DE3) expression host. Conjugated bile acids (GCA; GDCA; taurocholic acid, TCA; glycochenodeoxycholic acid, GCDCA; and taurochenodeoxycholic acid, TCDCA), guanidine hydrochloride (GdnHCl) and Pen V (potassium salt) were procured from Sigma (USA). The Sephadex200 (SEC-650 Bio-Rad, CA, USA) column for size exclusion chromatography and molecular weight markers were purchased from Bio-Rad. 2.2. Sequence analysis The gene sequence of EfBSH was retrieved from NCBI with GenBank ID EET97240.1 (http://www.ncbi.nlm.nih.gov). The global alignment of amino acid sequences and the percentage of identity were calculated using the EMBOSS pairwise alignment algorithms (http://www.ebi.ac.uk/emboss/). The theoretical pI of the protein was calculated from the ExPASy ProtParam tool server. 2.3. Molecular cloning, cell extract preparation and purification The open reading frame of bsh gene (978 bp) retrieved from genomic DNA of the E. faecalis, using the primers EfBSH For and EfBSH Rev, was cloned into the pET22b+ vector with restriction sites of NdeI and XhoI (Supplementary 1). The recombinant plasmid (pET22b-EfBSH) was mobilised into BL21* (DE)-competent cells. A single colony of the transformed BL21* (DE) possessing the pET22b-EfBSH construct was inoculated in Luria–Bertani (LB) medium containing 100 ␮g/ml of ampicillin and incubated at 37 ◦ C until an optical density (OD600 ) of 0.4–0.6 was attained. Further, isopropyl ␤-d-1-thiogalactopyranoside (IPTG) was added to the media up to a concentration of 0.5 mM for induction followed by shaking incubation (180 rpm) at 16 ◦ C overnight (16–18 h). Cells were centrifuged at 4000 rpm for 10 min at 4 ◦ C. They were then suspended in 25 ml of lysis buffer (25 mM Tris–HCl of pH 7.5, 20 mM imidazole and 500 mM NaCl), sonicated at 50% amplitude (E-squire Biotech Pvt., Ltd.) and centrifuged at 12,000 rpm for 30 min at 4 ◦ C. Purification of EfBSH was performed by His-select Ni–NTA (Quiagen) followed by Sephadex200 size exclusion chromatography (SEC 650, Bio-rad). The purity of the protein was assessed on 12% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) by Coomassie brilliant blue (CBB) R-250 staining. 2.4. Molecular weight determination and western blot analysis The protein was subjected to SDS-PAGE on a 12% polyacrylamide gel with molecular weight markers (Bio-Rad, Hercules, CA, USA) and stained using CBB. For determining the subunit molecular weight of the purified protein, matrix-assisted laser desorption/ionisation (MALDI) TOF/TOFTM (time of flight, TOF) was performed. For sample preparation, 1 mg ml−1 of protein was used. Cinapinic acid was used as the matrix. The native molecular weight of EfBSH was determined by SEC 650 (Bio-Rad, Hercules, CA, USA) size exclusion chromatography. The purified protein was identified and confirmed using anti-His antibodies (Supplementary 2). 2.5. Measurement of enzyme activity For estimating BSH activity, the reaction mixture (50 ␮l) containing enzyme and substrate was incubated at 50 ◦ C for 10 min and then the reaction was terminated by adding equal volumes (50 ␮l) of 15% (w/v) TCA. The amount of amino acids released from conjugated bile acid by the enzyme activity was estimated by ninhydrin assay with some modifications to the original method [2]. One unit of BSH activity is defined as the amount of enzyme that can liberate

1 ␮mol of amino acid from the substrate per minute. A qualitative estimate of the products formed in the reaction was also conducted via TLC (thin-layer chromatography). Protein concentration was measured by the Bradford method [32]. 2.6. Effect of pH, temperature and GdnHCl on EfBSH activity and stability The BSH activity was assayed at different pH values in the range 3.0–11.0 and temperatures 25–80 ◦ C to determine the optimum conditions for enzyme activity. Enzyme activity and stability were also studied in the pH range 1.0–11.0 and temperatures 25–80 ◦ C. The effect of pH was studied by incubating the protein in 100 mM buffers of different pH (1.0–11.0) for 4 h and assaying the residual activity. In order to assess the effect of pH (1.0–11.0), temperature (25–80 ◦ C) and GdnHCl (0.25–4 M) on secondary and tertiary structures of protein, the far-UV and near-UV CD (circular dichroism) spectra were recorded using a Jasco J-810 spectrometer (Jasco Instruments, Tokyo, Japan) equipped with a Peltier thermostat cuvette holder under constant N2 purging. The purified EfBSH at concentrations of 0.1 and 2 mg ml−1 was used for farUV (190–250 nm) and near-UV (250–310 nm) measurements in a quartz cuvette of 1-mm path length at a scan speed of 100 nm min−1 at 25 ◦ C. Each spectrum recorded was an average of three scans. The results were expressed as the mean residue ellipticity (MRE) defined as [Â] =  obs /(10.C.l.n), where  obs is the corrected CD measurement in millidegrees, C the protein concentration (M), l the path length of the cuvette (cm) and n the number of amino acid residues. 2.7. Effect of additives Chemical modifiers and metal ions were also used to evaluate their effect on enzyme activity. EfBSH was dialysed against the buffer to remove any additives present. The enzyme was incubated with various modifiers at room temperature (25 ◦ C) for 30 min, and then its activity was assayed at the aforementioned standard conditions. The reaction carried out without any additive was used as the control. 2.8. Enzyme kinetic parameters The kinetic constants K0.5 and Vmax of EfBSH were estimated by assaying the enzyme activity with increasing concentrations of conjugated bile salts (Sigma–Aldrich, St. Louis, MO, USA) as substrates in the range 0.5–60 mM following the standard assay protocol. The assay was carried out in triplicates, and the kinetic constants were determined by non-linear regression by fitting the kinetic data in GraphPad Prism version 5.0 (GraphPad Inc., USA). The effect of the non-substrate ligand Pen V (0.5–200 mM) on the conjugated bile acid (GDCA) activity of EfBSH was also explored. Steady-state kinetics was performed by varying the GDCA concentration in the presence of 1 and 50 mM Pen V. 3. Results and Discussion The EfBSH gene was retrieved from NCBI with a GenBank ID of EET97240.1, which was annotated as cholylglycine hydrolase. The gene-specific primers were used for cloning the EfBSH gene. The cloning method is described in the Supplementary file (S1). The activity of the recombinant EfBSH enzyme was assessed using TLC and ninhydrin methods, and the enzyme was confirmed to be a BSH. Lambert et al. [33] and Panigarahi et al. [34] have conducted the sequence analysis and annotated E. faecalis BSH to include in the BSH cluster. The EfBSH sequence contained 324 amino acid

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residues, and theoretical pI value was calculated to be 4.9 (ExPASy ProtParam Server). The translated amino acid sequence of EfBSH when compared with other characterised BSH sequences exhibited 69, 44, 37 and 19% identity with Lactobacillus plantarum, C. perfringens, Bifidobacterium longum and Bacteroides thetaiotaomicron, respectively. Active-site residues such as Cys2, Arg16, Asp19, Asn79, Asn170 and Arg223 are conserved (numbering is according to EfBSH sequence) in all cholylglycine hydrolases, which are highlighted in the multiple sequence alignment and are important for the catalytic activity (Supplementary Fig. S1). EfBSH showed novel characteristics including high activity and unique cooperative kinetics. 3.1. Cell extract preparation, purification and molecular weight analysis The gene for EfBSH was cloned in the pET22b+ expression vector, and the protein was expressed in E. coli BL21* (DE3) cells with a C-terminal 6X His tag. The protein expression was optimised and overexpressed by inducing culture with 0.5 mM IPTG and incubation at 16 ◦ C overnight to obtain the cytoplasmic soluble fraction. The overexpressed protein was purified to homogeneity using Ni–NTA affinity column followed by a gel filtration column (SEC650-Bio-rad) to remove any contaminant protein and soluble aggregate present. The purity of the protein was assessed by 12% SDS-PAGE analysis where purified single bands corresponding to the ∼37-kDa protein were observed (Fig. 1a). The identity of the purified protein was confirmed by Western blot analysis (Fig. 1b). Protein yields were 80–100 mg/l of culture. The purified protein was subjected to MALDI–TOF–MS (mass spectrometry), where peaks were obtained with the observed mass value of 37 kDa (Supplementary Fig. S2a). The gel filtration (SEC-650 column, BioRad, Hercules, CA, USA) analysis of the purified protein confirmed the tetrameric form of EfBSH (Supplementary Fig. S2b). Qualitative analysis of EfBSH activity was performed by identifying the reaction products using the TLC method (Supplementary Fig. S2c) 3.2. Effect of pH, temperature and GdnHCl on EfBSH activity and stability The purified enzyme EfBSH was assayed for BSH activity with conjugated bile acids, and the reaction was carried out at pH 5.0 and temperature 50 ◦ C. One of the resultant by-products of the reaction, glycine or taurine, was estimated by ninhydrin assay by recording the developed colour at 570 nm. The EfBSH enzyme displayed optimal activity at pH 5.0 (0.1 M citrate phosphate buffer) in the assay condition (Fig. 2a) . The optimum temperature for enzyme activity was found to be 50 ◦ C (Fig. 2b). As the enzyme began to aggregate, its activity reduced at 60 ◦ C. The enzyme was stable in a pH range of 5.0–8.0. Seventy percent of the activity was retained even after 4 h of incubation in 0.1 M buffer of pH 5.0–7.0, while only 40% of the activity remained at pH 8.0 (Fig. 3a). Far-UV CD spectrum showed a significant change in MRE upon incubation at pH 1.0, indicating structural changes such as an increase in ␣-helix and a decrease in ␤-sheet fractions at alkaline pH 11.0 but the reverse at pH1.0 (Fig. 3b, Supplementary Table 1). The enzyme was fairly stable in the temperature range of 25–40 ◦ C and retained 80% of its activity. However, the enzyme activity sharply reduced after 3 h of incubation at 50 ◦ C (65% activity), and the enzyme aggregated beyond 60 ◦ C and significantly lost its activity (Fig. 3c). Thermally induced secondary and tertiary structural changes were also recorded. Fig. 3d shows the decrease in ellipticity near 190–210 nm and 220 nm, which corresponds to ␣-helices and ␤-sheets, respectively, indicating the loss of secondary structure (Supplementary Table 1). A change in tertiary structure was displayed by near-UV CD spectra, where changes in ellipticity near

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290–310 nm indicated the initial stages of the collapse of the tertiary structure accompanied by loss in enzyme activity (Fig. S3). Far-UV CD spectra of EfBSH with GdnHCl (0.25–2 M) showed a decrease in the secondary structure fraction, and unfolding of the structure started at 1.5 M GdnHCl (Fig. 4a, Supplementary Table 1). In addition, the activity profile of EfBSH with increasing concentrations (0.25–6 M) of GdnHCl demonstrated a gradual loss of activity, which in turn corroborated with the GdnHCl-induced unfolding of the protein structure (Fig. 4b). 3.3. Effect of additives The effects of divalent metal ions, chelating agent (ethylenediaminetetraacetic acid (EDTA)) and detergent (Triton X-100) on the activity of EfBSH are shown in Supplementary Table 2. The EfBSH activity was moderately enhanced by Mg2+ , Ca2+ , Co2+ , Ni2+ and Mn2+ . Metal ions such as Ag2+ and Hg2+ completely inhibited the activity, thereby indicating the crucial role of a sulfhydryl group in enzyme activity. In the presence of Cu2+ ion, ∼28% of the inhibition occurred. EDTA had no effect on the enzyme activity. However, Triton X had marginally enhanced activity, which could be due to the dissociation of enzyme molecules in the soluble aggregates. 3.4. Enzyme assay and steady-state kinetics of EfBSH The standard ninhydrin assay with some modification was used for the estimation of the hydrolyzed by-product, that is, glycine or taurine amino acid. BSH enzymes reported so far followed the Michaelis–Menten (MM) kinetics, while EfBSH showed very distinct kinetic properties in comparison to other BSHs. Positive cooperativity and enhancement were noted in the EfBSH activity towards bile acids in the presence of the non-substrate ligand Pen V. The values of K0.5 , h and turnover number (kcat ) for different substrates such as GCA, GDCA, TCA, GCDCA and TCDCA were determined by incubating the enzyme sample with a range of concentrations of each substrate (0.5–60 mM) under the standard assay conditions. The concentration of the enzyme used was 1.2 ␮g for all kinetic studies. Hill’s equation [35] was used for computing the parameters K0.5 , h and Vmax . EfBSH showed Hill’s coefficient <1, indicating an apparent positive cooperativity for all the six bile salts studied (Fig. 5). Cooperative behaviour has also been reported in Lactobacillus salivarius BSH (LsBSH), when dithiothreitol (DTT) was dialysed and removed from the reaction. However, the enzyme showed MM kinetics in the presence of DTT [36]. Avinash et al. [37] have also observed cooperative behaviour in one of the members of the cholylglycine hydrolase subfamily PVA from gram-negative bacteria Pectobacterium atrosepticum. All the kinetic studies with EfBSH were conducted in the presence of 1 mM DTT maintained in the buffer. The presence of DTT did not modulate the allosteric behaviour of the enzyme. Removal of the DTT (by PD10 column) from the enzyme solution showed a slight decrease in activity presumably due to the oxidation of the thiol group of the active site cysteine [18]. To date, among the BSH enzymes, the kcat value 3.013 × 103 −1 S and the catalytic efficiency (kcat /K0.5 ) 3.029 × 105 M−1 S−1 for GDCA determined for EfBSH have been the highest (Table 2) [17,10,38,2,6,36,39]. The kinetic parameters of EfBSH calculated for all bile acids are summarised in Table 1. The activity-modulating effect of the non-substrate ligand Pen V (0.5–200 mM) for EfBSH activity towards GDCA was studied. The concentration of the bile acid (GDCA) was kept constant, while that of Pen V was increased up to 200 mM. It is worth noting that EfBSH showed enhanced activity towards GDCA in the presence of Pen V and no inhibition was observed, contrary to the expected results (Supplementary Table

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Fig. 1. (A). SDS-PAGE analysis of overexpressed recombinant EfBSH protein at each purification step from E. coli BL21* (DE3). Lane 1: Supernatant of EfBSH, Lane 2: Protein purified by Ni–NTA affinity chromatography, Lane 3: Precession Plus proteinTM dual colour marker, Lane 4: Purified protein fraction from size exclusion chromatography (Bio-red SEC650 column). The ∼37 kDa molecular weight corresponds to monomer of EfBSH. (B). Western blot analysis of purified EfBSH protein with anti-His antibodies; Lane 1: Precession Plus proteinTM dual colour marker, Lane2: EfBSH protein.

Fig. 2. (A). Optimum pH and (B) optimum temperature determination of the recombinant EfBSH protein.

Table 1 Steady-state kinetic parameters. Substrates GCA GDCA GCDCA TCA TDCA TCDCA

K0.5 (mM) 11.065 9.94 16.97 13.16 9.28 4.49

± ± ± ± ± ±

h 0.69 0.004 0.736 0.915 0.405 0.177

2.363 1.99 1.929 1.745 2.22 2.77

Kcat (S−1 )

Vmax ± ± ± ± ± ±

0.14 0.008 0.112 0.088 0.173 0.201

1390 1289 970 1072 1115 265.8

± ± ± ± ± ±

56.56 6.36 13.93 23.33 24.74 12.58

Kcat /K0.5 (M−1 S−1 )

3.25 × 10 ± 132 3.01 × 103 ± 14 2.26 × 103 ± 32 2.50 × 103 ± 54 2.60 × 103 ± 57 6.21 × 102 ± 29 3

2.937 × 105 3.029 × 105 1.336 × 105 1.903 × 105 2.808 × 105 1.382 × 105

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Fig. 3. Effect of the pH and temperature on activity and stability of the recombinant EfBSH protein: (A) and (B) Activity and stability of the EfBSH with different pH (1.0–11.0) and temperature. (C) and (D) Far-UV (MRE) CD spectra of EfBSH protein at different pH and temperatures. Activity was calculated as relative% activity, the highest activity was taken as 100% and activities for other conditions were determined.

Fig. 4. Effect of GdnHCl denaturant on activity and stability of the recombinant EfBSH protein (A). Far-UV (MRE) CD spectra of EfBSH protein at different concentrations of GdnHCl (B). Effect of GdnHCl on activity profile of the EfBSH protein.

Table 2 Comparison of the EfBSH activity with other reported BSH.

1 2 3 4 5 6

Specific activity (U/mg)a

Km (mM)

Kcat (S−1 )

Substrate

Reference nos.

0.107 3.4 – 165.2 11.84 1390

0.5 – 0.022 18.2 – 11.06

– – 85 99 – 3250

GCA GCA GCA TDCA GDCA GCA

[17] [38] [6] [36] [2] This study

Bold values indicated the compared results from this study. a activity in ␮ mol/min/mg.

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Fig. 5. Steady-state kinetic graphs of recombinant EfBSH with all six conjugated bile salts: (a) GCA, (b) GDCA, (c) GCDCA, (d) TCA, (e) TDCA and (f) TCDCA.

4. Conclusion In conclusion, the present study has focussed on cloning, expression, purification and biophysical and detailed biochemical characterisation of a BSH from E. faecalis. To the best of our knowledge, the catalytic efficiency of EfBSH was found to be the highest for any BSH reported to date. The enzyme demonstrated unique kinetics and displayed apparent positive cooperativity. EfBSH displayed enhanced activity in the presence of the non-substrate ligand Pen V instead of inhibition as expected based on previous studies on other BSHs. Therefore, this enzyme is distinct and different from the previously reported BSHs. The present findings add further insight into the biochemical features and unique kinetic properties of EfBSH. Fig. 6. Steady-state kinetics of recombinant EfBSH with GDCA in the presence of 1 and 50 mM Pen V. Kinetics with GDCA substrate without Pen V in reaction is shown as control (red), with 1 mM Pen V (green) and with 50 mM Pen V (black). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Conflict of interest The authors declare that they have no conflicts of interest.

3). It may be noted that Pen V showed competitive inhibition in the case of B. longum BSH [6]. The steady-state kinetics was performed with varying concentrations (0.5–60 mM) of GDCA (substrate) in the presence of 1 mM and 50 mM Pen V concentrations (Fig. 6). The kinetic constants calculated indicated enhanced catalytic efficiency (kcat /k0.5 ) with values of 3.08 × 105 and 3.52 × 105 M−1 S−1 , respectively (Supplementary Table 4), when compared to the catalytic efficiency towards GDCA in the absence of Pen V (Table 1).

Acknowledgements DC is a senior research fellow, registered in AcSIR for PhD and thanks CSIR-UGC, New Delhi, for the fellowship; SKR is a DST Ramanujan Fellow. The work reported here is part of the CSIR network project ‘HUM’.

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