To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.
To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01.
The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l−1 isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel–nitrilotriacetic acid (Ni2+-NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco-conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety.
BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation.
This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes.
Bile salts produced by liver hepatocytes assist in dietary lipid absorption in the small intestine. These form micelles, which emulsify, solubilize and transport cholesterol and fats across the intestinal epithelium (Eyssen 1973; Begley et al. 2004; Kim and Lee 2005). This surfactant activity also has an antimicrobial effect through the disruption of bacterial membranes (Begley et al. 2006). Bile salt synthesis de novo begins with the formation of primary bile acids from dietary cholesterol, followed by conjugation via an amide bond to the C-24 position with either glycine or taurine (Elkins and Savage 1998; Corzo and Gilliland 1999; Russell 2003; Begley et al. 2004; McAuliffe et al. 2005). After absorption, more than 95% of bile salts are returned to the liver via the enterohepatic circulation (Hofmann 1989; McAuliffe et al. 2005); however, because of microbial deconjugation in the terminal ileum, unconjugated bile acids become less soluble and are excreted in faeces instead.
Intestinal bacteria are constantly exposed to bile acids in the guts of animals. Thus, for probiotic bacteria to have enhanced overall health-promoting effects, high bile acid biotransformation activity is necessary for effective colonization of the gut and increased competitiveness against pathogenic microflora (Moser and Savage 2001; Begley et al. 2006; Shin et al. 2011). Bile salt hydrolase (BSH) activity in Lactobacillus acidophilus and Lactobacillus plantarum has been extensively investigated in rats (Chikai et al. 1987; Tannock 1995; Park et al. 1996, 2007; Tanaka et al. 2000; Lee et al. 2010), pigs (De Smet et al. 1998) and humans (Anderson and Gilliland 1999; Larsen et al. 2000). These studies demonstrated that BSH activity is associated with lowered serum cholesterol levels, which may prevent hypercholesterolaemia. This occurs by reducing the effective absorption of cholesterol in the small intestine and inducing formation of new bile salts to maintain bile salt homoeostasis by offsetting those lost in faeces (Kim and Lee 2005; Nguyen et al. 2007).
However, there are potential harmful side effects of secondary bile acid production and excessive deconjugation of tauro-conjugated bile salts by some intestinal bacteria such as Clostridium perfringens (Eyssen 1973; Russell 2003; Ridlon et al. 2006; Ohara et al. 2010). These included health hazards such as gall stone formation (Low-Beer and Nutter 1978; Berr et al. 1996; Mamianett et al. 1999; Veysey et al. 1999) and their carcinogenic activity in the colon (Kandell and Bernstein 1991; Nagengast et al. 1995; Bernstein et al. 2005). Thus, whether this activity is a desirable probiotic trait is questionable (Begley et al. 2006). Nevertheless, some studies have correlated probiotics with cancer prevention through inhibition of intestinal bacteria that are associated with colorectal cancer (Liong 2008; Ohara et al. 2010).
The mechanism of BSH activity in probiotic strains remains unclear; therefore, further functional studies of these enzymes in vitro and comparisons with other characterized BSH enzymes are needed. Various BSH genes from lactic acid bacteria (LAB) strains have been cloned and characterized, including those from Bifidobacterium strains (Kim et al. 2004, 2005), Lact. plantarum (Christiaens et al. 1992; Ha et al. 2006), Lact. acidophilus (Corzo and Gilliland 1999; Jiang et al. 2010) and Lactobacillus johnsonii (Lundeen and Savage 1990). The characteristics of these BSH enzymes vary widely among strains of the same species. Analysis of Lact. acidophilus NCFM (McAuliffe et al. 2005) demonstrated that some strains possess more than one BSH enzyme, the characteristics of which differ.
Lactobacillus johnsonii PF01, previously known as Lact. acidophilus PF01 (Ahn et al. 2002; Oh et al. 2008), is a resident bacterium of the gastrointestinal tract that adheres specifically to the epithelium of the duodenum and jejunum (Ahn et al. 2002). It was isolated initially from piglet faeces and has high bile resistance activity. Whole genome shotgun sequencing revealed the presence of three types of BSH genes (Lee et al. 2011), which we designated BSH A, B and C. Oh et al. (2008) previously isolated and characterized a bsh enzyme from this strain, which was 99% identical to the enzyme designated BSH B. In this study, BSH A and C genes from Lact. johnsonii PF01 were cloned, and the recombinant BSH (rBSH) enzymes overexpressed in Escherichia coli were characterized. To our knowledge, this is the first report of isolation of different types of BSH enzymes from a single strain of Lact. johnsonii, which exhibited preferential hydrolysis of either glyco-conjugated or tauro-conjugated bile acids. Analysis of the three PF01 enzymes by multiple sequence alignment and comparison with the crystal structure of the conjugated bile acid hydrolase (CBAH-1) enzyme from Cl. perfringens (Coleman and Hudson 1995) presents an opportunity for understanding the substrate specificity of BSHs.
The materials and methods used in this study were similar to the protocol of Oh et al. (2008), with some modifications and added procedures. Lactobacillus acidophilus PF01 was renamed Lact. johnsonii after 16S rRNA gene sequence comparison following whole genome sequencing of the PF01 strain (Lee et al. 2011). The draft genome sequence of Lact. johnsonii PF01 is available in the National Center for Biotechnology Information (NCBI) database at http://www.ncbi.nlm.nih.gov/ (GenBank accession no. AFQJ00000000.1). Briefly, Lact. johnsonii PF01 were cultured in de Man–Rogosa–Sharpe (MRS) broth (Difco) and incubated overnight at 37°C. Recombinant DNA techniques were performed according to the methodologies of Sambrook and Russell (2001). pET21b (Novagen, Bilerica, MA, USA) was used as the vector for gene expression in E. coli BLR (DE3) cells. Escherichia coli strains were grown in Luria–Bertani (LB) medium at 37°C with ampicillin (100 μg ml−1). Restriction enzymes were purchased from Takara Biomedical (Shiga, Japan) and were used according to the manufacturer's specifications. The sodium salts of the bile acids used in this experiment were from Sigma.
BSH nucleotide and amino acid sequences were analysed using the Blast2 program (NCBI) and the ClustalW software package (Thompson et al. 1994). Multiple sequences were aligned using ClustalW, and the strains and species used for comparison of BSH enzyme sequences were limited to those with protein sequences deposited in GenBank and for which substrate specificity data were available (Table 2).
Chromosomal DNA was isolated from overnight cultures of Lact. johnsonii PF01. The BSH genes were amplified via standard polymerase chain reaction (PCR) using specific oligonucleotide primers that included endonuclease restriction sites (Table 1). Then, the gel-purified PCR products corresponding to each BSH enzyme were ligated to the appropriate double-digested pET21b plasmid by incubation at 16°C for 3 h. The plasmid vector was dephosphorylated using calf intestine alkaline phosphatase. Afterwards, E. coli BLR (DE3) cells were transformed by heat shock at 42°C for 90 s. Successful transformants were selected using LB agar plates containing ampicillin (100 μg ml−1) and were transferred to plates containing either 0·3% taurodeoxycholic acid (TDC) with isopropylthiogalactopyranoside (IPTG) at a concentration of 0·1 mmol l−1 or 0·3% glycodeoxycholic acid (GDC) with IPTG (0·1 mmol l−1) to evaluate bile salt hydrolysis activities.
|Name||Sequence (5′→3′)||Enzyme site|
|BSH A||Forward AAA CTG CAG CAT ATG TGT ACC TCA ATT GTT TA||Nde I, Hind III|
|Reverse AGC CTG CAG AAG CTT ATT TTG ATA ATT AAT TG|
|BSH C||Forward AAA CTG CAG CAT ATG TGT ACA TCA ATT TTA T||Nde I, Xho I|
|Reverse AAA CTG CAG CTC GAG ATT TTC AAA TTT AAT|
|Code||Bacterial strain||BSH enzyme||Preferential BSH activity||GenBank accession no.||References|
|CPERF_cbah1||Clostridium perfringens 13||CBAH-1||TC||P54965.3||Coleman and Hudson (1995); Rossocha et al. (2005)|
|LALA4_bshA||Lactobacillus acidophilus LA4||BSH A||GC||ACL98173.1||Jiang et al. (2010)|
|LALA4_bshB||Lact. acidophilus LA4||BSH B||GC||ACL98174.1||Jiang et al. (2010)|
|LALA11_bshA||Lact. acidophilus LA11||BSH A||GC||ACL98175.1||Jiang et al. (2010)|
|LALA11_bshB||Lact. acidophilus LA11||BSH B||GC||ACL98176.1||Jiang et al. (2010)|
|LANCFM_bshA||Lact. acidophilus NCFM||BSH A||TC/GC||YP_193782.1||McAuliffe et al. (2005)|
|LANCFM_bshB||Lact. acidophilus NCFM||BSH B||GC||AAV42923.1||McAuliffe et al. (2005)|
|LGAM1_bsh||Lactobacillus gasseri AM1||BSH||GC||ACL98172.1||Jiang et al. (2010)|
|LJ100_cbshalp||Lactobacillus johnsonii 100-100||CBSH-α||TC/GC||AAG22541.1||Lundeen and Savage (1990)|
|LJ100_cbshbet||Lact. johnsonii 100-100||CBSH-β||TC/GC||AAC34381.1||Lundeen and Savage (1990)|
|LJPF01_bshA||Lact. johnsonii PF01||BSH A||TC||EGP12224.1||Isolated in this study|
|LJPF01_bshB||Lact. johnsonii PF01||BSH B||TC||EGP13287.1||Oh et al. (2008)|
|LJPF01_bshC||Lact. johnsonii PF01||BSH C||GC||EGP12391.1||Isolated in this study|
|LP80_pcbh1||Lactobacillus plantarum 80||pCBH1||GC||AAB24746.1||Christiaens et al. (1992); De Smet et al. (1995)|
|LPWCSF1_bsh1||Lact. plantarum WCSF1||BSH1||GC||CCC80500.1||Lambert et al. (2008)|
The resulting transformants were inoculated into LB broth containing ampicillin (100 μg ml−1) and incubated at 30°C until the optical density at 600 nm (O.D.600) reached 0·5–0·6. Expression of the BSH genes was induced by the addition of IPTG (0·1 mmol l−1). Cells were harvested by centrifugation at 9000 g for 10 min. The cell pellet was washed and resuspended in lysis buffer (300 mmol l−1 NaCl, 50 mmol l−1 Na2HPO4, pH 7·0) and disrupted by sonication for 1·5 min (alternating pulses: on for 2 s, off for 10 s; 35% amplitude). The presence of the BSH enzymes was confirmed by SDS–PAGE, as described by Laemmli (1970). SDS–PAGE was carried out using 10% (w/v) polyacrylamide gels containing 0·1% (w/v) SDS.
The supernatant from the sonicated mixture was harvested after centrifugation at 13 000 g for 20 min at 4°C. This soluble fraction, which contained the BSH enzymes, was placed in a nickel–nitrilotriacetic acid (Ni2+-NTA) agarose column (Qiagen, Hilden, Germany). The same lysis buffer used to resuspend the cell pellet was used for equilibration of the column. Whole-cell lysate was allowed to bind to the resin. The column was then washed twice with lysis buffer containing 20 mmol l−1 imidazole. Bound BSH enzymes were eluted using lysis buffer containing 250 mmol l−1 imidazole.
The method used to determine the substrate specificity of the BSH enzymes was modified from Tanaka et al. (1999). Approximately 10 μl of each purified BSH enzyme was mixed with a 190 μl reaction mixture comprised of 100 mmol l−1 sodium phosphate (pH 6·0), 10 mmol l−1 conjugated bile acids (glyco- or tauro-conjugated bile salts: Sigma) and 10 mmol l−1 dithiothreitol (DTT). The reaction mixtures were incubated at 37°C for 10 min. Each reaction was stopped using 15% trichloroacetic acid, and the mixture was centrifuged at 20 000 g for 5 min to obtain the reaction sample. The samples were then incubated again at 37°C for 10 min. The ninhydrin assay was performed to measure the amount of amino acids released using a ninhydrin agent (1% ninhydrin in 0·2 ml of 0·5 mol l−1 citrate buffer, pH 5·5, 1·2 ml glycerol and 0·2 ml of 0·5 mol l−1 citrate buffer, pH 5·5). Next, the reaction sample was boiled for 15 min and allowed to cool for 3 min. Spectrophotometric analysis was conducted at A570 using a UV-1601PC spectrophotometer (Shimadzu, Kyoto, Japan), and enzyme activities were expressed as U ml−1. One unit of BSH activity was defined as the amount of enzyme that liberated 1 μmol of amino acids from the substrate per min. After unit values have been determined, they were transformed into relative activities, with the highest value designated as 100%. Substrate specificity assays were performed in triplicate in individual experiments to produce standard deviations.
BSH activity was tested at various temperatures (20–90°C, in intervals of 10°C) and pH values (pH 3–9) using a mixture of the BSH enzymes with 100 mmol l−1 sodium phosphate buffer (pH 6·0), 10 mmol l−1 bile acids and 10 mmol l−1 DTT. The buffer used depended on the pH range (sodium acetate buffer, pH 3–6; 100 mmol l−1 sodium phosphate buffer, pH 6–8; 100 mmol l−1 Tris buffer, pH 8–9). Each set-up was performed in triplicate in separate experiments to produce standard deviations.
Genetic characterization data of BSH B were reported by Oh et al. (2008). The initiation, termination, and protein sequence of BSH B are included in this study (Figs 2 and 3) for bioinformatic comparison and analysis. Nucleotide sequence analysis (Fig. 2) of BSH A and C from the genome sequence revealed that each contained a single complete open reading frame (ORF) of 981 and 978 nucleotides that encoded 326 and 325 amino acids, respectively. The single complete ORF of the two BSH enzymes each had a methionine start codon (ATG); however, BSH A had a TAG stop codon, whereas BSH C terminated with TAA. BSH A was preceded by a 5′-AGGAGG-3′ Shine–Dalgarno sequence, while BSH C was preceded by 5′-AGGAAA-3′. The distance of the promoter sequence elements −10 and −35 varied in each gene, being located upstream of the initiation codon at 38–61 nt in BSH A and at 74–97 nt in BSH C. One TG motif was identified in BSH C, but this was absent from the promoter region of BSH A (Fig. 2).
The deduced protein sequences of BSH A and C had theoretical molecular weights of 36·65 and 36·38 kDa and pI values of 5·19 and 4·70, respectively, as predicted by the Expasy tool available at http://web.expasy.org/compute_pi/ (Gasteiger et al. 2005). Protein Blast analysis of the amino acid sequences indicated that the BSHs exhibited homology to cholylglycine hydrolases from other LAB. Highest similarities for BSH A (98% similarity) were with the BSH alpha peptide of Lact. johnsonii 100-100 (96% identity) (GenBank accession no. AAG22541.1), the conjugated BSHs from Lact. johnsonii NCC 533 (95% identity) (NP_965212.1) and DPC 6026 (96% identity) (AEB93154.1), and the cholylglycine hydrolase of Lact. johnsonii ATCC 3320 (94% identity) (ZP_04007948.1). Likewise, it had 80% identity and 91% similarity to the cholylglycine hydrolase of Lactobacillus gasseri JV-V03 (ZP_07058051.1). BSH C, in contrast, was similar to the conjugated BSHs of Lact. johnsonii DPC 6026 (100% similarity, 99% identity) (AEB93337.1) and NCC 533 (99% similarity, 98% identity) (NP_965003.1).
ClustalW was used for further analysis of the protein sequences (Fig. 3). Interestingly, of the BSH enzymes present in Lact. johnsonii PF01, BSH A and C were similar to one another, with a 60% identity. However, in Blast analysis, BSH B was more identical to the cbsh-beta of Lact. johnsonii 100-100 (99% identity) than to other PF01 BSH enzymes. BSH C, however, did not have high similarity to the other BSH enzymes included in this study, compared with those exhibited by BSH A and BSH B of Lact. johnsonii PF01. BSH C of Lact. johnsonii PF01 only had 69% identity to the bsh B enzymes of Lact. acidophilus LA4 (ACL98174.1) and Lact. acidophilus NCFM (AAV42923.1).
Data from the crystal structure of penicillin V acylase (PVA) and the CBAH enzyme of Cl. perfringens (Coleman and Hudson 1995; Rossocha et al. 2005) identified conserved residues of the putative active sites (C, D, N, N, R) in different BSH sequences (Oh et al. 2008). Residues of the substrate pocket binding sites were also determined for the CBAH protein sequence (Rossocha et al. 2005). The active site residues are conserved in all of the BSH enzymes of Lact. johnsonii PF01 and also in those strains and species that were used to determine the difference in substrate specificity of the three BSH enzymes (Fig. 3). However, the substrate-binding pocket residues are not conserved, similar to the results in previous studies (Rossocha et al. 2005; Begley et al. 2006). The residues at position 68 (Ala-68), a substrate specificity binding site in the Cl. perfringens CBAH crystal structure, is noted because of its conservation of a polar residue (Cysteine, C and Tyrosine, Y) for tauro-conjugating-specific BSHs or a nonpolar residue (Phenylalanine, F or Alanine, A) in BSHs with preferential hydrolysis of glyco-conjugated bile salts, despite lower similarity of BSH A and B with each other than with BSH C.
PCR was performed to amplify the BSH-encoding genes of interest. Each amplified BSH-encoding gene was generated with primers containing specific restriction enzyme sites: BSH A (NdeI/HindIII) and BSH C (NdeI/XhoI), as shown in Table 1. Ampicillin-resistant transformants were selected using LB agar plates containing either 0·3% TDC with IPTG at 0·1 mmol l−1 or 0·3% GDC with IPTG at 0·1 mmol l−1. All plates were incubated anaerobically at 37°C for 24 h. Colonies expressing BSH A produced copious amounts of cholic acid on TDC plates, as did the colony expressing BSH C on GDC plates (Fig. 1). Restriction enzyme analysis indicated that all BSH-positive clones contained an c. 1-kb insert after PCR amplification (data not shown).
Each previously characterized BSH gene was inserted into a pET21b expression vector digested at the enzyme sites shown in Table 1. Escherichia coli BLR (DE3) was used for the introduction of the genes, and overexpression was induced with IPTG at 0·1 mmol l−1. Double digestion of the vector and gene with two restriction endonucleases were used to determine the orientation of the BSH gene. The primers were designed to be in the same direction as the T7 promoter in pET21b with no hanging ends, to prevent mutation. Cells grew normally up to an OD600 of 0·5–0·6.
Purification was performed using a Ni2+-NTA agarose column to extract rBSH enzymes from cell lysates. Overexpressed rBSH A and C enzymes, visualized by SDS–PAGE, had molecular weights of c. 36–37 kDa (Fig. 4), which were in agreement with those deduced from the amino acid sequences.
Enzyme assays were performed to determine the substrate specificities of the rBSH enzymes. Eight major human bile salts were used (Fig. 5), including both primary and secondary bile acids and both tauro- and glyco-conjugated bile salts. BSH A showed greater reactivity with tauro-conjugated bile salts, whereas BSH C showed greater hydrolysing activity with glyco-conjugated bile salts. Most BSH enzymes exhibit more efficient hydrolysis of glyco-conjugated bile salts (Coleman and Hudson 1995; Taranto et al. 1999; Tanaka et al. 2000; Kim et al. 2004; Liong and Shah 2005; Jiang et al. 2010); however, Lact. johnsonii PF01 had only one BSH enzyme that exhibited a preference for glycine (Fig. 5).
The substrate specificities of BSH enzymes among intestinal bacteria vary. Most LAB, including Bifidobacterium bifidum ATCC 11863 (Kim et al. 2004), Bifidobacterium longum SBT 2928 (Tanaka et al. 2000), Lact. gasseri Am1 (Jiang et al. 2010), and Lact. plantarum 80 CK102 and WCSF1 (Christiaens et al. 1992; Ha et al. 2006; Lambert et al. 2008), display preferential hydrolysis of glyco-conjugated bile salts. The majority of Lact. acidophilus, including 016, L1, ATCC 43121 (Corzo and Gilliland 1999), LA4 and LA11 (Jiang et al. 2010), display preferential reactivity with glyco-conjugated bile acids. However, the Lact. johnsonii 100-100 cbsh enzyme subunits α and β (Lundeen and Savage 1990; Elkins and Savage 1998) display preferential hydrolysis of tauro-conjugated bile salts. Lactobacillus acidophilus NCFM (McAuliffe et al. 2005) possesses both a tauro-conjugated BSH and a glyco-conjugated BSH. Similarly, PF01 hydrolysed both, with BSH A and BSH B (Oh et al. 2008) having specific affinity for tauro-conjugated bile salts, whereas BSH C had an affinity for glyco-conjugated bile salts (Fig. 5).
To further characterize the purified BSH enzymes, we determined the effects of various temperatures and pH values on their relative activities. The maximum hydrolysis activity of BSH A occurred at 55°C (Fig. 6a) and decreased rapidly at temperatures above 70°C. In contrast, the maximum activity of BSH C occurred at 70°C and decreased significantly above 80°C, indicating it to be more heat stable than BSH A and BSH B (Oh et al. 2008). The optimum pH values of the enzymes were identical; their activities peaked at pH 5·0 (Fig. 6b).
The substrate-hydrolysing capabilities of BSH enzymes are not yet fully understood because of the wide variety of those identified in a number of bacterial taxa. Thus, in this study, we characterized two purified recombinant Lact. johnsonii PF01 BSH enzymes, determined their hydrolysis activities towards specific amino acid moieties and highlighted the similarities and differences among the three BSH enzymes identified in this strain.
Nucleotide sequence analysis identified promoter sequence elements (Fig. 2), −35 and −10 hexamers with spacer regions of 16–17 nt, upstream of the start codon and were conserved in the DNA sequences of all three BSH enzymes, despite some variation. There was a TG motif immediately upstream of the −10 hexamer in both BSH B and C, but not in BSH A. Generally, the three BSH enzymes have the elements important for recognition and expression by E. coli (McCracken and Timms 1999; Oh et al. 2008), but the presence of the TG motif in BSH B and C can enhance the promoter strength (McCracken and Timms 1999; Burr et al. 2000). The presence of these important promoter elements make BSH B and C good candidate promoters for the construction of an E. coli–lactobacilli shuttle vector (Oh et al. 2008). Moreover, the larger spacer region between the ribosome-binding site (RBS) and the start codon may enhance translation. Preliminary experiments showed that expression of BSH C was the lowest, and BSH B the highest, in Lact. johnsonii PF01 under bile stress conditions (data not shown). The distances between the RBS and start codon in BSH B and BSH C were 12 and 7 nt, respectively. Even though the recommended spacer optima are 5–13 nt, as determined by statistical and genetic analysis, optima for efficient translation vary on a case-by-case basis, as was demonstrated by Berwal et al. (2010) using E. coli expression vectors. Further experimentation is required to determine the translational efficiency of these genes.
Blast search analysis of the BSH DNA sequences showed that these enzymes are part of the N-terminal nucleophile (Ntn) hydrolase superfamily, members of which have an N-terminal catalytic nucleophile that cleaves an amide bond (Oinonen and Rouvinen 2000). Multiple sequence alignment of BSH A, B and C demonstrated that they were homologous to other well-studied Ntn-hydrolase enzymes, such as PVA of Bacillus sphaericus and CBAH of Cl. perfringens (Coleman and Hudson 1995; Tikkanen et al. 1996; Kim et al. 2004; Rossocha et al. 2005). All three BSH enzymes possessed a Cys-2 active site (Figs 2 and 3), which serves as a nucleophile and proton donor and is important for catalysis (Kabsch et al. 1976; Rossocha et al. 2005).
DNA sequence similarities among the three BSH enzymes diminished downstream of the start codon, and the amino acid sequences varied to some extent outside of conserved regions. Although they had higher sequence similarities to BSH enzymes from other sources, the three enzymes showed significant homology to each other. Thus, all BSH enzymes in Lact. johnsonii PF01 were likely acquired through horizontal gene transfer because of their greater degree of similarity to other species, such as Lact. acidophilus and Lact. plantarum, than to each other (Elkins et al. 2001; McAuliffe et al. 2005; Begley et al. 2006). Similarly, Elkins et al. (2001) speculated that BSH genes from Lact. johnsonii 100-100 were acquired horizontally because of the presence of numerous BSH loci that encoded additional functionality (i.e. more effective gut colonization with broader BSH substrate specificity). Nevertheless, BSH C seems to also be common only among Lact. johnsonii strains. Our sequence alignment confirmed that active site residues were strictly conserved in all BSH enzymes, but residues for substrate recognition were less so (Rossocha et al. 2005). Leu-142, the function of which remains unknown (Kumar et al. 2004), was also conserved.
It has been suggested that most evolution occurs to broaden substrate specificity (Jiang et al. 2010). The divergence among genetic characteristics of BSH enzymes was revealed by the phylogenetic tree produced using ClustalW (Fig. 7). BSH enzymes with smaller genetic distances (indicating that they share a recent ancestor) clustered together and demonstrated similar amino acid specificities. Begley et al. (2006) and Patel et al. (2010) emphasized that substrate specificity may occur at amino acid or steroid cholyl moieties; however, available kinetic data suggest that these enzymes recognize their substrates predominantly at amino acid moieties (Coleman and Hudson 1995; Tanaka et al. 2000; Kim et al. 2004; Rossocha et al. 2005).
No BSH enzyme substrate specificity patterns could be deduced for cholyl moieties. Each had variable relative activity for cholic acid, chenodeoxycholic acid and deoxycholic acid; however, they also showed low deconjugation activity of the secondary bile acids, THDC and taurochenodeoxycholic acid (TCDC). Rossocha et al. (2005) explained that this broad substrate specificity is because of cholate binding primarily by hydrophobic interactions, wherein its hydroxyl substituents are not recognized by the enzyme through hydrogen bonding. Earlier studies using epimerized hydroxyl substituents of cholate reported similar results, with no effect on substrate-binding specificity (Batta et al. 1984; Rossocha et al. 2005). Nevertheless, the two BSH enzymes in this study showed distinct substrate specificities towards amino acid moieties. BSH A expressed high relative activity for TC, TDC and TCDC, while BSH C showed high relative activity for GC, GDC and GCDC (Fig. 5).
The box in Fig. 3 highlights Ala-68, a substrate-binding pocket residue of the CBAH enzyme of Cl. perfringens (Rossocha et al. 2005). Ala-68 lies within the isovaleric side chain of deoxycholate, where hydrogen bonds are formed (Rossocha et al. 2005). Ala-68 also seems to have close interaction with the β11 sheet, which is considered important for catalysis and substrate binding in Ntn-hydrolases because of its position within the αββα core structure (Oinonen and Rouvinen 2000). The substrate-binding pockets of the Lactobacillus BSH protein sequences exhibited a pattern in this region, in which all amino acids substituted in this site were neutral but varied in polarity. Selected BSH enzymes that hydrolyse glyco-conjugated bile salts had polar amino acids, Cys (C) or Tyr (Y), in this site, while those that hydrolysed tauro-conjugated bile salts had a nonpolar Phe (F) or Ala (A) residue. Various parameters can be evaluated, such as the polarity of specific residues, because addition of hydrophilic (polar) amino acids in the vicinity of hydroxyl substituents of the substrates can alter the affinities of BSH towards their respective substrates (Rossocha et al. 2005). Likewise, covarying sites may also be present, wherein when a change in one residue occurs, a similar change occurs in another site because of the reciprocal nature of the contact sites and the hydrogen bonding that occurs during stabilization of the enzyme (Rossocha et al. 2005; Yip et al. 2011).
The diversity of BSH enzymes and their amino acid sequences among genera, species and strains complicates the identification of the targets of the unique activities of these hydrolases. There seem to be no specific binding conditions, because substitution of amino acid moieties does not affect the hydrolytic activity of the amide group, as long as the cholate and amino acid moieties fit properly and are complementary to the substrate-binding pockets (Batta et al. 1984; Kumar et al. 2006). Similar to our results, even though preferential hydrolysis of particular bile salts was evident, those containing different amino acid moieties were also hydrolysed, albeit in smaller quantities.
Nevertheless, among members of a family, three-dimensional protein structures are generally more conserved among homologues than are protein sequences (Chothia and Lesk 1986). During evolution, protein structure is more stable and changes more slowly than the associated nucleotide sequence; similar sequences can maintain an identical structure, and distantly related sequences can fold into similar structures (Chothia and Lesk 1986; Sander and Schneider 1991; Krieger et al. 2003; Kaczanowski and Zielenkiewicz 2010) and maintain functionality. Thus, in terms of BSH enzyme diversity among LAB, the sequences diverged greatly despite significant similarities. Homology modelling is applicable in this case, as only sequences with <20% similarities have completely different structures (Chothia and Lesk 1986; Krieger et al. 2003).
The optimum temperatures and pH values for BSH of Lact. johnsonii PF01 were comparable to those of other cloned BSH enzymes, such as those of Bifidobacterium spp. (Kim et al. 2004). The maximum activity at pH 5·0 suggests that these enzymes can hydrolyse bile salts efficiently in the small intestine. Furthermore, they were stable at high temperatures. Other factors complicate the analysis of these enzymes. For example, BSH activity in Lactobacilli is regulated by the growth phase of the bacteria and the type of conjugated bile salt present in the growth medium (Lundeen and Savage 1990, 1992). BSH activity may also decrease in the presence of excessive amounts of cholic acid, which may induce the bacterium to protect itself rather than deconjugate the salts (Jiang et al. 2010), as occurred in bsh mutant Lact. acidophilus NCFM strains (McAuliffe et al. 2005). Further investigation under optimum conditions is required to rule out other factors that affect BSH activity.
The BSH enzymes of Lact. johnsonii PF01 exhibited diverse activities and characteristics. Each BSH enzyme showed higher similarity to those from other sources but relatively lower sequence identity among themselves. Nevertheless, the BSH enzymes were homologous, indicating evolutionary broadening of their substrate specificities. Identification of the specificity-determining residues of BSH enzymes is complex because of the low conservation of these areas among the available cocrystal structures. Our isolation of homologous but amino acid-specific BSH enzymes from this strain will facilitate the identification of the components that determine their substrate specificities and the sites that can be targeted by PCR-based site-directed mutagenesis and structure-driven computational and theoretical approaches (Yip et al. 2011). Construction of bsh mutants may allow prediction of the structures of PF01 BSH enzymes and facilitate more precise determination of their substrate specificities. Further studies are important for the selection of probiotic bacteria, because excessive deconjugation of tauro-conjugated salts has been implicated in negative health outcomes (Ridlon et al. 2006; Jiang et al. 2010). Information regarding the deconjugation properties and specificities of these BSH enzymes will enable their manipulation for use in probiotic supplements that lower total serum cholesterol with few or no adverse health consequences.
This work was supported by a grant from the Next-Generation BioGreen 21 Program (PJ00812701), Rural Development Administration, Republic of Korea.