Abstract

Taurine aminotransferases catalyze the first step in taurine catabolism in many taurine-degrading bacteria and play an important role in bacterial taurine metabolism in the mammalian gut. Here, we report the biochemical and structural characterization of a new taurine:2-oxoglutarate aminotransferase from the human gut bacterium Bifidobacterium kashiwanohense (BkToa). Biochemical assays revealed high specificity of BkToa for 2-oxoglutarate as the amine acceptor. The crystal structure of BkToa in complex with pyridoxal 5′-phosphate (PLP) and glutamate was determined at 2.7 Å resolution. The enzyme forms a homodimer, with each monomer exhibiting a typical type I PLP-enzyme fold and conserved PLP-coordinating residues interacting with the PLP molecule. Two glutamate molecules are bound in sites near the predicted active site and they may occupy a path for substrate entry and product release. Molecular docking reveals a role for active site residues Trp21 and Arg156, conserved in Toa enzymes studied to date, in interacting with the sulfonate group of taurine. Bioinformatics analysis shows that the close homologs of BkToa are also present in other anaerobic gut bacteria.

Introduction

Aminoethylsulfonate (taurine) is highly abundant in the human gut, originating both from food and from the cleavage of taurine-conjugated bile salts by bacterial bile salt hydrolases [1]. Taurine is thought to serve as a source of carbon, nitrogen and sulfur for certain gut bacteria, particularly for strict anaerobes residing in the nutrient-poor environment of the distal gut. A prominent example is the anaerobic sulfite-reducing bacterium Bilophila wadsworthia, which ferments taurine into ammonia, acetate and H2S, and is associated with inflammation and chronic human diseases [2,3]. Other gut anaerobes are thought to utilize taurine as a nitrogen source, converting it into hydroxyethylsulfonate (isethionate) as a byproduct. Although the exact species responsible have not yet been isolated, this reaction has been observed in cultures of anaerobic gut bacteria, and is thought to be the source of isethionate in mammalian tissues [4].

The biochemistry of bacterial taurine degradation has been extensively studied over the past decades [5]. However, the knowledge of the enzymes and biochemical pathways for taurine metabolism by diverse bacteria inhabiting the anaerobic mammalian gut is still lacking. A key enzyme in bacterial taurine metabolism is taurine aminotransferase, which catalyzes the conversion of taurine to sulfoacetaldehyde, with either pyruvate [Taurine:pyruvate aminotransferase (Tpa)] or 2-oxoglutarate [Taurine:2-oxoglutarate aminotransferase (Toa)] as the amine acceptor. This is the first chemical step in many of the taurine degradation pathways reported to date [5]. Taurine aminotransferases are classified as ω-aminotransferases, belonging to the class III aminotransferase family [6], a large and functionally diverse family containing 48 480 sequences to date (in the Pfam family PF00202).

Tpa was first characterized and sequenced from B. wadsworthia (BwTpa) [7]. Tpa sequences were later verified in several environmental bacteria, including Silicibacter pomeroyi (SpTpa) and Rhodococcus opacus (RoTpa), and found to be involved in diverse pathways for taurine degradation [8,9]. Tpa activity has also been detected in the lysate of the strict anaerobe Clostridium pasteurianum C1, which utilizes taurine as a sulfur source [10].

Toa has been characterized and identified in Klebsiella oxytoca TauN1 (KoToa), and Chromohalobacter salexigens (CsToa) [11,12], where it is involved in taurine nitrogen assimilation (Figure 1). In this pathway, taurine is imported by taurine ABC transporter, converted to sulfoacetaldehyde by Toa, and then reduced to isethionate by sulfoacetaldehyde reductase IsfD, a member of the short chain dehydrogenase/reductase family. Isethionate is exported as a waste product by the putative isethionate exporter IsfE. This pathway is also present in Klebsiella species from the human gut [12].

Proposed taurine nitrogen assimilation pathway and related gene clusters.

Figure 1.
Proposed taurine nitrogen assimilation pathway and related gene clusters.

(A) Proposed taurine nitrogen assimilation pathway in Klebsiella oxytoca TauN1, and Chromohalobacter salexigens. (B) Comparison of the BkToa, KoToa and CsToa genome neighborhoods.

Figure 1.
Proposed taurine nitrogen assimilation pathway and related gene clusters.

(A) Proposed taurine nitrogen assimilation pathway in Klebsiella oxytoca TauN1, and Chromohalobacter salexigens. (B) Comparison of the BkToa, KoToa and CsToa genome neighborhoods.

Because of their key role in taurine degradation pathways, taurine aminotransferases have the potential to serve as genetic markers for the ability to metabolize taurine. However, the systematic identification of taurine aminotransferases by bioinformatics is complicated by the large size of the aminotransferase family, and by the high level of diversity among taurine aminotransferase sequences (e.g. 33% identity between BwTpa and KoToa). Furthermore, none of the taurine aminotransferases has been structurally characterized. Determination of the crystal structure of a taurine aminotransferase would facilitate the identification and classification of new taurine aminotransferases and taurine-metabolizing bacteria.

In this work, we report the biochemical characterization and the crystal structure of a new Toa from Bifidobacterium kashiwanohense PV20-2 (BkToa, UniProt accession A0A0A7I435), an isolate from human infant feces. Bifidobacteria are common gastrointestinal bacteria inhabiting the distal gut, and BkToa is part of a gene cluster putatively involved in taurine nitrogen assimilation (Figure 1). In this pathway, isethionate is formed by a newly identified sulfoacetaldehyde reductase TauF, a member of the metal-dependent alcohol dehydrogenase family, described in detail in an accompanying paper [13]. In addition, we studied the phylogenetic relationship of BkToa to other reported taurine aminotransferases, with which it shares low homology (e.g. 44% identity with KoToa; 33% identity with BwTpa).

Materials and methods

General

Lysogeny broth (LB) medium was purchased from Oxoid Limited (Hampshire, U.K.). Water used in this work was ultrapure deionized water from Millipore Direct-Q. Acetonitrile was purchased from Concord Technology (MN, U.S.A.). Formic acid was purchased from Merck (NJ, U.S.A.). TALON cobalt resins were purchased from Clontech Laboratories Inc. (CA, U.S.A.). All protein purification chromatographic experiments were performed on an ÄKTA pure FPLC machine (GE Healthcare, U.S.A.). Chemicals were purchased from Sigma/Aldrich, J&K and Solarbio.

Cloning, expression and purification of BkToa

The codon-optimized gene fragment of BkToa was synthesized by General Biosystems Inc., and inserted into pET-28a-HT at the SspI restriction site, using the Gibson Assembly® cloning protocol [New England Biolabs (NEB), Ipwich, MA, U.S.A.] [14]. The resulting plasmid HT-BkToa contains a His6-tag and a Tobacco Etch Virus (TEV) protease cleavage site followed by BkToa open reading frame.

The HT-BkToa plasmid was transformed into Escherichia coli BL21 (DE3) cells. The transformant was grown in LB medium containing kanamycin (50 μg/ml) at 37°C in flasks in a shaker incubator at 220 rpm, and BkToa expression was induced with 1 mM IPTG overnight at 16°C. The cells were harvested by centrifugation at 8000×g for 20 min at 4°C.

Cell paste from 2 l culture was resuspended in 100 ml lysis buffer containing 20 mM Tris–HCl, pH 7.5, 200 mM NaCl, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM dithiothreitol (DTT). Cells were then lysed by probe sonication. After cell debris was removed by centrifugation at 13 000×g, the cell lysate was filtered and loaded onto a 10 ml TALON column pre-equilibrated with buffer A [20 mM Tris–HCl (pH 7.5), 200 mM NaCl]. The column was washed with 50 ml of buffer A containing 50 mM imidazole. The column was further washed with 150 ml of buffer A containing a gradient of imidazole from 100 to 200 mM and eluted with 50 ml of buffer A containing 200 mM imidazole. The light yellow fractions containing BkToa were pooled, TEV protease was added (TEV/BkToa 1:25 molar ratio), and the mixture was immediately dialyzed twice against 2 l buffer A containing 1 mM DTT, to remove imidazole. The dialyzed protein was then loaded onto a 10 ml TALON column again to retain TEV protease, which contains a His6 tag. The flow-through containing untagged BkToa was collected and dialyzed against 2 l buffer B (20 mM Tris–HCl, pH 7.5) containing 1 mM DTT. The protein solution was then loaded onto a 10 ml anion exchange Q column, washed with a 200 ml of a gradient of 100–400 mM NaCl and eluted with 500 mM NaCl in buffer B. The eluate was then concentrated with a centrifugal concentrator YM30 (Millipore) to 14 mg/ml. The concentrations of purified BkToa were calculated from its absorption at 280 nm (ɛ280 nm =  57 300 M−1 cm−1), measured using a NanoDrop One (Thermo Fisher Scientific). The purified BkToa was analyzed on a 10% SDS polyacrylamide gel.

Oligomeric state determination

A 2 ml solution of 14 mg/ml BkToa was injected into a Superdex200 gel filtration column (∼120 ml) and eluted over 120 min with buffer C [10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 150 mM KCl] at 1 ml/min. The same conditions were used to analyze a solution of molecular mass markers including thyroglobulin bovine (669 kDa), horse apoferritin (443 kDa), sweet potato β-amylase (200 kDa), bovine serum albumin (66 kDa), and bovine carbonic anhydrase (29 kDa) (Sigma MWGF 1000-1KT). The molecular mass of BkToa was calculated from its elution volume, using a second-degree polynomial for the relationship between log(molecular mass) and retention time. BkToa collected from the gel filtration chromatography was concentrated to 15 mg/ml using a centrifugal concentrator YM30 and subjected to biochemical analyses and crystallization.

Fluorescence-based thermal shift assays

BkToa melting curves were measured using fluorescence-based thermal shift assays [15]. Samples contained 0.2 mg/ml protein and 1× Sypro Orange protein gel stain solution (Sigma) in a total volume of 50 μl. Melting curves were obtained in a QuantStudio 6 Flex real-time PCR machine (Life Technologies, CA, U.S.A.). The temperature was increased from 10 to 95°C with an incremental rate of 0.033°C/s. The melting temperatures were obtained as the midpoint of each transition. The experiments were performed in triplicate.

IsfD-coupled activity assay for BkToa

To determine the optimal pH for BkToa, a 200 μl mixture containing 0.1 mM pyridoxal 5′-pyrophosphate (PLP), 5 mM 2-oxoglutarate (2-ketoglutarate, 2-KG), 5 mM taurine, 0.5 mM NADPH, 0.5 μM Klebsiella oxytoca TauN1 IsfD (sulfoacetaldehyde reductase) [16] in 100 mM of different buffers [2-(N-morpholino)ethanesulfonic acid (MES), pH 6.0; HEPES, pH 7.0; Tris–HCl, pH 8.0 and 9.0; 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO), pH 10.0] was pre-mixed in a 96-well plate and incubated at room temperature (RT) for 5 min, followed by the addition of 0.1 μM BkToa to initiate the reaction. Absorbance at 340 nm was monitored for 3 min. Negative controls omitting either taurine, Toa, or 2-KG were included.

To determine the amine acceptor specificity of BkToa, assays were performed exactly as described above at pH 8.0 with 0.3 μM IsfD and 2 μM BkToa using either 2-KG or pyruvate as amine acceptor.

To determine the kinetic parameters of BkToa, assays were performed exactly as described above at pH 8.0 with 0.1 μM BkToa in the presence of excess IsfD (0.5 μM). The concentration of one substrate (taurine or 2-KG) was fixed at 50 mM, while varying the concentration of the other substrate.

Glutamate dehydrogenase/alanine dehydrogenase-coupled activity assays for BkToa

To carry out the transaminase reaction, a 200 μl reaction mixture containing 100 mM Tris–HCl, pH 8.5, 0.1 mM PLP, 5 mM 2-KG, 5 mM taurine was pre-warmed in a 35°C water bath for 3 min, followed by the addition of 10 μg BkToa and incubation for 20 min. The enzyme was then heat-inactivated in a boiling water bath for 2 min. To detect the product, 100 μl of the reaction mixture was taken out and mixed with 100 μl of 10 mM NAD+, 70 mUnits bovine liver GLDH (glutamate dehydrogenase) (Sigma Catalog# 2626) in the same reaction buffer. Absorbance at 340 nm was monitored over 5 min using a plate reader (Tecan M200). Negative controls omitting either taurine, 2-KG, or Toa were included. An analogous assay was also performed with pyruvate replacing 2-KG as an amine acceptor, and 70 mUnits ALD (alanine dehydrogenase) for product detection.

LC–MS detection of sulfoacetaldehyde formation

The sulfoacetaldehyde product was detected by derivatization with 2,4-dinitrophenylhydrazine (DNPH) (J&K, Beijing, China) [16]. A 200 μl reaction mixture, containing 100 mM Tris–HCl, pH 8.0, 0.1 mM PLP, 5 mM 2-KG, 5 mM taurine, 10 μg BkToa was incubated for 5 min at RT. 100 μl of reaction solution was mixed with 1.1 ml of 0.73 M sodium acetate buffer (pH 5.0), followed by 800 μl of freshly prepared DNPH solution (40 mg dissolved in 100 ml methanol). The mixture was incubated at 50°C for 1 h and then filtered prior to LC–MS analysis.

LC–MS analysis was performed as previously described [16] on an Agilent 6420 Triple Quadrupole LC/MS instrument (Agilent Technologies, CA, U.S.A.). The samples (20 μl) were analyzed on an Agilent ZORBAX SB-C18 column (4.6 × 250 mm, product number 880975-902), equilibrated with 75% of 0.1% formic acid in H2O, 25% of 0.1% formic acid in CH3CN, and developed from 25% to 65% CH3CN at a flow rate of 1.0 ml/min. UV detection was set at 360 nm.

Co-crystallization of BkToa with PLP and glutamate

Initial screening of BkToa crystals was performed using an automated liquid handling robotic system (Gryphon, Art Robbins, CA, U.S.A.) in a 96-well format by the sitting-drop vapor diffusion method. The screens were set up at 295 K using various sparse matrix crystal screening kits from Hampton Research (CA, U.S.A.) and Molecular Dimensions (OH, U.S.A.). Several crystallization conditions gave plate-shaped crystals. After further optimization using the hanging-drop vapor diffusion method in 24-well plates, we obtained crystals large enough for single crystal X-ray diffraction studies (Supplementary Figure S4). The best condition yielding large crystals was 0.1 M MES, pH 5.0, 0.2 M ammonium chloride, 20% W/V PEG 6000, 0.5 mM PLP, 100 mM glutamate. Crystals were flash-cooled in liquid nitrogen using reservoir solution containing 25% glycerol as cryoprotectant. Diffraction data were collected on BL18U1 at Shanghai Synchrotron Radiation Facility (SSRF) to a resolution of 2.7 Å. The dataset was indexed, integrated and scaled using HKL3000 suite [17]. Molecular replacement was performed by PHENIX [18] using the crystal structure of an amine transaminase (PDB: 5LH9) as a searching model. The structure was manually built according to the modified experimental electron density using Coot [19] and further refined by PHENIX [18] in iterative cycles. The statistics for data collection and final refinement is presented in Table 1. All structural figures were generated using UCSF Chimera [20]. The atomic co-ordinates and structure factor amplitudes (accession code 6JIX) have been deposited into the protein data bank (http://www.rcsb.org).

Table 1
Data collection and refinement statistics for the BkToa crystal (PDB ID: 6JIX)
 Bifidobacterium kashiwanohense Toa (PDB ID: 6JIX) 
Data collection 
 Beamline SSRF 
 Wavelength (Å) 0.9795 
 Space group P 1 21 1 
 Cell dimensions 
  a, b, c (Å) 68.4, 164.5, 109.2 
  α, β, γ (°) 90.0, 105.8, 90.0 
 Number of unique reflections 66 598(6386) 
 Resolution (Å) 42.72–2.647 (2.742–2.647) 
 R-meas 0.133 (0.578) 
 R-pim 0.071 (0.304) 
 CC (1/2) 0.984 (0.837) 
 Average I/σ (I7.7 (3.7) 
 Completeness (%) 98.52 (94.49) 
 Redundancy 3.4 (3.5) 
Refinement 
 Ligand PLP, Glutamate 
 Resolution (Å) 44.29–2.65 
 Number of reflections 66 582(6385) 
Rwork/Rfree 0.1661 / 0.2262 
 Average B factor (Å231.61 
 R. m. s. deviations 
  Bond length (Å) 0.007 
  Bond angle (°) 0.90 
 Number of atoms 
  Protein 13 704 
  Ligands 144 
  Water 365 
 Number of protein residues 1785 
 MolProbity 
  Ramachandran favored (%) 95.27 
  Ramachandran allowed (%) 4.50 
  Ramachandran outliers 0.23 
  Rotamer outliers 0.00 
  Clashscore 9.68 
 Bifidobacterium kashiwanohense Toa (PDB ID: 6JIX) 
Data collection 
 Beamline SSRF 
 Wavelength (Å) 0.9795 
 Space group P 1 21 1 
 Cell dimensions 
  a, b, c (Å) 68.4, 164.5, 109.2 
  α, β, γ (°) 90.0, 105.8, 90.0 
 Number of unique reflections 66 598(6386) 
 Resolution (Å) 42.72–2.647 (2.742–2.647) 
 R-meas 0.133 (0.578) 
 R-pim 0.071 (0.304) 
 CC (1/2) 0.984 (0.837) 
 Average I/σ (I7.7 (3.7) 
 Completeness (%) 98.52 (94.49) 
 Redundancy 3.4 (3.5) 
Refinement 
 Ligand PLP, Glutamate 
 Resolution (Å) 44.29–2.65 
 Number of reflections 66 582(6385) 
Rwork/Rfree 0.1661 / 0.2262 
 Average B factor (Å231.61 
 R. m. s. deviations 
  Bond length (Å) 0.007 
  Bond angle (°) 0.90 
 Number of atoms 
  Protein 13 704 
  Ligands 144 
  Water 365 
 Number of protein residues 1785 
 MolProbity 
  Ramachandran favored (%) 95.27 
  Ramachandran allowed (%) 4.50 
  Ramachandran outliers 0.23 
  Rotamer outliers 0.00 
  Clashscore 9.68 

Docking of the taurine-PLP aldimine

The taurine-PLP aldimine ligand was prepared using the LigPrep module in Schrödinger software [21], and docked into the crystal structure of BkToa using the induced fit docking (IFD) module in Schrödinger [22]. The IFD method allows minor changes in the protein backbone and conformational changes in the amino acid side chains surrounding the binding pockets. We defined Lys279, a key residue in the pocket, as the receptor box center. Ligands within 30 kcal/mol of the best solution were re-docked into every refined low-energy protein structure using Glide XP [23,24]. The best solution was chosen based on the docking score.

Site-directed mutagenesis

Three single amino acid point mutations, W21A, D154A, and R156A, were introduced by site-directed mutagenesis using primers listed in Supplementary Table S1, and confirmed by sequencing. A 25 μl of PCR reaction contained 50 ng HT-BkToa plasmid as template, 0.4 μM forward and reverse primers, and the Fast Alteration DNA Polymerase (KM101 from TIANGEN, Beijing, China). The 18-cycle PCR reaction mixture was digested by DpnI to remove the template before transformed into FDM competent cells (TIANGEN).

Phylogenetic tree construction

A BLAST search of the UniRef50 database was conducted using BkToa as a query sequence. Representative sequences in this database share a maximal 50% identity [25]. The top 150 hits were compiled, and multiple sequence alignments were constructed using Clustal Omega [26]. A maximum likelihood phylogenetic tree was constructed using PHYML [27] with the LG substitution model [28]. The unequal rate of variation among amino acid sites was modeled with a γ-distribution with a shape parameter [29] of 1.3. The level of confidence for the branches was determined based on 100 bootstrap replicates [30]. The resulting consensus tree was rendered using the web-based program iTOL [31]. Sequence logos were plotted using WebLogo [32].

Results

Characterization of purified recombinant BkToa

BkToa was recombinantly produced and purified to near homogeneity (Figure 2A). Purified recombinant BkToa exhibited a characteristic absorption spectrum of a PLP-enzyme (Figure 2B). The gel filtration elution profile of purified BkToa shows a single symmetric peak centered at 73.8 ml (Figure 2C). The observed molecular mass for BkToa was 109.7 kDa (Figure 2C and Supplementary Figure S1), whereas the calculated molecular mass for BkToa monomer is 49.6 kDa. This suggests that BkToa exists as a dimer in solution, which is consistent with the oligomeric state of many other ω-aminotransferases in class III aminotransferase family [6]. Thermal stability of BkToa was measured by a fluorescence-based thermal shift assay, and the melting temperature was determined to be 47.5°C (Figure 2D).

SDS–PAGE, UV–Vis, gel filtration and thermostability analyses of purified BkToa.

Figure 2.
SDS–PAGE, UV–Vis, gel filtration and thermostability analyses of purified BkToa.

(A) 10% SDS gel with: lane 1, molecular mass marker; and lane 2–4, 1, 2, 4 µg of BkToa. (B) UV–Vis spectrum of purified BkToa. Inset: enlargement of the 300–500 nm range, showing the absorbance of the sub-stoichiometric PLP cofactor. The two tautomeric forms of the internal almidine are labeled for the two characteristic peaks. (C) Elution profile of BkToa using Superdex 200 gel filtration chromatography to determine BkToa molecular mass, estimated to be 109.7 kDa. Based on the gel filtration elution profile data in Supplementary Figure S1, the molecular mass of BkToa was calculated from its elution volume, using a second-degree polynomial for the relationship between log(molecular mass) and retention time. Black dots represent the five standard proteins and red dot represents BkToa (Inset). (D) Fluorescence-based melting temperature measurement.

Figure 2.
SDS–PAGE, UV–Vis, gel filtration and thermostability analyses of purified BkToa.

(A) 10% SDS gel with: lane 1, molecular mass marker; and lane 2–4, 1, 2, 4 µg of BkToa. (B) UV–Vis spectrum of purified BkToa. Inset: enlargement of the 300–500 nm range, showing the absorbance of the sub-stoichiometric PLP cofactor. The two tautomeric forms of the internal almidine are labeled for the two characteristic peaks. (C) Elution profile of BkToa using Superdex 200 gel filtration chromatography to determine BkToa molecular mass, estimated to be 109.7 kDa. Based on the gel filtration elution profile data in Supplementary Figure S1, the molecular mass of BkToa was calculated from its elution volume, using a second-degree polynomial for the relationship between log(molecular mass) and retention time. Black dots represent the five standard proteins and red dot represents BkToa (Inset). (D) Fluorescence-based melting temperature measurement.

BkToa is a taurine:2-KG aminotransferase

Taurine:2-KG aminotransferase activity of BkToa was detected using a coupled spectrophotometric assay with the NADPH-dependent sulfoacetaldehyde reductase IsfD (Figure 3A). The optimal pH was determined to be 8.0 for BkToa (Supplementary Figure S2). No reaction was observed in negative controls omitting either taurine, 2-KG, or BkToa (Figure 3A). No activity was detected with pyruvate replacing 2-KG as the amine acceptor, suggesting that the physiological acceptor is 2-KG (Figure 3A). Kinetic parameters were obtained from the IsfD-coupled assays (kcat= 3.7 s−1, KM for taurine = 1.1 mM, and KM for 2-KG = 1.8 mM, Supplementary Figure S3, Table 2).

BkToa activity assays.

Figure 3.
BkToa activity assays.

(A) Coupled enzyme activity assays for BkToa, as shown in the reaction equation. Transamination from taurine catalyzed by BkToa generates sulfoacetaldehyde. The assays then monitor NADPH consumption accompanying sulfoacetaldehyde reduction by IsfD. (B) Coupled enzyme activity assays for BkToa, as shown in the reaction equation. Transamination from taurine to 2-KG catalyzed by BkToa generates glutamate, which is detected by assays monitoring NADH formation accompanying oxidation by GLDH. Control assays omitting either substrate or enzyme are labeled. The assay with pyruvate as amine acceptor was performed with ALD instead of GLDH as coupled enzyme.

Figure 3.
BkToa activity assays.

(A) Coupled enzyme activity assays for BkToa, as shown in the reaction equation. Transamination from taurine catalyzed by BkToa generates sulfoacetaldehyde. The assays then monitor NADPH consumption accompanying sulfoacetaldehyde reduction by IsfD. (B) Coupled enzyme activity assays for BkToa, as shown in the reaction equation. Transamination from taurine to 2-KG catalyzed by BkToa generates glutamate, which is detected by assays monitoring NADH formation accompanying oxidation by GLDH. Control assays omitting either substrate or enzyme are labeled. The assay with pyruvate as amine acceptor was performed with ALD instead of GLDH as coupled enzyme.

Table 2
Kinetic parameters of BkToa
kcat (s−1KM (taurine, mM) kcat/KM(taurine) (M−1·s−1KM (2-KG, mM) kcat/KM(2-KG) (M−1·s−1
3.7 ± 0.1 1.1 ± 0.1 3364 1.8 ± 0.1 2056 
kcat (s−1KM (taurine, mM) kcat/KM(taurine) (M−1·s−1KM (2-KG, mM) kcat/KM(2-KG) (M−1·s−1
3.7 ± 0.1 1.1 ± 0.1 3364 1.8 ± 0.1 2056 

Formation of glutamate as a reaction product was confirmed by an assay with GLDH (Figure 3B). Replacement of 2-KG with pyruvate as the amine acceptor did not lead to the formation of alanine, as detected in an analogous assay with ALD (Figure 3B), demonstrating that BkToa is not a Tpa. Formation of sulfoacetaldehyde was confirmed by LC–MS (Figure 4). No sulfoacetaldehyde was detected in negative controls omitting either taurine, 2-KG or BkToa.

LC–MS analysis of product formation in BkToa-catalyzed reaction.

Figure 4.
LC–MS analysis of product formation in BkToa-catalyzed reaction.

(A) Elution profiles of the LC–MS assays monitoring absorbance at 360 nm. (B) The ESI (−) m/z spectrum of the sulfoacetaldehyde-DNPH and 2-KG-DNPH peaks in (A).

Figure 4.
LC–MS analysis of product formation in BkToa-catalyzed reaction.

(A) Elution profiles of the LC–MS assays monitoring absorbance at 360 nm. (B) The ESI (−) m/z spectrum of the sulfoacetaldehyde-DNPH and 2-KG-DNPH peaks in (A).

Crystal structure of BkToa in complex with PLP and glutamate

The crystal structure of BkToa in complex with PLP and glutamate was solved at 2.7 Å resolution. The asymmetric unit contains two BkToa dimers (Figure 5A). Each monomer exhibits a typical type I PLP-enzyme fold [33], consisting of a small domain formed by two discontinuous residue stretches (residues 1–63 and 334–447), and a large domain (residues 64–333), similar to previously characterized aminotransferases (Figure 5B). The PLP-binding site is located at the interface of the large and small domains (Figure 5B). Structure-based sequence alignment of BkToa with KoToa, CsToa, BwTpa, and four other structurally characterized ω-aminotransferases (Figure 5C) showed that the PLP-binding residues mentioned above are conserved in these aminotransferases, consistent with previous analyses [6].

Crystal structure of BkToa.

Figure 5.
Crystal structure of BkToa.

(A) The quaternary structure of the BkToa homodimer is shown in ribbon with two subunits (blue and grey). The PLP cofactor and glutamate are displayed in stick representation. (B) Domain structure of a BkToa monomer is shown in ribbon. Small domain shown in blue is composed of an N terminal segment (sky blue) and a C terminal segment (light blue) and the large domain is shown in green. (C) Structure-based sequence alignments between BkToa, KoToa, CsToa and selected ω-aminotransferases of known structures with high sequence identities including 5G09 (S-selective ω-aminotransferase from Bacillus megaterium), 4GRX (ω-aminotransferase from Paracoccus denitrificans), 4A6T (ω-aminotransferase from Chromobacterium violaceum) and 5TI8 (putrescine aminotransferase from Pseudomonas sp.). Conserved key residues involved in PLP binding are highlighted in yellow, and the glycine loop with amino acid backbone involved in PLP interaction is boxed. Catalytic lysine residue is shown in blue. The key W residues and the DXR motif involved in taurine binding and substrate specificity, present in Toa, but not in other aminotransferases are highlighted in purple.

Figure 5.
Crystal structure of BkToa.

(A) The quaternary structure of the BkToa homodimer is shown in ribbon with two subunits (blue and grey). The PLP cofactor and glutamate are displayed in stick representation. (B) Domain structure of a BkToa monomer is shown in ribbon. Small domain shown in blue is composed of an N terminal segment (sky blue) and a C terminal segment (light blue) and the large domain is shown in green. (C) Structure-based sequence alignments between BkToa, KoToa, CsToa and selected ω-aminotransferases of known structures with high sequence identities including 5G09 (S-selective ω-aminotransferase from Bacillus megaterium), 4GRX (ω-aminotransferase from Paracoccus denitrificans), 4A6T (ω-aminotransferase from Chromobacterium violaceum) and 5TI8 (putrescine aminotransferase from Pseudomonas sp.). Conserved key residues involved in PLP binding are highlighted in yellow, and the glycine loop with amino acid backbone involved in PLP interaction is boxed. Catalytic lysine residue is shown in blue. The key W residues and the DXR motif involved in taurine binding and substrate specificity, present in Toa, but not in other aminotransferases are highlighted in purple.

The electron density for the PLP cofactor is well defined in all four monomers in the asymmetric unit (Figure 6A). In the structure, PLP does not form an internal Schiff base with the conserved Lys279. The distance between the Nε-atom of Lys279 and the aldehyde C-atom of PLP is 3.1 Å. The phosphate group of PLP is stabilized by the backbone of Gly113, Gly114 and Ala115. The hydrophobic side chains of Tyr141 and Val252 stack against the pyridine ring of PLP. The pyridine nitrogen forms a hydrogen bond with Asp250 (Figure 6A).

PLP in the active site of the crystal structure and docking of taurine-PLP external aldimine to the active site.

Figure 6.
PLP in the active site of the crystal structure and docking of taurine-PLP external aldimine to the active site.

(A) PLP-binding site. Interacting residues are displayed and labeled. The hydrogen bonds involved in PLP binding are indicated by dashed lines. 2Fo-Fc electron densities for PLP are shown at 1.0σ. (B) Docking of taurine-PLP external aldimine to the active site. Key residues involved in substrate binding are displayed and labeled. The hydrogen bonds and/or salt bridges contributing to the protein–substrate interaction are indicated by dashed lines. The Thr311 residue from the second monomer is colored in blue.

Figure 6.
PLP in the active site of the crystal structure and docking of taurine-PLP external aldimine to the active site.

(A) PLP-binding site. Interacting residues are displayed and labeled. The hydrogen bonds involved in PLP binding are indicated by dashed lines. 2Fo-Fc electron densities for PLP are shown at 1.0σ. (B) Docking of taurine-PLP external aldimine to the active site. Key residues involved in substrate binding are displayed and labeled. The hydrogen bonds and/or salt bridges contributing to the protein–substrate interaction are indicated by dashed lines. The Thr311 residue from the second monomer is colored in blue.

Docking of the taurine-PLP aldimine intermediate suggested a possible role for Trp21, Tyr141, Arg156 and Thr311 in coordinating the taurine sulfonate group (Figure 6B). Among these residues, Tyr141 and Thr311 are also involved in interacting with PLP, as described above. Arg156 forms salt bridges, with the sulfonate group of taurine and the carboxylate group of Asp154. Sequence alignment revealed that Trp21, Asp154 and Arg156 are present in previously characterized taurine aminotransferases, but not in other structurally characterized ω-aminotransferases, suggesting that they may contribute to substrate specificity for taurine (Figure 5C). Structural comparison between BkToa and other ω-aminotransferases demonstrates high similarity in protein overall folding and PLP interacting residues (Supplementary Figure S5). In contrast, Trp21, Asp154 and Arg156 in BkToa that may contribute to substrate specificity for taurine were replaced by various amino acid residues in these ω-aminotransferases (Supplementary Figure S5). To further test our hypothesis, site-directed mutagenesis was performed. The W21A, D154A and R156A mutant BkToA were purified to homogeneity (Supplementary Figure S6). The IsfD-coupled assays for these mutant enzymes exhibited a complete loss of enzyme activity, consistent with the hypothesis that these residues are indeed interacting with the substrate (Figure 3A).

The crystal structure also contains two glutamate molecules, occupying two sites in a pocket adjacent to the active site. These two sites connect the PLP-binding site to the surface of the protein and might constitute a channel for substrate entry and product release (Figure 7).

Surface diagram and zoomed-in view revealing PLP and glutamate-binding pocket.

Figure 7.
Surface diagram and zoomed-in view revealing PLP and glutamate-binding pocket.

The second monomer is colored in blue. Inset: Zoomed-in view of the binding pocket. 2FoFc electron densities for both glutamate molecules are shown at 1.0σ.

Figure 7.
Surface diagram and zoomed-in view revealing PLP and glutamate-binding pocket.

The second monomer is colored in blue. Inset: Zoomed-in view of the binding pocket. 2FoFc electron densities for both glutamate molecules are shown at 1.0σ.

Phylogenetic analysis of BkToa

To explore the relationship between BkToa and other characterized taurine aminotransferases, a phylogenetic tree was constructed from the 150 closest homologs of BkToa among the representative sequences in the UniRef50 database (Figure 8A). Known taurine aminotransferases were located in three of the UniRef clusters on distant branches of the phylogenetic tree (Figure 8A). The cluster UniRef50_A0A0H4P1N9 contains 199 sequences including BkToa. The cluster UniRef50_A0A0N9XFD2 contains 1968 sequences including KoToa [11] and CsToa [12]. The cluster UniRef50_Q9APM5 contains 715 sequences including BwTpa [7] and the Tpa from Rhodobacter capsulatus [34], Silicibacter pomeroyi [8] and Neptuniibacter caesariensis [35]. None of the other clusters in the phylogenetic tree contains taurine aminotransferases that were previously characterized.

Phylogenetic tree of ω-aminotransferases and conservation of active site residues.

Figure 8.
Phylogenetic tree of ω-aminotransferases and conservation of active site residues.

(A) Maximum likelihood phylogenetic tree of 150 closest homologs of BkToa in the UniRef50 database. The labels indicate the representative sequence for each UniRef50 cluster. The clusters containing characterized taurine aminotransferases are UniRef50_A0A0H4P1N9 (blue, BkToa), UniRef50_A0A0N9XFD2 (yellow, Toa from Klebsiella oxytoca and Chromohalobacter salexigens) and UniRef50_Q9APM5 (green, Tpa from B. wadsworthia, Rhodobacter capsulatus, Silicibacter pomeroyi, Neptuniibacter caesariensis). (B) Sequence logos indicating the conservation of putative PLP- and taurine-coordinating active-site residues (BkToa: Tyr141, Asp250, Val252, Lys279 and Thr311). From top to bottom: the 150 representative sequences used in phylogenetic tree construction, the 199 sequences in UniRef50_A0A0H4P1N9 (BkToa), and the 1968 sequences in UniRef50_A0A0N9XFD2 (KoToa).

Figure 8.
Phylogenetic tree of ω-aminotransferases and conservation of active site residues.

(A) Maximum likelihood phylogenetic tree of 150 closest homologs of BkToa in the UniRef50 database. The labels indicate the representative sequence for each UniRef50 cluster. The clusters containing characterized taurine aminotransferases are UniRef50_A0A0H4P1N9 (blue, BkToa), UniRef50_A0A0N9XFD2 (yellow, Toa from Klebsiella oxytoca and Chromohalobacter salexigens) and UniRef50_Q9APM5 (green, Tpa from B. wadsworthia, Rhodobacter capsulatus, Silicibacter pomeroyi, Neptuniibacter caesariensis). (B) Sequence logos indicating the conservation of putative PLP- and taurine-coordinating active-site residues (BkToa: Tyr141, Asp250, Val252, Lys279 and Thr311). From top to bottom: the 150 representative sequences used in phylogenetic tree construction, the 199 sequences in UniRef50_A0A0H4P1N9 (BkToa), and the 1968 sequences in UniRef50_A0A0N9XFD2 (KoToa).

We next investigated the conservation of residues putatively involved in interaction with taurine and PLP. Among the representative sequences used for the construction of the phylogenetic tree, the PLP-coordinating residues (Tyr141, Asp250, Val252, Lys279 and Thr311) are widely conserved. However, the putative taurine-coordinating residues (Trp21 and Arg156) are poorly conserved, suggesting variations in the substrate-binding pocket among the sequences in the tree (Figure 8B). Within the two Toa clusters UniRef50_A0A0H4P1N9 (BkToa) and UniRef50_A0A0N9XFD2 (KoToa), both PLP- and taurine-coordinating residues are conserved, suggesting a similar mode of substrate binding between these two clusters, and allowing us to hypothesize that enzymes within these two clusters possess taurine aminotransferase activity or catalyze the transamination of substrates with similar chemical structures. Due to the low homology between BkToa and BwTpa, we were unable to confidently ascertain whether Trp21 and Arg156 are conserved in BwTpa, or whether it has a different mode of substrate binding.

Discussion

The characterization of BkToa adds to the diversity of enzymes involved in taurine metabolism, particularly in anaerobic human gut bacteria. Its genome context suggests a role in nitrogen assimilation, and biochemical assays demonstrate that the amine acceptor BkToa is 2-KG and not pyruvate, similar to previously studied taurine aminotransferases involved in nitrogen assimilation [11,12]. The metabolic functions of Toa in Bifidobacteria and other anaerobic bacteria are currently being investigated in our laboratory. Some Bifidobacteria, including B. kashiwanohense, also encode a bile salt hydrolase (UniProt A0A0A7I2N8) that cleaves taurine-conjugated bile salts, although the metabolic function of this reaction is not yet understood [36].

The 2.7 Å crystal structure of BkToa is, to our knowledge, the only structure of a taurine aminotransferase reported to date. Molecular docking and sequence alignments led us to propose that Trp21 and Arg156, conserved in Toa, are involved in coordinating the sulfonate group and may facilitate future bioinformatics prediction of more taurine aminotransferases. Sulfonate metabolism by gut bacteria is receiving increased attention due to its relevance to H2S production human disease, and the crystal structure will greatly facilitate further studies of taurine metabolism in the microbiome.

Abbreviations

     
  • 2-KG

    2-ketoglutarate

  •  
  • ALD

    Bacillus subtilis alanine dehydrogenase

  •  
  • BkToa

    Bifidobacterium kashiwanohense Toa

  •  
  • BwTpa

    B. wadsworthia Tpa

  •  
  • CAPSO

    3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid

  •  
  • CsToa

    Chromohalobacter salexigens Toa

  •  
  • DNPH

    2,4-dinitrophenylhydrazine

  •  
  • DTT

    dithiothreitol

  •  
  • GLDH

    glutamate dehydrogenase

  •  
  • HEPES

    4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

  •  
  • IsfD

    sulfoacetaldehyde reductase

  •  
  • KoToa

    Klebsiella oxytoca TauN1 Toa

  •  
  • LB

    Lysogeny broth

  •  
  • MES

    2-(N-morpholino)ethanesulfonic acid

  •  
  • NEB

    New England Biolabs

  •  
  • PLP

    pyridoxal 5′-phosphate

  •  
  • PMSF

    phenylmethylsulfonyl fluoride

  •  
  • RoTpa

    Rhodococcus opacus Tpa

  •  
  • RT

    room temperature

  •  
  • SpTpa

    Silicibacter pomeroyi Tpa

  •  
  • SSRF

    Shanghai Synchrotron Radiation Facility

  •  
  • TEV

    tobacco etch virus

  •  
  • Toa

    taurine:2-oxoglutarate aminotransferase

  •  
  • Tpa

    taurine:pyruvate aminotransferase

Author Contribution

M.L., J.Y., Y.Z. (Zhou) and G.H. designed and carried out experiments with BkToa cloning, expression, purification, crystallization and enzyme activity assays. Y.W. designed and carried out experiments with bioinformatics and was involved in conceptualizing the project and writing the manuscript. L.L. was involved in collecting and analyzing the X-Ray diffraction data. P.C. was involved in structure modeling. E.L.A., H.Z., Z.Y. and Y.Z. (Zhang) were involved in conceptualizing the project, getting grants for the project, overall supervision of the project and writing the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China, Grant 31870049 and 31570060 (Y.Z.), National Key Research and Development Program of China (2017YFD0201400, 2017YFD0201403) (Z.Y.), and the Agency for Science, Research and Technology of Singapore Visiting Investigator Program (H.Z.).

Acknowledgements

We thank the instrument analytical center of School of Pharmaceutical Science and Technology at Tianjin University for providing the LC–MS analysis and Mr Zhi Li and Prof. Xiangyang Zhang for their helpful discussion. We thank Dr Jun Xu for the assistance in using the in-house X-ray diffraction machine at Tianjin University, and the staff at SSRF for the assistance in using the beamline BL18U1.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Author notes

*

These authors contribute to this work equally.