Skip to main content
NCBI home page
The Journal of Biological Chemistry logoLink to The Journal of Biological Chemistry
. 2024 May 13;300(6):107371. doi: 10.1016/j.jbc.2024.107371

Bacterial cysteate dissimilatory pathway involves a racemase and d-cysteate sulfo-lyase

Chunxiu Liu 1, Kailiang Ma 1, Li Jiang 1, Xumei Liu 1, Yang Tong 1, Sen Yang 1, Xinghua Jin 2, Yifeng Wei 3,∗, Yan Zhang 1,4,5,6,∗
PMCID: PMC11193023  PMID: 38750791

Abstract

The sulfite-reducing bacterium Bilophila wadsworthia, a common human intestinal pathobiont, is unique in its ability to metabolize a wide variety of sulfonates to generate sulfite as a terminal electron acceptor (TEA). The resulting formation of H2S is implicated in inflammation and colon cancer. l-cysteate, an oxidation product of l-cysteine, is among the sulfonates metabolized by B. wadsworthia, although the enzymes involved remain unknown. Here we report a pathway for l-cysteate dissimilation in B. wadsworthia RZATAU, involving isomerization of l-cysteate to d-cysteate by a cysteate racemase (BwCuyB), followed by cleavage into pyruvate, ammonia and sulfite by a d-cysteate sulfo-lyase (BwCuyA). The strong selectivity of BwCuyA for d-cysteate over l-cysteate was rationalized by protein structural modeling. A homolog of BwCuyA in the marine bacterium Silicibacter pomeroyi (SpCuyA) was previously reported to be a l-cysteate sulfo-lyase, but our experiments confirm that SpCuyA too displays a strong selectivity for d-cysteate. Growth of B. wadsworthia with cysteate as the electron acceptor is accompanied by production of H2S and induction of BwCuyA. Close homologs of BwCuyA and BwCuyB are present in diverse bacteria, including many sulfate- and sulfite-reducing bacteria, suggesting their involvement in cysteate degradation in different biological environments.

Keywords: cysteate, sulfo-lyase, racemase, Bilophila wadsworthia, terminal electron acceptor

Sulfonates, characterized by a C-SO3− moiety with sulfur in the +4 oxidation state, encompass a range of natural products and are an important class of organosulfur compounds found in many environments (1, 2, 3). The unique physical and chemical properties of sulfonates include their membrane impermeability, ionizability, and relatively inert C-S bond, making them well suited for roles as osmolytes (e.g., taurine), membrane lipids (e.g., capnine and sulfoquinovosyl-diacylglycerol), and coenzymes (e.g., coenzyme M) in biological systems (4, 5, 6). l-cysteate (2-amino-3-sulfopropionic acid) is a common sulfonate natural product, first discovered in 1946 as a product of cysteine oxidation in weathered wool and later as a component of spider webs (7, 8). In cats, dietary l-cysteate has been shown to serve as a precursor for the biosynthesis of taurine (9) (Fig. S1A). This zwitterionic sulfonate plays a vital role as an osmolyte in various mammalian cell types. In marine phytoplankton, l-cysteate has been proposed as an intermediate in the biosynthesis of various sulfonate osmolytes [taurine, isethionate, dihydroxypropanesulfonate (DHPS), and sulfolactate] (Fig. S1A) (10). In certain Bacteroidetes bacteria, l-cysteate also serves as an intermediate in the biosynthesis of the sulfonolipid capnine (Fig. S1B) (11), essential for gliding motility and virulence (12).

Many bacteria are known to be capable of degrading sulfonates, including l-cysteate, as sources of sulfur, carbon, nitrogen, and energy for growth, and the associated enzymes and metabolic pathways play important roles in organosulfur mineralization in different envvironments (13, 14, 15, 16). Two l-cysteate dissimilatory pathways have been studied in detail, involving sulfonate C-S cleavage by either a Fe(II)-containing (R)-sulfolactate sulfo-lyase (SuyAB) (16), or a PLP-dependent “l-cysteate sulfo-lyase” (CuyA) (13). In the nitrate-reducing facultative aerobic bacterium Paracoccus pantotrophus NKNCYSA, l-cysteate is first converted to 3-sulfopyruvate by an (R)-cysteate: 2-oxoglutarate aminotransferase, followed by reduction to 3-sulfolactate by the NADH-dependent sulfolactate dehydrogenase and C-S bond cleavage by SuyAB to yield sulfite and pyruvate (Fig. S1C) (15, 16). Alternatively, in the aerobic marine bacterium Silicibacter pomeroyi DSS-3T, the C-S bond of l-cysteate is reported to be directly cleaved by CuyA to yield sulfite and 2-aminoacrylate, which undergoes tautomerization to 2-iminopropanoate and subsequent hydrolysis to pyruvate and ammonium (13). l-cysteate is also degraded by anaerobic bacteria, including many sulfate- and sulfite-reducing bacteria (SSRB), although the enzymes involved have not been studied in detail (16). SSRB play important roles in sulfonate sulfur mineralization in anaerobic environments including the human digestive tract. They utilize the sulfonate-derived sulfite as a terminal electron acceptor (TEA) to generate H2S (17), which has been linked to damage to the mucosal barrier, inflammatory conditions such as colitis, and colon cancer (18, 19).

Among the human gut SSRB, the pathobiont Bilophila wadsworthia has attracted interest for its unique metabolism and disease association, closely linked to its metabolism of sulfonates. B. wadsworthia lacks ATP sulfurylase and adenosine 5′-phosphosulfate (APS) reductase, and is thus unable to derive its TEA from sulfate (20, 21). Instead, it derives sulfite as a TEA by degrading a wide range of sulfonate substrates. Recent research on B. wadsworthia have led to the identification of enzymes involved in the degradation of isethionate (22) and taurine (derived from taurine-conjugated bile salts) (23) as well as DHPS (24), sulfolactate (25), and sulfoacetate (derived from bacterial metabolism of dietary sulfoquinovose) (26). Previous studies also showed that B. wadsworthia RZATAU can degrade l-cysteate generating TEA (23). Growth on l-cysteate was accompanied by induction of a 35 kDa protein proposed to be a CuyA homolog (13, 16), although the sequence was not identified.

In this study, we present the identification and biochemical characterization of B. wadsworthia CuyA (BwCuyA). Contrary to previous reports, our enzymatic assays with recombinant BwCuyA show 50 times higher activity for d-cysteate than for l-cysteate. We further report that B. wadsworthia contains a novel cysteate racemase (BwCuyB) that catalyzes the conversion of l-cysteate to d-cysteate. CuyA is found in association with CuyB in many bacteria, including SSRB, suggesting a similar mechanism of l-cysteate degradation.

Results

Enzymes of the PLP-dependent family in B. wadsworthia

The previously characterized S. pomeroyi DSS-3T “l-cysteate sulfo-lyase” (SpCuyA) belongs to the type II PLP-dependent enzymes family (Pfam PF00291). To identify candidate B. wadsworthia CuyA (BwCuyA) sequences, we analyzed the eight PF00291 enzymes in its genome of the sequenced strain B. wadsworthia (strain 3_1_6). Among them, E5Y7Z0 was excluded due to its large size (52 kDa). The seven remaining enzymes, annotated as d-cysteine desulfhydrase, tryptophan synthase, diaminopropionate ammonia-lyase, threonine synthase, or cysteine synthase, were ranked in order of sequence identity to SpCuyA, and their genome neighborhoods were analyzed (Fig. 1A). We initially investigated E5Y294, annotated as threonine synthase, as it is located adjacent to a “tricarboxylate transporter” that may serve as the transporter for l-cysteate. However, the recombinant enzyme displayed no cysteate sulfo-lyase activity (data not shown). Therefore, we next investigated E5Y343, annotated as d-cysteine desulfhydrase, as a candidate BwCuyA, as it exhibits the highest sequence identity (51.8%) to SpCuyA.

Figure 1.

Figure 1

Open in a new tab

Genome neighborhoodsof CuyA in S. pomeroyi DSS-3Tand CuyA homologs in B. wadsworthia (strain 3_1_6) with the proposedl-cysteate metabolism pathway in B. wadsworthia.A, arrangement of CuyA in S. pomeroyi DSS-3T and genes encoding PLP-dependent family (Pfam PF00291) enzymes (∼35 kDa) in B. wadsworthia (strain 3_1_6). The Uniprot IDs of these enzymes are Q5LL69, E5Y343, E5Y9P0, E5YAD5 E5Y899, E5Y4G8, E5Y294 and E5Y5R3, respectively. The percentage values indicate percent identities of amino acid sequences to SpCuyA. TR, transcriptional regulator. B, the proposed pathway for import and metabolism of l-cysteate in B. wadsworthia.

Identification of BwCuyA as a d-cysteate sulfo-lyase

To facilitate biochemical assays, we recombinantly produced the candidate BwCuyA (Fig. S2A) and PpALD (Pelosinus propionicus alanine dehydrogenase, previously characterized by our laboratory) (Fig. S2B) (27) as a coupling enzyme for the spectrophotometric assay. The UV-Vis absorbance spectrum of BwCuyA exhibited characteristic features of the PLP cofactor (Fig. S2C). Incubation of BwCuyA with l-cysteate, PpALD, NADH, and NH4Cl led to a time-dependent decrease in absorbance at 340 nm, indicating that BwCuyA catalyzes the conversion of l-cysteate to pyruvate, which is subsequently reduced by PpALD, with the consumption of NADH (27). However, despite efforts to optimize the assay conditions, the activity of BwCuyA remained low (kobs = 0.06 s−1). Examination of the genome neighborhood of BwCuyA revealed the presence of a dicarboxylate: cation symporter family (SDF) transporter, a member of the PLP-independent aspartate racemase family (CuyB, E5Y345, Pfam PF01177), and a homolog of 2-iminobutanoate/2-iminopropanoate deaminase RidA, which usually exhibits enamine/imine deaminase activity, capable of accelerating the release of ammonia from reactive enamine/imine intermediates of the PLP-dependent enzymes (Fig. 1A) (28). We thus hypothesized that CuyB is a cysteate racemase, and that the physiological substrate of CuyA is d-cysteate. In the newly proposed pathway, l-cysteate is imported by the SDF transporter, isomerized by CuyB to d-cysteate, cleaved by CuyA to release sulfite, ammonium, and pyruvate (Fig. 1B).

d-cysteate of high purity was synthesized by oxidation of d-cysteine as previously described (29), confirmed by analyses with 1H and 13C NMR (Fig. S3, A–D) and assessment of the specific rotations (l-cysteate: +5.2, d-cysteate: −3.6). Over 99% purity was estimated using quantitative 1H NMR and a benzoic acid as an internal control (Fig. S3E). The PpALD-coupled spectrophotometric assay was repeated, showing a significantly higher activity for BwCuyA with d-cysteate as the substrate. Addition of PLP was required for optimal activity (Fig. S4A) and the optimal reaction pH was determined to be 9.0 (Fig. S4B). Activity was directly proportional to the amount of BwCuyA added (Figs. 2A and S4C). The apparent Michaelis-Menten kinetic parameters were also determined (kcat = 4.6 ± 0.1 s−1, KM = 0.6 ± 0.0 mM for d-cysteate) (Fig. S4D). No activity was observed when d-cysteate was replaced with several C3 amino acids and their derivatives, including l-cysteine, l-serine, d-alanine, l-alanine, and O-phospho-l-serine, while partial activities were detected, 17% for d-serine, 4% for d-cysteine and 2% l-cysteate (Fig. 2B). Incubation of BwCuyA with d-cysteate and PLP led to the release of sulfite as detected by a Fuchsin colorimetric assay (30). No reaction was observed in negative controls omitting BwCuyA or d-cysteate (Fig. 2C). LC-MS analysis of the reaction mixture derivatized with DNPH showed the appearance of two peaks with ESI (−) m/z 267, corresponding to two stereoisomers of the product pyruvate-DNPH (Fig. 2, D–G).

Figure 2.

Figure 2

Open in a new tab

BwCuyA activity assays.A, linear dependence of PLP-dependent d-cysteate sulfo-lyase activity on 0, 25, 50 and 100 nM of BwCuyA, as measured using a PpALD-coupled spectrophotometric assay. Error bars reflect the SD values for three replicates. B, substrate specificity of BwCuyA determined by PpALD-coupled assay. Error bars reflect the SD values for three replicates. Significance was determined by one-way ANOVA test, ∗∗∗∗p < 0.0001. C, quantification of sulfite formed in the complete assay and negative controls by Fuchsin assay. Error bars reflect the SD values for three replicates. Significance was determined by a Two-tailed unpaired t test, ∗∗∗∗p < 0.0001. D, LC-MS analyses of pyruvate formation in the reaction of BwCuyA and d-cysteate, following derivatization with DNPH, monitoring the ion counts of ESI negative mode, showing the presence of two pyruvate-DNPH isomer peaks in the complete assay but not in negative controls. E, elution profiles of pyruvate-DNPH isomers for HPLC analysis, monitoring the UV absorbance at 360 nm. F and G, the ESI (−) m/z ionization mass spectra of pyruvate-DNPH peaks in (D).

BwCuyB is a l-cysteate racemase

Next, BwCuyB was recombinantly produced and assayed (Fig. S5A). The catalytic mechanism of PLP-independent amino acid racemases involves a pair of Cys residues that carry out α-deprotonation of the l-amino acid, followed by re-protonation on the opposing face to give the d-amino acid, resulting in the exchange of the amino acid α-proton with the solvent (31). To assay for proton exchange, CuyB was incubated with l-cysteate in the presence of 50% D2O, followed by LC-MS analysis (Fig. 3). In the full reaction mixture, a prominent peak was observed in the extracted ion chromatogram (EIC) at ESI (−) m/z 169 (Fig. 3A), and its corresponding mass spectrum showed two peaks at m/z 168 and 169 (Fig. 3B), corresponding to a mixture of undeuterated and mono-deuterated cysteate as expected for active CuyB. In the negative control omitting CuyB, a much smaller peak was observed in the (EIC), and its corresponding mass spectrum showed a major peak at m/z 168 (Fig. 3C), corresponding to undeuterated cysteate. Similar observations were made when the assay was repeated with d-cysteate, which confirms that CuyB also catalyzes the reverse reaction (Fig. S5, B–D).

Figure 3.

Figure 3

Open in a new tab

Deuterium exchange assays for BwCuyB using LC–MS.A, extracted ion chromatographs of reaction mixture with l-cysteate as the substrate, showing peaks for l-cysteate and d-cysteate in assays conducted with 50% D2O [ESI (−) m/z 169]. B, the mass spectrum of deuterated d-cysteate [ESI (−) m/z 169] and undeuterated cysteate [ESI (−) m/z 168] in the complete assay conducted with 50% D2O. C, the mass spectrum of undeuterated cysteate [ESI (−) m/z 168] in 50% D2O w/o CuyB assay.

BwCuyB activity for isomerization of l-cysteate to d-cysteate was also determined in a coupled spectrophotometric assay with excess BwCuyA and PpALD. The activity was directly proportional to the quantity of BwCuyB added (Fig. 4A). Considering the partial activities of CuyA with d-cysteine and d-serine, we also conducted the same coupling assay with l-cysteate replaced with l-cysteine or l-serine, but no activity was detected (Fig. 4B). Incubation of 10 mM l-cysteate with 50 μM PLP, BwCuyB and BwCuyA led to the release of sulfite as detected using the Fuchsin assay, while no sulfite was detected in negative controls (Fig. 4C). LC-MS analysis of the BwCuyB-BwCuyA reaction mixture derivatized with DNPH showed the appearance of two peaks with ESI (−) m/z 267, corresponding to two stereoisomers of the product pyruvate-DNPH (Fig. 4, D–G).

Figure 4.

Figure 4

Open in a new tab

BwCuyB activity assays.A, linear dependence of l-cysteate racemase activity on different concentrations of BwCuyB (0, 20, 40 and 80 nM), as measured by BwCuyA-PpALD-coupled spectrophotometric assay. Error bars reflect the SD values for three replicates. B, substrate specificity of BwCuyB determined by BwCuyA-PpALD coupled assay. C, quantification of sulfite formed in the complete assay and the negative controls by Fuchsin assay. Error bars reflect the SD values for three replicates. Significance was determined by one-way ANOVA test, ∗∗∗∗p < 0.0001. D, LC-MS analysis of pyruvate formation in the reaction involving BwCuyA, BwCuyB and l-cysteate, following derivatization with DNPH. The ion counts were monitored in ESI negative mode, revealing the presence of two pyruvate-DNPH peaks in the full reaction mixture, while absent in the negative controls. E, elution profiles of pyruvate-DNPH products for HPLC analysis, monitoring the UV absorbance at 360 nm. F and G, the ESI (−) m/z ionization mass spectra of pyruvate-DNPH peaks in (D).

l-Cysteate dissimilation by B. wadsworthia

To validate the physiological function of CuyA, B. wadsworthia RZATAU DSM 11045 was cultivated anaerobically in a defined medium with various sources of TEA. Growth of B. wadsworthia was supported with pyruvate (as the carbon and electron source) plus l-cysteate, and also in the positive control with pyruvate plus thiosulfate, while no growth was observed in the negative control with pyruvate alone (Fig. 5A). The growth of pyruvate plus l-cysteate was accompanied by the formation of H2S in the headspace gas as detected using a methylene blue assay (Fig. 5B) (22). SDS-PAGE analysis revealed the induction of a prominent 35 kDa protein band in l-cysteate-grown cells but not thiosulfate-grown cells (Fig. 5C), similar to previous reports. Through protein mass spectrometry, the 35 kDa protein band in l-cysteate-grown cells was confirmed to be CuyA (E5Y343) (Supplemental Dataset 1).

Figure 5.

Figure 5

Open in a new tab

l-cysteate-dependent growth of B. wadsworthia and H2S formation.A, growth of B. wadsworthia supported by 20 mM l-cysteate plus 20 mM pyruvate, 20 mM l-cysteate, 20 mM Na2S2O3 plus 20 mM pyruvate (positive control) or 20 mM pyruvate (negative control). The OD600 of each culture was measured, with un-inoculated medium serving as the blank. Error bars reflect the SD values for three replicates. Significance was determined by one-way ANOVA test, ∗∗∗∗p < 0.0001. B, formation of H2S in the headspace gas accompanying reduction of various sulfur-containing electron acceptors by B. wadsworthia, as detected by a methylene blue assay. Error bars indicate the SD values for three replicates. Significance was determined by a Two-tailed unpaired t test, ∗∗∗∗p < 0.0001. C, SDS-PAGE analysis of B. wadsworthia grown on Na2S2O3 plus pyruvate (lane 1) or l-cysteate plus pyruvate (lane 2). The labeled protein was identified through in-gel digestion followed by LUMOS MS analysis.

Analysis of CuyA structural models

To gain insights into the substrate binding and catalytic mechanism of CuyA, the AlphaFold models of BwCuyA and the previously characterized SpCuyA (13) were obtained from the AlphaFold protein structure database (32), and overlaid with the crystal structure of Salmonella typhimurium d-cysteine desulfhydrase (StDcyD, PDB accession 4D96) (33), which contains an external aldimine complex with the substrate analog 1-amino-1-carboxy cyclopropane (ACC) (Fig. S6B). The three enzymes share several conserved amino acid residues in the substrate binding pocket, including Lys 51 (StDcyD numbering), which forms the internal Schiff base with PLP, Asn 50, which forms a hydrogen bond with the PLP phosphate group, and Ser 78, Asn 79, Tyr 287 and Thr 315, which form other contacts with the PLP-substrate complex (Fig. S6, A and C). The catalytic mechanism of StDcyD involves formation of the external aldimine complex with d-cysteine, followed by α-deprotonation catalyzed by Tyr 287, and elimination of HS− to form a PLP-2-aminoacrylate intermediate. Similarities between the active sites of the three enzymes suggest a common reaction mechanism involving the PLP-2-aminoacrylate intermediate. Furthermore, conservation of Tyr 287 in BwCuyA and SpCuyA is consistent with the observation that BwCuyA favors d-cysteate and suggested that SpCuyA may also favor d-cysteate over l-cysteate.

Generality of the CuyA and CuyB system

We next recombinantly produced SpCuyA (Fig. S7A) and assessed its stereochemical preference. SpCuyA displayed catalytic activity for d-cysteate (kobs = 5 s−1), a 100-fold lower activity for l-cysteate (kobs = 0.05 s−1), with partial activity for d-cysteine (kobs = 0.2 s−1), and no activity for l-cysteine (Fig. S7B), similar to observations for BwCuyA, which exhibits nearly 50-fold higher catalytic activity for d-cysteate (kobs = 3 s−1) than for l-cysteate (kobs = 0.06 s−1), and partial activity for d-cysteine (kobs = 0.1 s−1), no activity for l-cysteine (Fig. 2B). As previously noted, the SpCuyA genome neighborhood contains the transcriptional regulator CuyR and putative sulfite exporter CuyZ. The genome of S. pomeroyi also contains a homolog of BwCuyB (Q5LLG5, 56% sequence identity) although not in the genome neighborhood of SpCuyA.

A sequence similarity network (SSN) of 16,421 homologs of BwCuyA in the UniRef50_E8U390 (UniRef50 contains sequences clustered at ≥50% identity) (34) was constructed using the Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) (35), and plotted using Cytoscape (36) (Fig. S8). The highlighted cluster in Fig. S8, which contains 1720 putative CuyA sequences, was replotted with each node representing a group of proteins sharing over 80% sequence identity (Fig. 6A). Their accession codes and origins are listed in Table S2. The EFI genome neighborhood tool was used to identify sequences associated with homologs of CuyB (belonging to the family PF01177) within a 10-open reading frame window, and nodes representing sequences with CuyB in the neighborhood were labeled in blue (Fig. 6A). Among 1720 putative CuyAs, only 668 contain a CuyB homolog within their genome neighborhood (Table S3). Many of the sequences belong to SSRB, of which representative genome neighborhoods were displayed (Fig. 6B). Among these, Desulfovibrio desulfuricans IC1, Desulfomicrobium norvegicum DSM 1741 and Desulfitobacterium dehalogenans contain homologs of CuyA and CuyB present in the same genome neighborhood. Desulforapulum autotrophicum, and Desulfotalea psychrophila strain LSv54 contain CuyA but lack CuyB in the genome neighborhood, with the latter containing two copies of CuyA as noted by Dengar et al. (13) D. norvegicum was previously reported to grow on l-cysteate (13), while D. desulfuricans, D. dehalogenans and D. autotrophicum were reported to grow on cysteate of unspecified stereochemistry (37). Conversely, Desulfitobacterium hafniense (16) and D. desulfuricans ATCC 29577 (37), which lack homologs of CuyA, were previously reported not to grow on cysteate. Interestingly, it was also noted that D. psychrophila was unable to grow on cysteate (13), although the stereochemistry was also not specified. The discovery of CuyB and the stereochemical preference of CuyA indicates that more work is required to ascertain the ability of these strains to grow on each individual enantiomer of cysteate.

Figure 6.

Figure 6

Open in a new tab

Sequence similarity network and genome neighborhood analysis.A, sequence similarity network of 1720 putative CuyA homologs. Sequences associated with CuyB (cysteate racemase, PF01177) were labeled in dark blue. B, representative genome neighborhood network analysis of CuyAs from the SSN cluster. TR, transcriptional regulator.

Discussion

Our biochemical investigations into cysteate metabolism in B. wadsworthia have provided further insights into the mechanism of cysteate degradation in this model anaerobic sulfonate-degrading bacterium. A deeper comprehension of the metabolism of this pathogenic organism holds the potential to advance the development of preventive measures and treatments for gut inflammation and colon cancer. In the proposed pathway, l-cysteate is first isomerized to d-cysteate by a novel dithiol cysteate isomerase CuyB, then cleaved by d-cysteate sulfo-lyase CuyA, which, contrary to previous assumptions for S. pomeroyi CuyA (13), exhibits a strong selectivity for d-cysteate over l-cysteate. The stereoselectivity of CuyA was rationalized by comparing its active site model with the crystal structure of the well-studied d-cysteine desulfhydrase, revealing the conservation of several substrate-binding residues and a critical Tyr residue catalyzing stereoselective α-deprotonation. Likewise, re-testing S. pomeroyi CuyA with both l- and d-cysteate demonstrated a strong selectivity for d-cysteate, and the presence of a CuyB homolog in the S. pomeroyi genome supports the generality of the CuyB-CuyA system.

Clarification of the stereochemical details of the cysteate metabolic enzymes highlights the importance of considering stereochemistry in the metabolism of C3 sulfonates, which unlike C2 sulfonates, often contain a stereogenic center (38, 39, 40). For instance, sulfolactate and DHPS, products of bacterial degradation of the prevalent sulfo-sugar sulfoquinovose (SQ), are believed to primarily occur as (S)-isomers, due to retention of the stereogenic center from SQ. In Chromohalobacter salexigens, the stereocenter of (S)-sulfolactate is inverted through oxidation by the NAD+ dependent (S)-sulfolactate dehydrogenase SlcC, and subsequent reduction by the NADHP-dependent (R)-sulfolactate dehydrogenase, then cleaved by SuyAB (38). In Cupriavidus pinatubonensis, the stereocenter of (S)-DHPS is inverted through oxidation by the NADP+-dependent (S)-DHPS 2-dehydrogenase HpsO, and subsequent reduction by the NADH-dependent (R)-DHPS 2-dehydrogenase HpsP, then oxidized to (R)-sulfolactate and cleaved by SuyAB (40). The specificity of CuyA for d-cysteate is puzzling given that l-cysteate derived from l-cysteine oxidation is expected to be the predominant isomer in nature. Given the close homology mechanistic similarity between CuyA and d-cysteine desulfhydrase (DcyD), one plausible explanation could be that CuyA evolved from DcyD, preserving its stereochemical preference. This would necessitate a separate cysteate racemase CuyB, to convert l-cysteate to d-cysteate.

CuyA is present in phylogenetically diverse terrestrial, marine and microbiome bacteria, often accompanied by CuyB, suggesting that the ability to degrade cysteate is widespread among bacteria in different environments. This indicates that cysteate is likely a common metabolite in these environments, although the major natural sources of cysteate and the relative abundance of l- and d-cysteate are not well understood. Marine microorganisms are known to utilize the abundant sulfate in seawater to synthesize various sulfonate osmolytes, which may add to the organosulfur content of aerobic seawater and anaerobic sediments. Cysteate of undetermined stereochemistry has been detected at micromolar to sub-millimolar levels in various eukaryotic phytoplankton and cyanobacteria (41), although its physiological significance and biosynthetic mechanism remain unknown. Cysteate may also form through abiotic oxidation of proteinogenic cysteine, which is expected to occur only in aerobic environments and to yield l-cysteate. A possible source of d-cysteate may be α-epimerization of peptide-bound l-cysteine or l-cysteate.

Especially noteworthy is the presence of BwCuyA homologs in other human-associated SSRB, including the intestinal commensal Desulfovibrio piger, the intestinal pathobiont Desulfovibrio fairfieldensis, and the oral pathobiont Desulfobulbus oralis, with the latter two also containing CuyB, suggesting the ability of these major gut SSRB genera to produce H2S from cysteate degradation. The fermenting bacterium Fusobacterium nucleatum is known to produce H2S from cysteine, a process linked to inflammatory conditions. Further studies are needed to determine the content of cysteate in different diets, and its contribution to H2S production by SSRB in different regions of the digestive tract.

Experimental procedures

Media and chemicals

Lysogeny Broth (LB) medium components, yeast extract and tryptone, were purchased from Oxoid, while the dry powder used for preparing Anaerobe Basal Broth (ABB) was purchased from Rishui Biotech. High-purity solvents, acetonitrile and formic acid, were purchased from Concord Technology and Merck, respectively. Water used in this work was ultrapure deionized water from Millipore Direct-Q. The sources of chemicals, NADH, l-cysteate, l/d amino acids, and PLP were Genview, Aladdin, Solarbio, and Shyuanye, respectively. O-phospho-l-serine and benzoic acid were obtained from Meryer locally in Tianjin.

d-cysteate was synthesized in our laboratory as previously described in the literature (29). 10 mg of the product and commercial l-cysteate dissolved in D2O were used for 1H-NMR (400 MHz, Bruker) and 13C-NMR (100 MHz) analysis, and 5 mg each in 2 ml ddH2O was subjected to specific rotation measurements with an MCP 200 (Anton Paar). To determine the purity of the synthesized d-cysteate, benzoic acid was used as an internal standard. 11.25 mg of d-cysteate spiked with 11.00 mg of commercial benzoic acid was dissolved in DMSO-d6 for 1H-NMR (400 MHz) analysis. The content of the sample was assessed by peak integration in reference to the internal standard.

Gene synthesis, cloning, and plasmid construction

The BwCuyA and BwCuyB genes were amplified by PCR from B. wadsworthia RZATAU genomic DNA, using the primer pairs listed in Table S1. Amplified DNA fragments were then inserted into a modified pET28 vector at the SspI site by Gibson assembly to form HT-BwCuyA and HT-BwCuyB plasmids, encoding a His6-tag and a tobacco etch virus (TEV) protease cleavage site at the N-terminus of the protein. Escherichia coli codon-optimized DNA fragment encoding S. pomeroyi DSS-3T CuyA (Q5LL69) was synthesized and inserted into the SspI site of the HT vector by Tsingke.

Protein expression, purification, and quantitation

BwCuyA, BwCuyB, PpALD, and SpCuyA were heterologously expressed in E. coli BL21 (DE3) cells harboring the corresponding plasmids. A single colony was inoculated into 5 ml LB medium containing 50 μg/ml kanamycin as a starter culture. The culture was incubated at 37 °C with shaking at 220 rpm for 5 h, followed by dilution into 1 L of same medium for further incubation. When OD600 reached 0.6 to 0.8, the temperature was then reduced to 18 °C and 0.3 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to induce protein expression. After 16 h, the cells were harvested by centrifugation (5000g for 10 min at 4 °C), and the cell pellets were re-suspended in 35 ml of lysis buffer [50 mM Tris-HCl, pH 8.0, 150 mM KCl, 1 mM phenylmethanesulfonyl fluoride (PMSF), 0.2 mg/ml lysozyme, 0.03% Triton X-100, and 0.02 mg/ml DNase I]. The cell suspensions were then frozen in a −80 °C freezer for further processing.

The protein purification chromatographic experiments were conducted using GE Healthcare’s ÄKTA pure or ÄKTA prime plus FPLC machines equipped with the appropriate columns. The cell suspension was thawed and incubated at room temperature (RT) for 30 min to allow lysis. Upon cell lysis, 5 mM β-mercaptoethanol (BME) was added, and nucleic acid was precipitated with 1% streptomycin sulfate and removed by centrifugation (8000g for 10 min at 4 °C). The supernatant through a 0.45-μm filter was loaded onto a 5-ml TALON Co2+ column (Clontech Laboratories), pre-equilibrated with buffer A (20 mM Tris-HCl, pH 7.5, 0.2 M KCl and 5 mM BME). The column was washed with 10 column volumes (CV) of buffer A. The protein eluate in 5 CV of buffer B (20 mM Tris-HCl, pH 7.5, 5 mM BME, 0.2 M KCl, and 150 mM imidazole) was dialyzed against 2 L of buffer A at 4 °C for 3 h, concentrated and frozen in aliquots in liquid N2, and stored at −80 °C.

The purified proteins were examined by SDS-PAGE on a commercial gel (Bis-Tris, 4–20%, YoungPAGE, Genscript). The protein concentrations were determined by measuring the absorption at 280 nm using a Nanodrop (Thermo Scientific) referring to the extinction coefficients calculated with the ProtParam tool (https://www.expasy.org/): BwCuyA, ε280 = 24,870 M−1 cm−1, BwCuyB, ε280 = 5960 M−1 cm−1, PpALD, ε280 = 27,390 M−1 cm−1 and SpCuyA, ε280 = 27,390 M−1 cm−1.

ALD-coupled kinetic assays for CuyA activity

The sulfo-lyase activity of BwCuyA was measured using an ALD-coupled spectrophotometric assay in the presence of excess PpALD enzyme. A 200-μl reaction mixture containing 200 mM HCO3−/CO32−, pH 9.0, 5 μM PpALD, 20 mM NH4Cl, 0.4 mM NADH, 10 mM d-cysteate,1 μM BwCuyA with or without 10 μM PLP was prepared, and the decrease in absorbance at 340 nm was monitored using an M200 plate reader (Tecan, Switzerland) over 1.5 min at RT. The negative control without BwCuyA was also included. The ΔA340 nm and the extinction coefficient of NADH (ε340 = 6220 M−1 cm−1) were used to calculate the reaction rates. The addition of PLP was found required for the maximal activity, indicating the sub-stoichiometric content of PLP in the enzyme as isolated, and 10 molar excess of PLP was added in the subsequent experiments. The optimal pH of BwCuyA was determined by carrying out the activity assay with 100 nM BwCuyA in 200 mM of different buffers: HEPES, pH 6.0, 7.0; Tris-HCl, pH 8.0, 8.5; HCO3−/CO32−, pH 9.0, 9.5, 10.0, 11.0, 12.0. Enzyme dose-dependent assays were carried out by varying the enzyme concentrations from 25 to 100 nM. To measure the apparent Michaelis-Menten kinetic constants, the concentration of d-cysteate was varied from 0 to 15 mM in the presence of 100 nM BwCuyA. To investigate the substrate specificity of BwCuyA and SpCuyA, 100 mM of all or some of the structurally similar compounds, including l-cysteate, d-cysteine, l-cysteine, d-serine, l-serine, d-alanine, l-alanine, and O-phospho-l-serine, were tested in the PpALD-coupled assays. The reaction was initiated by the addition of 1 μM CuyA.

End-point Fuchsin assays for BwCuyA activity detecting the formation of sulfite

Sulfite was detected using a colorimetric assay involving the formation of a colored complex between sulfite and Fuchsin dye in an acidic solution. Serial dilutions of sodium sulfite (0.7, 0.6, 0.5, 0.4, 0.2, 0.1 and 0 mM) were used to establish a standard curve. A 200-μl reaction mixture containing 200 mM HCO3−/CO32−, pH 9.0, 50 mM KCl, 1 μM BwCuyA, 10 μM PLP, and 10 mM d-cysteate was incubated at RT for 30 min. Two negative controls omitting either BwCuyA or d-cysteate were also performed. While the reaction was incubated, stock solution A (0.8 M H2SO4, 0.08% Fuchsin, and 1.6% formaldehyde, mixed 7: 2: 1) was freshly prepared. A 50 μl portion per reaction sample was mixed with 950 μl of solution A, incubated for 10 min at RT, and the UV-Vis absorbances at 580 nm were recorded, and the concentrations of sulfite were calculated referring to the standard curve.

End-point LC-MS assays for BwCuyA activity detecting the formation of pyruvate

The end-point reaction sample was also derivatized with 2,4-dinitrophenylhydrazine (DNPH) in order to detect pyruvate formation using LC-MS assay. 50 μl of reaction solution was mixed with 550 μl of 0.73 M sodium acetate buffer, pH 5.0, followed by 400 μl of freshly prepared DNPH solution (10 mg dissolved in 25 ml methanol). The mixture was incubated at 50 °C for 1 h and then filtered prior to LC-MS analysis. 4 mM of commercial pyruvate dissolved in ddH2O was also prepared as the standard. LC-MS analysis was performed with 20 μl sample on an Agilent 6420 triple quadrupole LC-MS instrument (Agilent Technologies) equipped with an Agilent ZORBAX SB-C18 column (4.6 × 250 mm). The drying gas temperature was maintained at 330 °C with a flow rate of 10 l/min and a nebulizer pressure of 45 psi. The HPLC conditions were as follows: linear gradient from 4% to 96% solvent B in 18 min, where solvent A was ddH2O and 0.1% formic acid, and solvent B was acetonitrile and 0.1% formic acid. The flow rate was set to 1 ml/min. UV detection was set at 360 nm. The mass spectrometer was run in ESI negative mode.

Deuterium exchange assays for BwCuyB activity

A 100 μl reaction mixture containing 1 μM BwCuyB, and 10 mM substrate (l-cysteate or d-cysteate) in 200 mM HCO3−/CO32−, pH 9.0, 50 mM KCl and 50% D2O was prepared, and incubated at RT for 30 min. LC-MS analysis was similar to that for CuyA activity except that a linear gradient from 4 to 20% solvent B in 18 min and no UV detection were applied.

CuyA-ALD-coupled kinetic assays for BwCuyB activity

The racemase activity of BwCuyB was measured using a coupled assay in the presence of excess PpALD and BwCuyA enzymes. A 200-μl reaction mixture containing 200 mM HCO3−/CO32−, pH 9.0, 5 μM BwCuyA, 50 μM PLP, 5 μM PpALD, 20 mM NH4Cl, 0.4 mM NADH and 10 mM l-cysteate was prepared. Enzyme dose-dependent assays were carried out by varying the BwCuyB concentrations from 20 to 80 nM. The ΔA340 nm was monitored using a Tecan M200 plate reader over 1.5 min at RT. 100 mM of l-cysteine and l-serine were also tested as the substrates of BwCuyB. In these cases, the reaction was initiated by the addition of 1 μM BwCuyB.

End-point CuyA-coupled assays for BwCuyB activity

A 200 μl reaction mixture containing 200 mM HCO3−/CO32−, pH 9.0, 50 mM KCl, 1 μM BwCuyB, 5 μM BwCuyA, 50 μM PLP, and 10 mM l-cysteate was incubated at RT for 30 min. Four negative controls omitting l-cysteate, BwCuyA, BwCuyB, or both enzymes were also performed. Sulfite and pyruvate formation were detected using the Fuchsin and LC-MS assays respectively as described above.

Growth of B. wadsworthia on l-cysteate

B. wadsworthia strain RZATAU (DSM 11045) was purchased from DSMZ. All empty bottles and vials used for anaerobic cultures were stored for 3 days before use in a nitrogen glovebox (Lab2000, Etelux), which maintained an atmosphere of N2 with oxygen levels below 10 ppm. These bottles were sealed in the glove box and autoclaved outside. The ABB powder and the heat-insensitive DSM 503 medium components were dissolved in degasified ddH2O contained in a 500 ml bottle, sealed in the glovebox, and autoclaved outside. Heat-sensitive components were dissolved in degasified ddH2O and filter-sterilized in the glove box. Cells were first inoculated anaerobically in a 5 ml ABB medium contained in a 10-ml vial and incubated at 37 °C for 3 to 5 days. Cells in 100 μl ABB culture were spun down and transferred into four anaerobic vials each containing 5 ml DSM 503 medium supplemented with 60 mM Na-formate and 200 μg/l 1,4-naphthochinone. This inoculum corresponds to an initial OD600 ∼0.1 in the defined media. To test whether l-cysteate can be utilized to provide TEA and as a carbon and electron source, sodium pyruvate, sodium pyruvate plus Na2S2O3, l-cysteate only, or l-cysteate plus pyruvate each 20 mM was added to one of the four vials. Sodium pyruvate was added as a carbon and electron source, while Na2S2O3, as a known TEA, was added as a positive control. The OD600 of each culture was monitored over 6 days. 1 ml headspace gas was transferred using a syringe to a mixture containing 800 μl of 0.75% zinc acetate and 50 μl of 7% NaOH in an Eppendorf tube. The mixture was incubated at RT for 15 min, followed by sequential addition of 150 μl of 0.1% N,N-dimethyl-p-phenylenediamine dihydrochloride (DPD, 1 mg DPD dissolved in 1 ml 5 N HCl) and 10 mM FeCl3 (dissolved in 1 N HCl). The reaction mixture was incubated at 30 °C for 20 min and centrifuged at 14,000g for 5 min. The supernatant was subjected to absorbance measurement at 670 nm using a Tecan M200 plate reader.

Protein identification by SDS/PAGE and mass spectrometry

B. wadsworthia cells were boiled in Laemmli loading buffer for cell lysis and proteins were separated on a 10% SDS/PAGE gel which was stained with Coomassie Blue R-250 staining solution, followed by a destaining process. The most prominent band induced by growth on l-cysteate and pyruvate was manually excised. After in-gel digestion with trypsin and extraction, the mixture of peptides was loaded onto Orbitrap Fusion LUMOS MS equipped with a 150 μm × 2 cm self-packed C18 trap column (particle size 3 μm, Dr. MASCH GmbH, Germany) and a 150 μm × 30 cm self-packed C18 analytical column (particle size 1.9 μm, Dr. MASCH GmbH). The HPLC conditions were as follows: linear gradient from 8 to 12% mobile phase B over 10 min, 12 to 27% over 69 min, 27 to 45% over 28 min, 45 to 95% over 3 min, and 95% over 10 min, where mobile phase A was ddH2O and 0.1% formic acid, and mobile phase B was 80% acetonitrile and 0.1% formic acid. The MS/MS spectra from each LC-MS/MS run were searched against the B. wadsworthia protein database GCF_000185705.2 (Bilo_- wads_3_1_6_V2) from UniProt (release date of March 2022; 68,406 entries) using an in-house Proteome Discoverer (Version PD 2.2, ThermoFisher Scientific). Protein identification was performed based on Sequest HT. The raw data of this experment have been deposited at the Mendeley Data public repository (https://data.mendeley.com/datasets/94w5swwdvt/1).

Bioinformatics method

A sequence similarity network (SSN) of close homologs of BwCuyA in the cluster UniRef50_E8U390 was constructed using EFI-EST. The sequence similarity network (SSN) of a total of 16,421 PLP-dependent enzyme sequences was constructed with the E-value cut-off of 10−90. The 80% similarity representative node (RepNode) network was displayed using Cytoscape v3.519. The information regarding the genome neighborhood within a 10 open reading frame (10-ORF) window was inserted using EFI-GNT.

Data availability

All data described in this study are contained within the main manuscript and Supporting information.

Supporting information

This article contains supporting information (9, 10, 11, 16, 33).

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We thank the instrument analytical center of the School of Pharmaceutical Science and Technology at Tianjin University, Zhi Li and Yan Gao, for providing the LC-MS and Protein Mass Spectrometry analysis.

Author contributions

Y. T., Y. Z., Y. W., C. L., and K. M. investigation; S. Y., X. L., Y. Z., X. J., Y. W., C. L., and K. M. methodology; L. J., Y. Z., Y. W., and C. L. validation; L. J., Y. Z., Y. W., C. L., and K. M. data curation; X. L. and Y. Z. resources; Y. Z., Y. W., and C. L. writing–review & editing; Y. Z. and C. L. writing–original draft; Y. Z., Y. W., and C. L. visualization; Y. Z. and Y. W. supervision; Y. Z. project administration; Y. Z. funding acquisition; Y. Z., Y. W., and C. L. formal analysis; Y. Z., C. L., and Y. W. conceptualization.

Funding and additional information

This work was supported by the National Natural Science Foundation of China (NSFC) Distinguished Young Scholar of China Program 32125002 (Y. Z.), the New Cornerstone Science Foundation NCI202321 (Y. Z.), the National Key R&D Program of China 2019YFA0905700 (Y. Z.) and the National Key R&D Program of China 2020YFA0907900 (Y. Z.).

Reviewed by members of the JBC Editorial Board. Edited by Chris Whitfield

Contributor Information

Yifeng Wei, Email: wei_yifeng@sifbi.a-star.edu.sg.

Yan Zhang, Email: yan.zhang@tju.edu.cn.

Supporting information

Supplemental dataset 1
mmc1.xlsx (370.7KB, xlsx)
Supplementray data
mmc2.docx (1.8MB, docx)

References

  • 1.Cook A.M., Laue H., Junker F. Microbial desulfonation. FEMS Microbiol. Rev. 1998;22:399–419. doi: 10.1111/j.1574-6976.1998.tb00378.x. [DOI] [PubMed] [Google Scholar]
  • 2.Cook A.M., Smits T.H.M., Denger K. In: Microbial Sulfur Metabolism. Dahl C., Friedrich C.G., editors. Springer; Berlin, Heidelberg: 2008. Sulfonates and organotrophic sulfite metabolism; pp. 170–183. [Google Scholar]
  • 3.Kertesz M.A. Riding the sulfur cycle--metabolism of sulfonates and sulfate esters in gram-negative bacteria. FEMS Microbiol. Rev. 2000;24:135–175. doi: 10.1016/S0168-6445(99)00033-9. [DOI] [PubMed] [Google Scholar]
  • 4.Geiger O., González-Silva N., López-Lara I.M., Sohlenkamp C. Amino acid-containing membrane lipids in bacteria. Prog. Lipid Res. 2010;49:46–60. doi: 10.1016/j.plipres.2009.08.002. [DOI] [PubMed] [Google Scholar]
  • 5.Huxtable R.J. Physiological actions of taurine. Physiol. Rev. 1992;72:101–163. doi: 10.1152/physrev.1992.72.1.101. [DOI] [PubMed] [Google Scholar]
  • 6.Westheimer F.H. Why nature chose phosphates. Science. 1987;235:1173–1178. doi: 10.1126/science.2434996. [DOI] [PubMed] [Google Scholar]
  • 7.Consden R., Gordon A.H., Martin A.J. The identification of amino-acids derived from cystine in chemically modified wool. Biochem. J. 1946;40:580–582. [PMC free article] [PubMed] [Google Scholar]
  • 8.Vollrath F., Fairbrother W.J., Williams R.J.P., Tillinghast E.K., Bernstein D.T., Gallagher K.S., et al. Compounds in the droplets of the orb spider's viscid spiral. Nature. 1990;345:526–528. [Google Scholar]
  • 9.Edgar S.E., Hickman M.A., Marsden M.M., Morris J.G., Rogers Q.R. Dietary cysteic acid serves as a precursor of taurine for cats. J. Nutr. 1994;124:103–109. doi: 10.1093/jn/124.1.103. [DOI] [PubMed] [Google Scholar]
  • 10.Williams B.T., Todd J.D. A day in the life of marine sulfonates. Nat. Microbiol. 2019;4:1610–1611. doi: 10.1038/s41564-019-0576-5. [DOI] [PubMed] [Google Scholar]
  • 11.Liu Y., Wei Y., Teh T.M., Liu D., Zhou Y., Zhao S., et al. Identification and characterization of the biosynthetic pathway of the sulfonolipid capnine. Biochemistry. 2022;61:2861–2869. doi: 10.1021/acs.biochem.2c00102. [DOI] [PubMed] [Google Scholar]
  • 12.Abbanat D.R., Leadbetter E.R., Godchaux W.I., Escher A.P. Sulphonolipids are molecular determinants of gliding motility. Nature. 1986;324:367–369. [Google Scholar]
  • 13.Denger K., Smits T.H., Cook A.M. l-cysteate sulpho-lyase, a widespread pyridoxal 5'-phosphate-coupled desulphonative enzyme purified from Silicibacter pomeroyi DSS-3T. Biochem. J. 2006;394:657–664. doi: 10.1042/BJ20051311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Eichhorn E., Leisinger T. Escherichia coli utilizes methanesulfonate and l-cysteate as sole sulfur sources for growth. FEMS Microbiol. Lett. 2001;205:271–275. doi: 10.1111/j.1574-6968.2001.tb10960.x. [DOI] [PubMed] [Google Scholar]
  • 15.Mikosch C., Denger K., Schäfer E.M., Cook A.M. Anaerobic oxidations of cysteate: degradation via l-cysteate:2-oxoglutarate aminotransferase in Paracoccus pantotrophus. Microbiology. 1999;145:1153–1160. doi: 10.1099/13500872-145-5-1153. [DOI] [PubMed] [Google Scholar]
  • 16.Rein U., Gueta R., Denger K., Ruff J., Hollemeyer K., Cook A.M. Dissimilation of cysteate via 3-sulfolactate sulfo-lyase and a sulfate exporter in Paracoccus pantotrophus NKNCYSA. Microbiology. 2005;151:737–747. doi: 10.1099/mic.0.27548-0. [DOI] [PubMed] [Google Scholar]
  • 17.Ridlon J.M., Wolf P.G., Gaskins H.R. Taurocholic acid metabolism by gut microbes and colon cancer. Gut Microbes. 2016;7:201–215. doi: 10.1080/19490976.2016.1150414. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Attene-Ramos M.S., Nava G.M., Muellner M.G., Wagner E.D., Plewa M.J., Gaskins H.R. DNA damage and toxicogenomic analyses of hydrogen sulfide in human intestinal epithelial FHs 74 Int cells. Environ. Mol. Mutagen. 2010;51:304–314. doi: 10.1002/em.20546. [DOI] [PubMed] [Google Scholar]
  • 19.Attene-Ramos M.S., Wagner E.D., Plewa M.J., Gaskins H.R. Evidence that hydrogen sulfide is a genotoxic agent. Mol. Cancer Res. 2006;4:9–14. doi: 10.1158/1541-7786.MCR-05-0126. [DOI] [PubMed] [Google Scholar]
  • 20.Wei Y., Zhang Y. Glycyl radical enzymes and sulfonate metabolism in the microbiome. Annu. Rev. Biochem. 2021;90:817–846. doi: 10.1146/annurev-biochem-080120-024103. [DOI] [PubMed] [Google Scholar]
  • 21.Neukirchen S., Pereira I.A.C., Sousa F.L. Stepwise pathway for early evolutionary assembly of dissimilatory sulfite and sulfate reduction. ISME J. 2023;17:1680–1692. doi: 10.1038/s41396-023-01477-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Xing M., Wei Y., Zhou Y., Zhang J., Lin L., Hu Y., et al. Radical-mediated C-S bond cleavage in C2 sulfonate degradation by anaerobic bacteria. Nat. Commun. 2019;10:1609. doi: 10.1038/s41467-019-09618-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Laue H., Denger K., Cook A.M. Taurine reduction in anaerobic respiration of Bilophila wadsworthia RZATAU. Appl. Environ. Microbiol. 1997;63:2016–2021. doi: 10.1128/aem.63.5.2016-2021.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Liu J., Wei Y., Lin L., Teng L., Yin J., Lu Q., et al. Two radical-dependent mechanisms for anaerobic degradation of the globally abundant organosulfur compound dihydroxypropanesulfonate. Proc. Natl. Acad. Sci. U. S. A. 2020;117:15599–15608. doi: 10.1073/pnas.2003434117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Burrichter A., Denger K., Franchini P., Huhn T., Müller N., Spiteller D., et al. Anaerobic degradation of the plant sugar sulfoquinovose concomitant with H2S production: Escherichia coli K-12 and Desulfovibrio sp. strain DF1 as co-culture model. Front. Microbiol. 2018;9:2792. doi: 10.3389/fmicb.2018.02792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Liu X., Wei Y., Zhang J., Zhou Y., Du Y., Zhang Y. Isethionate is an intermediate in the degradation of sulfoacetate by the human gut pathobiont Bilophila wadsworthia. J. Biol. Chem. 2023;299 doi: 10.1016/j.jbc.2023.105010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Duan Y., Wei Y., Xing M., Liu J., Jiang L., Lu Q., et al. Anaerobic hydroxyproline degradation involving C-N cleavage by a glycyl radical enzyme. J. Am. Chem. Soc. 2022;144:9715–9722. doi: 10.1021/jacs.2c01673. [DOI] [PubMed] [Google Scholar]
  • 28.Lambrecht J.A., Flynn J.M., Downs D.M. Conserved YjgF protein family deaminates reactive enamine/imine intermediates of pyridoxal 5'-phosphate (PLP)-dependent enzyme reactions. J. Biol. Chem. 2012;287:3454–3461. doi: 10.1074/jbc.M111.304477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Ye X., Wang Z., Zhang J., Wan X. Noncovalently functionalized commodity polymers as tailor-made additives for stereoselective crystallization. Angew. Chem. Int. Ed. Engl. 2021;60:20243–20248. doi: 10.1002/anie.202106603. [DOI] [PubMed] [Google Scholar]
  • 30.Denger K., Ruff J., Rein U., Cook A.M. Sulphoacetaldehyde sulpho-lyase (EC 4.4.1.12) from Desulfonispora thiosulfatigenes: purification, properties and primary sequence. Biochem. J. 2001;357:581–586. doi: 10.1042/0264-6021:3570581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Ohtaki A., Nakano Y., Iizuka R., Arakawa T., Yamada K., Odaka M., et al. Structure of aspartate racemase complexed with a dual substrate analogue, citric acid, and implications for the reaction mechanism. Proteins. 2008;70:1167–1174. doi: 10.1002/prot.21528. [DOI] [PubMed] [Google Scholar]
  • 32.Varadi M., Anyango S., Deshpande M., Nair S., Natassia C., Yordanova G., et al. AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Res. 2022;50:D439–D444. doi: 10.1093/nar/gkab1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Bharath S.R., Bisht S., Harijan R.K., Savithri H.S., Murthy M.R. Structural and mutational studies on substrate specificity and catalysis of Salmonella typhimurium d-cysteine desulfhydrase. PLoS One. 2012;7 doi: 10.1371/journal.pone.0036267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Suzek B.E., Huang H., McGarvey P., Mazumder R., Wu C.H. UniRef: comprehensive and non-redundant UniProt reference clusters. Bioinformatics. 2007;23:1282–1288. doi: 10.1093/bioinformatics/btm098. [DOI] [PubMed] [Google Scholar]
  • 35.Gerlt J.A., Bouvier J.T., Davidson D.B., Imker H.J., Sadkhin B., Slater D.R., et al. Enzyme function initiative-enzyme similarity tool (EFI-EST): a web tool for generating protein sequence similarity networks. Biochimi. Biophys. Acta Proteins Proteom. 2015;1854:1019–1037. doi: 10.1016/j.bbapap.2015.04.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Shannon P., Markiel A., Ozier O., Baliga N.S., Wang J.T., Ramage D., et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13:2498–2504. doi: 10.1101/gr.1239303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Lie T.J., Leadbetter J.R., Leadbetter E.R. Metabolism of sulfonic acids and other organosulfur compounds by sulfate-reducing bacteria. Geomicrobiol. J. 1998;15:135–149. [Google Scholar]
  • 38.Denger K., Cook A.M. Racemase activity effected by two dehydrogenases in sulfolactate degradation by Chromohalobacter salexigens: purification of (S)-sulfolactate dehydrogenase. Microbiology. 2010;156:967–974. doi: 10.1099/mic.0.034736-0. [DOI] [PubMed] [Google Scholar]
  • 39.Chen X., Liu L., Gao X., Dai X., Han Y., Chen Q., et al. Metabolism of chiral sulfonate compound 2,3-dihydroxypropane-1-sulfonate (DHPS) by Roseobacter bacteria in marine environment. Environ. Int. 2021;157 doi: 10.1016/j.envint.2021.106829. [DOI] [PubMed] [Google Scholar]
  • 40.Mayer J., Huhn T., Habeck M., Denger K., Hollemeyer K., Cook A.M. 2,3-Dihydroxypropane-1-sulfonate degraded by Cupriavidus pinatubonensis JMP134: purification of dihydroxypropanesulfonate 3-dehydrogenase. Microbiology. 2010;156:1556–1564. doi: 10.1099/mic.0.037580-0. [DOI] [PubMed] [Google Scholar]
  • 41.Durham B.P., Boysen A.K., Carlson L.T., Groussman R.D., Heal K.R., Cain K.R., et al. Sulfonate-based networks between eukaryotic phytoplankton and heterotrophic bacteria in the surface ocean. Nat. Microbiol. 2019;4:1706–1715. doi: 10.1038/s41564-019-0507-5. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental dataset 1
mmc1.xlsx (370.7KB, xlsx)
Supplementray data
mmc2.docx (1.8MB, docx)

Data Availability Statement

All data described in this study are contained within the main manuscript and Supporting information.

Articles from The Journal of Biological Chemistry are provided here courtesy of American Society for Biochemistry and Molecular Biology

RESOURCES