A glycyl radical enzyme enables hydrogen sulfide production by the human intestinal bacterium Bilophila wadsworthia

Abstract

Hydrogen sulfide (H₂S) production in the intestinal microbiota has many contributions to human health and disease. An important source of H₂S in the human gut is anaerobic respiration of sulfite released from the abundant dietary and host-derived organic sulfonate substrate in the gut, taurine (2-aminoethanesulfonate). However, the enzymes that allow intestinal bacteria to access sulfite from taurine have not yet been identified. Here we decipher the complete taurine desulfonation pathway in Bilophila wadsworthia 3.1.6 using differential proteomics, in vitro reconstruction with heterologously produced enzymes, and identification of critical intermediates. An initial deamination of taurine to sulfoacetaldehyde by a known taurine:pyruvate aminotransferase is followed, unexpectedly, by reduction of sulfoacetaldehyde to isethionate (2-hydroxyethanesulfonate) by an NADH-dependent reductase. Isethionate is then cleaved to sulfite and acetaldehyde by a previously uncharacterized glycyl radical enzyme (GRE), isethionate sulfite-lyase (IslA). The acetaldehyde produced is oxidized to acetyl-CoA by a dehydrogenase, and the sulfite is reduced to H₂S by dissimilatory sulfite reductase. This unique GRE is also found in Desulfovibrio desulfuricans DSM642 and Desulfovibrio alaskensis G20, which use isethionate but not taurine; corresponding knockout mutants of D. alaskensis G20 did not grow with isethionate as the terminal electron acceptor. In conclusion, the novel radical-based C-S bond-cleavage reaction catalyzed by IslA diversifies the known repertoire of GRE superfamily enzymes and enables the energy metabolism of B. wadsworthia This GRE is widely distributed in gut bacterial genomes and may represent a novel target for control of intestinal H₂S production.

Keywords: anaerobic degradation; carbon-sulfur bond-cleaving glycyl radical enzyme; human gut microbiome; human health; organosulfonate respiration.

Fig. 1.

Metabolism of taurine and isethionate by the human gut bacterium B. wadsworthia and by Desulfovibrio spp. (A) B. wadsworthia and other intestinal bacteria degrade dietary and host-derived organosulfonates to access sulfite as an electron acceptor for their anaerobic respiration. The desulfonation reaction in B. wadsworthia has not yet been identified. (B) Summary of the pathways investigated in this study (no single organism represented). B. wadsworthia uses taurine and isethionate as electron acceptors, and the two Desulfovibrio spp. strains can use isethionate, but not taurine. All three strains lack the Xsc enzyme known in many aerobic bacteria (dashed line on the left). Instead, the GRE IslA with its GRE-activase component (IslB) are found in both B. wadsworthia and the Desulfovibrio spp., and a novel NADH-coupled, isethionate-forming sulfoacetaldehyde reductase (SarD) is found only in B. wadsworthia. The sulfite released by the GRE is reduced to sulfide by Dsr and coupled to proton translocation for ATP synthesis (symbolized as [H]) when using electrons from oxidation of an alternative electron donor, such as lactate (gray box). AckA, acetate kinase; Pta, phosphotransacetylase. (C) The gene clusters identified in this study. In B. wadsworthia, two separate clusters encode for taurine transport and conversion to isethionate (ald, tpa, and sarD), and also for isethionate desulfonation and the conversion of acetaldehyde to acetyl-CoA (adhE, islA, and islB). The Desulfovibrio strains harbor only a gene cluster for isethionate transport (dctP and fused dctMQ) and desulfonation (islA and islB), and acetaldehyde dehydrogenases are encoded elsewhere in their genomes (not depicted in C). Single-gene knockouts in D. alaskensis G20 that result in an inability to use isethionate (but not free sulfite) are indicated by squared gene symbols.

Fig. 2.

Proteomics experiments with cell-free extracts reveal a putative desulfonating GRE. (A and B) Proteins encoded by the taurine and isethionate utilization gene clusters, including the predicted isethionate GRE (IslA) and GRE-activase (IslB) components, are specifically and strongly expressed during growth with taurine (B. wadsworthia) and isethionate (B. wadsworthia, Desulfovibrio strains). Constitutively expressed proteins for, for example, sulfite reduction (Dsr) and metabolism of acetyl-CoA (Pta and AckA), are shown for comparison. IMG locus tag numbers are given; their prefixes are shown in Fig. 1C. Shown are results of representative total proteomic analyses replicated at least twice with extracts prepared from independent growth experiments.

Fig. 3.

The B. wadsworthia GRE is a C-S bond-cleaving IslA. (A) The GRE-activating radical SAM enzyme IslB installs a protein-centered, stable glycyl radical within the GRE IslA. Activated IslA then catalyzes a C-S bond-cleavage reaction with isethionate as the substrate. The mechanistic hypothesis is illustrated in SI Appendix, Fig. S1B. (B) Electron paramagnetic resonance spectroscopy demonstrating glycyl radical formation on recombinantly produced and purified IslA after its activation by recombinant purified and reconstituted GRE-activase IslB. (C) Conversion of isethionate to sulfite by IslA was observed only in the absence of molecular oxygen and in the presence of all reaction components, as was acetaldehyde formation (D). The data in C are shown as the mean ± SD of three technical replicates, and data in D are shown as representative HPLC chromatograms of a minimum of two technical replicates. (E) The Michaelis–Menten kinetics of isethionate cleavage were determined by using a coupled spectrophotometric assay with yeast alcohol dehydrogenase and NADH. The k_cat was 14 ± 0.4 s⁻¹, and the K_m was 8.1 ± 0.8 mM. Data are shown as the mean ± SD of four technical replicates.

Fig. 4.

Anaerobic organosulfonate metabolism has an unexpectedly broad distribution in sequenced bacteria. (A) A maximum likelihood phylogenetic tree illustrating that the IslAs are phylogenetically distinct from previously characterized GREs. The listed sequence identifiers are GenBank accession codes. (B) Illustration of the distribution of 115 putative IslAs, as retrieved from the National Center for Biotechnology Information database at a threshold of 62% identity across sequenced bacterial genomes (SI Appendix, Table S1). The pie chart showing their relative taxonomic distribution on the phylum level (outer ring) demonstrates that the majority are found in genomes of Proteobacteria and Firmicutes, and at the class level (inner circle), that they are distributed predominantly across Deltaproteobacteria, Clostridia, and Negativicute genomes. The percentages shown in brackets indicate (at the class level) the number of genomes with putative IslAs relative to all known genome sequences for this class, confirming the enrichment of IslAs in the Deltaproteobacteria (encoded in 25% of all known genomes).