Investigation Of Enzymes And Pathways For The Degradation Of C2 Sulfonates In Anaerobic Intestinal Bacteria

The C2 sulfonates taurine(2-aminoethylsulfonate)and isethionate(2-hydroxyethylsulfonate)are of particular importance to human health,and are produced by the microbial degradation of taurine-conjugated bile salts in the gut.There,a consortium of anaerobic intestinal bacteria converts them into toxic H₂S,which has been implicated in inflammation,colorectal cancer and other diseases.While the rich enzymology of taurine and isethionate degradation has been studied over the past five decades,the enzymes and pathways involved in their metabolism in strict anaerobes,including intestinal bacteria,are poorly understood.This research presents the discovery of a novel O₂-sensitive glycyl radical enzyme(GRE),which is involved in the degradation of C2 sulfonates in strict anaerobes.Sequence similarity network(SSN)analysis of the GRE superfamily and the following genome neighborhood analysis of phylogenetically-diverse sulfate-and sulfite-reducing bacteria(SSRB)led to the identification of a particular GRE of unknown function(GUF),which was assigned as a putative isethionate sulfo-lyase(IseG).IseG from Desulfovibrio vulgaris Hildenborough was recombinantly produced and activated to analyze its catalytic activity in vitro.The results demonstrated that IseG catalyzes the radical-mediated C-S bond cleavage of isethionate,and is kinetically competent in converting isethionate to acetaldehyde and sulfite.Further structural characterization of the isethionate-bound IseG crystal explained the radical mechanism of isethionate C-S bond cleavage by IseG.Phylogenetic analysis demonstrated that IseG is present in metabolically diverse environmental and human-associated anaerobic bacteria.This research further investigated IseG-dependent pathways for C2 sulfonates degradation in human intestinal SSRB.In Desulfovibrio piger,a prevalent human intestinal SSRB,IseG induction and H₂S production were detected when using isethionate as the sole terminal electron acceptor(TEA),and a specific transporter for isethionate import was confirmed by isothermal calorimetry.These results demonstrate the presence of IseG-dependent pathways specifically for the uptake and dissimilation of isethionate in SSRB,constituting the first example of a role for a GRE in generating TEA for anaerobic respiration.In Bilophila wadsworthia,a disease-associated SSRB,IseG induction was detected in cells grown on either isethionate or taurine as the sole carbon source and TEA.In this characterized sulfonate dissimilation pathway,taurine is converted by a pyruvate aminotransferase(Tpa)to sulfoacetaldehyde,which is reduced by a sulfoacetaldehyde reductase(TauF)to isethionate.Subsequent C-S cleavage of isethionate by Bw IseG generates sulfite as a TEA for anaerobic respiration,which releases toxic H₂S.IseG-dependent pathways for isethionate and taurine dissimilation serve as a paradigm for the microbial conversion of a host metabolite into a toxin in the unique metabolic niche of Bilophila.Apart from SSRB,IseG is also present in some fermenting bacteria.In a strain of Clostridium butyricum,an anaerobic pathway for taurine sulfur assimilation was biochemically characterized.In vitro reconstitution of the pathway demonstrated that taurine was first converted to isethionate by CbTpa and CbTauF,followed by C-S cleavage of isethionate to form acetaldehyde and sulfite by IseG.This IseG-dependent pathway enables taurine and isethionate to be used as a sulfur source for cell growth.In summary,the identification and characterization of IseG and associated proteins uncovered a suite of new metabolic pathways for the degradation of C2 sulfonates in anaerobic bacteria.Assimilative pathways for the utilization of sulfur from taurine and isehionate may play an important role in bacteria inhabiting the densely populated and competitive environment of the colon.Dissimilative pathways in SSRB constitute an important part of the sulfur cycle in both the environment and the human intestinal tract,and are of great relevance to human health.Production of toxic levels of H₂S by SSRB impacts other intestinal bacteria and host tissue.Pathways involving IseG are envision to be new antibiotic drug targets specific for disease-linked SSRB and therapeutic targets for the treatment of human diseases.