scholarly journals A glycyl radical enzyme enables hydrogen sulfide production by the human intestinal bacteriumBilophila wadsworthia

2019 ◽  
Vol 116 (8) ◽  
pp. 3171-3176 ◽  
Author(s):  
Spencer C. Peck ◽  
Karin Denger ◽  
Anna Burrichter ◽  
Stephania M. Irwin ◽  
Emily P. Balskus ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Bond Cleavage ◽  
Anaerobic Respiration ◽  
Desulfovibrio Desulfuricans ◽  
Intestinal Bacteria ◽  
Differential Proteomics ◽  
Terminal Electron Acceptor ◽  
Glycyl Radical ◽  
Glycyl Radical Enzyme

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 inBilophila wadsworthia3.1.6 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 inDesulfovibrio desulfuricansDSM642 andDesulfovibrio alaskensisG20, which use isethionate but not taurine; corresponding knockout mutants ofD. alaskensisG20 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 ofB. wadsworthia. This GRE is widely distributed in gut bacterial genomes and may represent a novel target for control of intestinal H2S production.

scholarly journals Molecular Basis of C–S Bond Cleavage in the Glycyl Radical Enzyme Isethionate Sulfite-Lyase

2020 ◽  
Author(s):  
Christopher D. Dawson ◽  
Stephania Irwin ◽  
Lindsey Backman ◽  
Catherine Drennan ◽  
Emily Balskus
Keyword(s):  
Molecular Basis ◽  
Bond Cleavage ◽  
Glycyl Radical ◽  
Glycyl Radical Enzyme

Metabolism of Hydrocarbons in n-Alkane-Utilizing Anaerobic Bacteria

2016 ◽  
Vol 26 (1-3) ◽  
pp. 138-151 ◽  
Author(s):  
Heinz Wilkes ◽  
Wolfgang Buckel ◽  
Bernard T. Golding ◽  
Ralf Rabus
Keyword(s):  
Bond Cleavage ◽  
Anaerobic Bacteria ◽  
Sulfate Reducer ◽  
Alkane Degradation ◽  
Carbon Centers ◽  
Glycyl Radical ◽  
Kinetic Isotope ◽  
Dead End ◽  
Bacterium Strain ◽  
Glycyl Radical Enzyme

The glycyl radical enzyme-catalyzed addition of <i>n</i>-alkanes to fumarate creates a C-C-bond between two concomitantly formed stereogenic carbon centers. The configurations of the two diastereoisomers of the product resulting from <i>n</i>-hexane activation by the <i>n</i>-alkane-utilizing denitrifying bacterium strain HxN1, i.e. (1-methylpentyl)succinate, were assigned as (2<i>S</i>,1′<i>R</i>) and (2<i>R</i>,1′<i>R</i>). Experiments with stereospecifically deuterated <i>n</i>-(2,5-<sup>2</sup>H<sub>2</sub>)hexanes revealed that exclusively the pro-<i>S</i> hydrogen atom is abstracted from C2 of the <i>n</i>-alkane by the enzyme and later transferred back to C3 of the alkylsuccinate formed. These results indicate that the alkylsuccinate-forming reaction proceeds with an inversion of configuration at the carbon atom (C2) of the <i>n</i>-alkane forming the new C-C-bond, and thus stereochemically resembles a S<sub>N</sub>2-type reaction. Therefore, the reaction may occur in a concerted manner, which may avoid the highly energetic hex-2-yl radical as an intermediate. The reaction is associated with a significant primary kinetic isotope effect (kH/kD ≥3) for hydrogen, indicating that the homolytic C-H-bond cleavage is involved in the first irreversible step of the reaction mechanism. The (1-methylalkyl)succinate synthases of <i>n</i>-alkane-utilizing anaerobic bacteria apparently have very broad substrate ranges enabling them to activate not only aliphatic but also alkyl-aromatic hydrocarbons. Thus, two denitrifiers and one sulfate reducer were shown to convert the nongrowth substrate toluene to benzylsuccinate and further to the dead-end product benzoyl-CoA. For this purpose, however, the modified β-oxidation pathway known from alkylbenzene-utilizing bacteria was not employed, but rather the pathway used for <i>n</i>-alkane degradation involving CoA ligation, carbon skeleton rearrangement and decarboxylation. Furthermore, various <i>n</i>-alkane- and alkylbenzene-utilizing denitrifiers and sulfate reducers were found to be capable of forming benzyl alcohols from diverse alkylbenzenes, putatively via dehydrogenases. The thermophilic sulfate reducer strain TD3 forms <i>n</i>-alkylsuccinates during growth with <i>n</i>-alkanes or crude oil, which, based on the observed patterns of homologs, do not derive from a terminal activation of <i>n</i>-alkanes.

scholarly journals Molecular Basis of C–N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase

2016 ◽  
Vol 23 (10) ◽  
pp. 1206-1216 ◽  
Author(s):  
Smaranda Bodea ◽  
Michael A. Funk ◽  
Emily P. Balskus ◽  
Catherine L. Drennan
Keyword(s):  
Molecular Basis ◽  
Bond Cleavage ◽  
Glycyl Radical ◽  
Glycyl Radical Enzyme

scholarly journals The Stickland fermentation precursor trans-4-hydroxyproline differentially impacts the metabolism of Clostridioides difficile and commensal Clostridia

2021 ◽  
Author(s):  
Casey M Theriot ◽  
Amber D Reed ◽  
Joshua R Fletcher ◽  
Yue (Yolanda) Huang ◽  
Rajani Thanissery ◽  
...  
Keyword(s):  
Amino Acid ◽  
Gut Microbiota ◽  
Clostridioides Difficile ◽  
Glycyl Radical ◽  
Metabolic Gene Expression ◽  
Hydroxyproline Metabolism ◽  
Mouse Model Of Infection ◽  
Glycyl Radical Enzyme ◽  
Significant Disease

An intact gut microbiota confers colonization resistance against Clostridioides difficile through a variety of mechanisms, likely including competition for nutrients. Recently, proline was identified as an important environmental amino acid that C. difficile uses to support growth and cause significant disease. The ability to dehydrate trans-4-hydroxyproline via the HypD glycyl radical enzyme is widespread amongst gut microbiota, including C. difficile and members of the commensal Clostridia, suggesting that this amino acid is an important nutrient in the host environment. Therefore, we constructed a C. difficile ΔhypD mutant and found that it was modestly impaired in fitness in a mouse model of infection, and was associated with an altered microbiota when compared to mice challenged with the wild type strain. Changes in the microbiota between the two groups were largely driven by members of the Lachnospiraceae family and the Clostridium genus. We found that C. difficile and type strains of three commensal Clostridia had significant alterations to their metabolic gene expression in the presence of trans-4-hydroxyproline in vitro. The proline reductase (prd) genes were elevated in C. difficile, consistent with the hypothesis that trans-4-hydroxyproline is used by C. difficile to supply proline for fermentation. Similar transcripts were also elevated in some commensal Clostridia tested, although each strain responded differently. This suggests that the uptake and utilization of other nutrients by the commensal Clostridia may be affected by trans-4-hydroxyproline metabolism, highlighting how a common nutrient may be a signal to each organism to adapt to a unique niche.

scholarly journals Molecular basis of C-S bond cleavage in the glycyl radical enzyme isethionate sulfite-lyase

2021 ◽  
Author(s):  
Christopher D. Dawson ◽  
Stephania M. Irwin ◽  
Lindsey R.F. Backman ◽  
Chip Le ◽  
Jennifer X. Wang ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Bond Cleavage ◽  
Glycyl Radical ◽  
Glycyl Radical Enzyme

Endogenous hydrogen sulfide sulfhydrates IKKβ at cysteine 179 to control pulmonary artery endothelial cell inflammation

2019 ◽  
Vol 133 (20) ◽  
pp. 2045-2059 ◽  
Author(s):  
Da Zhang ◽  
Xiuli Wang ◽  
Siyao Chen ◽  
Selena Chen ◽  
Wen Yu ◽  
...  
Keyword(s):  
Hydrogen Sulfide ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Cell Model ◽  
Site Directed Mutagenesis ◽  
Critical Event ◽  
Hypertensive Rats ◽  
Pulmonary Artery Endothelial Cell

Abstract Background: Pulmonary artery endothelial cell (PAEC) inflammation is a critical event in the development of pulmonary arterial hypertension (PAH). However, the pathogenesis of PAEC inflammation remains unclear. Methods: Purified recombinant human inhibitor of κB kinase subunit β (IKKβ) protein, human PAECs and monocrotaline-induced pulmonary hypertensive rats were employed in the study. Site-directed mutagenesis, gene knockdown or overexpression were conducted to manipulate the expression or activity of a target protein. Results: We showed that hydrogen sulfide (H2S) inhibited IKKβ activation in the cell model of human PAEC inflammation induced by monocrotaline pyrrole-stimulation or knockdown of cystathionine γ-lyase (CSE), an H2S generating enzyme. Mechanistically, H2S was proved to inhibit IKKβ activity directly via sulfhydrating IKKβ at cysteinyl residue 179 (C179) in purified recombinant IKKβ protein in vitro, whereas thiol reductant dithiothreitol (DTT) reversed H2S-induced IKKβ inactivation. Furthermore, to demonstrate the significance of IKKβ sulfhydration by H2S in the development of PAEC inflammation, we mutated C179 to serine (C179S) in IKKβ. In purified IKKβ protein, C179S mutation of IKKβ abolished H2S-induced IKKβ sulfhydration and the subsequent IKKβ inactivation. In human PAECs, C179S mutation of IKKβ blocked H2S-inhibited IKKβ activation and PAEC inflammatory response. In pulmonary hypertensive rats, C179S mutation of IKKβ abolished the inhibitory effect of H2S on IKKβ activation and pulmonary vascular inflammation and remodeling. Conclusion: Collectively, our in vivo and in vitro findings demonstrated, for the first time, that endogenous H2S directly inactivated IKKβ via sulfhydrating IKKβ at Cys179 to inhibit nuclear factor-κB (NF-κB) pathway activation and thereby control PAEC inflammation in PAH.

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