Bacterial microcompartments for isethionate desulfonation in the taurine-degrading human-gut bacterium Bilophila wadsworthia

Background: Bilophila wadsworthia, a strictly anaerobic, sulfite-reducing bacterium and common member of the human gut microbiota, has been associated with diseases such as appendicitis and colitis. It is specialized on organosulfonate respiration for energy conservation, i.e., utilization of dietary and host-derived organosulfonates, such as taurine (2 aminoethansulfonate), as sulfite donors for sulfite respiration, producing hydrogen sulfide (H2S), an important intestinal metabolite that may have beneficial as well as detrimental effects on the colonic environment. Its taurine desulfonation pathway involves a glycyl radical enzyme (GRE), isethionate sulfite-lyase (IslAB), which cleaves isethionate (2 hydroxyethane sulfonate) into acetaldehyde and sulfite. Results: We demonstrate that taurine metabolism in B. wadsworthia 3.1.6 involves bacterial microcompartments (BMCs). First, we confirmed taurine-inducible production of BMCs by proteomic, transcriptomic and ultra-thin sectioning and electron-microscopical analyses. Then, we isolated BMCs from taurine-grown cells by density-gradient ultracentrifugation and analyzed their composition by proteomics as well as by enzyme assays, which suggested that the GRE IslAB and acetaldehyde dehydrogenase are located inside of the BMCs. Finally, we are discussing the recycling of cofactors in the IslAB-BMCs and a potential shuttling of electrons across the BMC shell by a potential iron-sulfur (FeS) cluster-containing shell protein identified by sequence analysis. Conclusions: We characterized a novel subclass of BMCs and broadened the spectrum of reactions known to take place enclosed in BMCs, which is of biotechnological interest. We also provided more details on the energy metabolism of the opportunistic pathobiont B. wadsworthia and on microbial H2S production in the human gut.

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Bacterial microcompartments for isethionate desulfonation in the taurine-degrading human-gut bacterium Bilophila wadsworthia

BMC Microbiology ◽

10.1186/s12866-021-02386-w ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Anna G. Burrichter ◽

Stefanie Dörr ◽

Paavo Bergmann ◽

Sebastian Haiß ◽

Anja Keller ◽

...

Keyword(s):

Enzyme Assays ◽

Strictly Anaerobic ◽

Human Gut ◽

Bacterial Microcompartments ◽

Shell Protein ◽

Glycyl Radical ◽

Iron Sulfur ◽

Thin Sectioning ◽

Electron Microscopical ◽

Glycyl Radical Enzyme

Abstract Background Bilophila wadsworthia, a strictly anaerobic, sulfite-reducing bacterium and common member of the human gut microbiota, has been associated with diseases such as appendicitis and colitis. It is specialized on organosulfonate respiration for energy conservation, i.e., utilization of dietary and host-derived organosulfonates, such as taurine (2-aminoethansulfonate), as sulfite donors for sulfite respiration, producing hydrogen sulfide (H2S), an important intestinal metabolite that may have beneficial as well as detrimental effects on the colonic environment. Its taurine desulfonation pathway involves the glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslAB), which cleaves isethionate (2-hydroxyethanesulfonate) into acetaldehyde and sulfite. Results We demonstrate that taurine metabolism in B. wadsworthia 3.1.6 involves bacterial microcompartments (BMCs). First, we confirmed taurine-inducible production of BMCs by proteomic, transcriptomic and ultra-thin sectioning and electron-microscopical analyses. Then, we isolated BMCs from taurine-grown cells by density-gradient ultracentrifugation and analyzed their composition by proteomics as well as by enzyme assays, which suggested that the GRE IslAB and acetaldehyde dehydrogenase are located inside of the BMCs. Finally, we are discussing the recycling of cofactors in the IslAB-BMCs and a potential shuttling of electrons across the BMC shell by a potential iron-sulfur (FeS) cluster-containing shell protein identified by sequence analysis. Conclusions We characterized a novel subclass of BMCs and broadened the spectrum of reactions known to take place enclosed in BMCs, which is of biotechnological interest. We also provided more details on the energy metabolism of the opportunistic pathobiont B. wadsworthia and on microbial H2S production in the human gut.

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Glycyl Radical Enzyme-Associated Microcompartments: Redox-Replete Bacterial Organelles

mBio ◽

10.1128/mbio.02327-18 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Bryan Ferlez ◽

Markus Sutter ◽

Cheryl A. Kerfeld

Keyword(s):

Substrate Specificity ◽

Pathogenic Bacteria ◽

External Source ◽

Redox Proteins ◽

Physiological Importance ◽

Bacterial Microcompartments ◽

Glycyl Radical ◽

Self Assembling ◽

Glycyl Radical Enzyme ◽

Protein Shell

ABSTRACTAn increasing number of microbes are being identified that organize catabolic pathways within self-assembling proteinaceous structures known as bacterial microcompartments (BMCs). Most BMCs are characterized by their singular substrate specificity and commonly employ B12-dependent radical mechanisms. In contrast, a less-well-known BMC type utilizes the B12-independent radical chemistry of glycyl radical enzymes (GREs). Unlike B12-dependent enzymes, GREs require an activating enzyme (AE) as well as an external source of electrons to generate an adenosyl radical and form their catalytic glycyl radical. Organisms encoding these glycyl radical enzyme-associated microcompartments (GRMs) confront the challenge of coordinating the activation and maintenance of their GREs with the assembly of a multienzyme core that is encapsulated in a protein shell. The GRMs appear to enlist redox proteins to either generate reductants internally or facilitate the transfer of electrons from the cytosol across the shell. Despite this relative complexity, GRMs are one of the most widespread types of BMC, with distinct subtypes to catabolize different substrates. Moreover, they are encoded by many prominent gut-associated and pathogenic bacteria. In this review, we will focus on the diversity, function, and physiological importance of GRMs, with particular attention given to their associated and enigmatic redox proteins.

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Molecular basis for catabolism of the abundant metabolite trans-4-hydroxy-L-proline by a microbial glycyl radical enzyme

eLife ◽

10.7554/elife.51420 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Lindsey RF Backman ◽

Yolanda Y Huang ◽

Mary C Andorfer ◽

Brian Gold ◽

Ronald T Raines ◽

...

Keyword(s):

Active Site ◽

Metabolic Pathways ◽

Molecular Basis ◽

Radical Mechanism ◽

Human Gut ◽

Clostridioides Difficile ◽

Glycyl Radical ◽

Biochemical Studies ◽

Glycyl Radical Enzyme ◽

Insight Into

The glycyl radical enzyme (GRE) superfamily utilizes a glycyl radical cofactor to catalyze difficult chemical reactions in a variety of anaerobic microbial metabolic pathways. Recently, a GRE, trans-4-hydroxy-L-proline (Hyp) dehydratase (HypD), was discovered that catalyzes the dehydration of Hyp to (S)-Δ1-pyrroline-5-carboxylic acid (P5C). This enzyme is abundant in the human gut microbiome and also present in prominent bacterial pathogens. However, we lack an understanding of how HypD performs its unusual chemistry. Here, we have solved the crystal structure of HypD from the pathogen Clostridioides difficile with Hyp bound in the active site. Biochemical studies have led to the identification of key catalytic residues and have provided insight into the radical mechanism of Hyp dehydration.

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Bioinformatic Characterization of Glycyl Radical Enzyme-Associated Bacterial Microcompartments

Applied and Environmental Microbiology ◽

10.1128/aem.02587-15 ◽

2015 ◽

Vol 81 (24) ◽

pp. 8315-8329 ◽

Cited By ~ 29

Author(s):

Jan Zarzycki ◽

Onur Erbilgin ◽

Cheryl A. Kerfeld

Keyword(s):

Active Site ◽

Metabolic Pathways ◽

Experimental Investigations ◽

Vitamin B ◽

Bacterial Microcompartments ◽

Glycyl Radical ◽

Shell Composition ◽

Glycyl Radical Enzyme ◽

Assembly Pathways

ABSTRACTBacterial microcompartments (BMCs) are proteinaceous organelles encapsulating enzymes that catalyze sequential reactions of metabolic pathways. BMCs are phylogenetically widespread; however, only a few BMCs have been experimentally characterized. Among them are the carboxysomes and the propanediol- and ethanolamine-utilizing microcompartments, which play diverse metabolic and ecological roles. The substrate of a BMC is defined by its signature enzyme. In catabolic BMCs, this enzyme typically generates an aldehyde. Recently, it was shown that the most prevalent signature enzymes encoded by BMC loci are glycyl radical enzymes, yet little is known about the function of these BMCs. Here we characterize the glycyl radical enzyme-associated microcompartment (GRM) loci using a combination of bioinformatic analyses and active-site and structural modeling to show that the GRMs comprise five subtypes. We predict distinct functions for the GRMs, including the degradation of choline, propanediol, and fuculose phosphate. This is the first family of BMCs for which identification of the signature enzyme is insufficient for predicting function. The distinct GRM functions are also reflected in differences in shell composition and apparently different assembly pathways. The GRMs are the counterparts of the vitamin B12-dependent propanediol- and ethanolamine-utilizing BMCs, which are frequently associated with virulence. This study provides a comprehensive foundation for experimental investigations of the diverse roles of GRMs. Understanding this plasticity of function within a single BMC family, including characterization of differences in permeability and assembly, can inform approaches to BMC bioengineering and the design of therapeutics.

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