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An 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 B 12 -dependent radical mechanisms. An 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 B 12 -dependent radical mechanisms. In contrast, a less-well-known BMC type utilizes the B 12 -independent radical chemistry of glycyl radical enzymes (GREs). Unlike B 12 -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.

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.

2(S)-dihydroxypropanesulfonate (DHPS) is a microbial degradation product of 6-deoxy-6-sulfo-d-glucopyranose (sulfoquinovose), a component of plant sulfolipid with an estimated annual production of 1010 tons. DHPS is also at millimolar levels in highly abundant marine phytoplankton. Its degradation and sulfur recycling by microbes, thus, play important roles in the biogeochemical sulfur cycle. However, DHPS degradative pathways in the anaerobic biosphere are not well understood. Here, we report the discovery and characterization of two O₂-sensitive glycyl radical enzymes that use distinct mechanisms for DHPS degradation. DHPS-sulfolyase (HpsG) in sulfate- and sulfite-reducing bacteria catalyzes C–S cleavage to release sulfite for use as a terminal electron acceptor in respiration, producing H₂S. DHPS-dehydratase (HpfG), in fermenting bacteria, catalyzes C–O cleavage to generate 3-sulfopropionaldehyde, subsequently reduced by the NADH-dependent sulfopropionaldehyde reductase (HpfD). Both enzymes are present in bacteria from diverse environments including human gut, suggesting the contribution of enzymatic radical chemistry to sulfur flux in various anaerobic niches.

Radical enzymes, including glycyl radical enzymes (GREs) and B12-dependent enzymes, catalyze a wide range of biochemical transformations through radical-based mechanisms. An unusual property—conditional resistance to chymotrypsin digestion—has previously been reported for two GREs. However, whether this feature is broadly conserved among related radical enzymes and what factors trigger it has remained unclear. In this study, we investigated five radical enzymes: four GREs and one B12-dependent diol dehydratase. Proteolytic assays demonstrated that substrate binding significantly enhances resistance to chymotrypsin degradation, suggesting a conserved conformational shift from an open, protease-sensitive state to a closed, protease-resistant form. X-ray crystallographic analysis of a GRE-type 1,2-propanediol dehydratase from Raoultella planticola confirmed that active site occupancy correlates with increased protease resistance. Importantly, non-substrate analogs such as 1,3-propanediol and β-methylcholine failed to induce protection, underscoring the specificity of ligand-induced stabilization. These findings reveal a broadly conserved mechanism of substrate-induced conformational stabilization in GREs and B12-dependent radical enzymes and offer a scalable strategy for ligand identification with potential applications in enzyme engineering.

An important microbial enzymatic pathway in the gut is choline/phosphatidylcholine degradation in which intestinal microbes utilize dietary choline to produce TMA via the choline utilization cluster polypeptide C (CutC) enzyme, a member of the glycyl radical enzyme family. Our goal in this theoretical work was to study the reaction mechanism and elucidate how the enzyme environment (steric and electronic) modulates the reaction path. Dissecting the effect of the enzyme environment on the reaction mechanism and shedding light on how steric and electronic effects affect the reaction path is an insightful and significant contribution of this work. Our theoretical results suggest that the final product of enzyme catalysis might be carbinolamine and not TMA and acetaldehyde. In addition, we found out that Glu491 plays the role of a base in this reaction (a disputed fact) by temporarily abstracting a proton from the hydroxylic group of choline sometime during the reaction—with the proton transfer being critical for the reaction to proceed to completion. We also found that the choice of computational protocol not only alters the reaction energetics but can change the reaction path by creating new intermediates and transition states or eliminating existing ones.

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.

AbstractGlycyl radical enzymes (GREs) utilize a glycyl radical cofactor to carry out a diverse array of chemically challenging enzymatic reactions in anaerobic bacteria. Although the glycyl radical is a powerful catalyst, it is also oxygen sensitive such that oxygen exposure causes cleavage of the GRE at the site of the radical. This oxygen sensitivity presents a challenge to facultative anaerobes dwelling in areas prone to oxygen exposure. Once GREs are irreversibly oxygen damaged, cells either need to make new GREs or somehow repair the damaged one. One particular GRE, pyruvate formate lyase (PFL), can be repaired through the binding of a 14.3 kDa protein, termed YfiD, which is constitutively expressed in E. coli. Herein, we have solved a solution structure of this ‘spare part’ protein using nuclear magnetic resonance spectroscopy. These data, coupled with data from circular dichroism, indicate that YfiD has an inherently flexible N-terminal region (residues 1–60) that is followed by a C-terminal region (residues 72–127) that has high similarity to the glycyl radical domain of PFL. Reconstitution of PFL activity requires that YfiD binds within the core of the PFL barrel fold; however, modeling suggests that oxygen-damaged, i.e. cleaved, PFL cannot fully accommodate YfiD. We further report that a PFL variant that mimics the oxygen-damaged enzyme is highly susceptible to proteolysis, yielding additionally truncated forms of PFL. One such PFL variant of ~ 77 kDa makes an ideal scaffold for the accommodation of YfiD. A molecular model for the rescue of PFL activity by YfiD is presented.Graphic abstract

The anaerobic degradation pathway of toluene is initiated by the addition of the methyl group of toluene to the double bond of fumarate. This reaction is catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase (BSS). The enzyme consists of three subunits, α, β, and γ, and differs from most other glycyl-radical enzymes in having additional cofactors. We have purified a Strep-tagged nonactivated BSS from recombinant Escherichia coli and identified the additional cofactors as FeS clusters by UV/vis, EPR, and Mössbauer spectroscopy. Analysis of the metal content as well as the EPR and Mössbauer spectra indicated that BSS contains magnetically coupled low-potential [4Fe–4S] clusters. Several enzyme preparations showed differing amounts of [3Fe–4S] clusters that could be reconstituted to [4Fe–4S] clusters, indicating that they arise from partial decay of the initial [4Fe–4S] clusters. The most likely location of these FeS clusters in the enzyme are the small β and γ subunits, which are unique for the BSS subfamily of glycyl-radical enzymes and contain conserved cysteines as potential ligands.

The anaerobic degradation pathways of toluene and m-xylene are initiated by addition of a fumarate cosubstrate to the methyl group of the hydrocarbon, yielding (R)-benzylsuccinate and (3-methylbenzyl)succinate, respectively, as first intermediates. These reactions are catalyzed by a novel glycyl-radical enzyme, (R)-benzylsuccinate synthase. Substrate specificities of benzylsuccinate synthases were analyzed in Azoarcus sp. strain T and Thauera aromatica strain K172. The enzyme of Azoarcus sp. strain T converts toluene, but also all xylene and cresol isomers, to the corresponding succinate adducts, whereas the enzyme of T. aromatica is active with toluene and all cresols, but not with any xylene isomer. This corresponds to the capabilities of Azoarcus sp. strain T to grow on either toluene or m-xylene, and of T. aromatica to grow on toluene as sole hydrocarbon substrate. Thus, differences in the substrate spectra of the respective benzylsuccinate synthases of the two strains contribute to utilization of different aromatic hydrocarbons, although growth on different substrates also depends on additional determinants. We also provide direct evidence by electron paramagnetic resonance (EPR) spectroscopy that glycyl radical enzymes corresponding to substrate-induced benzylsuccinate synthases are specifically detectable in anoxically prepared extracts of toluene- or m-xylene-grown cells. The presence of the EPR signals and the determined amount of the radical are consistent with the respective benzylsuccinate synthase activities. The properties of the EPR signals are highly similar to those of the prototype glycyl radical enzyme pyruvate formate lyase, but differ slightly from previously reported parameters for partially purified benzylsuccinate synthase.

A small, nucleotide-binding domain, the ATP-cone, is found at the N-terminus of most ribonucleotide reductase (RNR) catalytic subunits. By binding adenosine triphosphate (ATP) or deoxyadenosine triphosphate (dATP) it regulates the enzyme activity of all classes of RNR. Functional and structural work on aerobic RNRs has revealed a plethora of ways in which dATP inhibits activity by inducing oligomerisation and preventing a productive radical transfer from one subunit to the active site in the other. Anaerobic RNRs, on the other hand, store a stable glycyl radical next to the active site and the basis for their dATP-dependent inhibition is completely unknown. We present biochemical, biophysical, and structural information on the effects of ATP and dATP binding to the anaerobic RNR from Prevotella copri . The enzyme exists in a dimer–tetramer equilibrium biased towards dimers when two ATP molecules are bound to the ATP-cone and tetramers when two dATP molecules are bound. In the presence of ATP, P. copri NrdD is active and has a fully ordered glycyl radical domain (GRD) in one monomer of the dimer. Binding of dATP to the ATP-cone results in loss of activity and increased dynamics of the GRD, such that it cannot be detected in the cryo-EM structures. The glycyl radical is formed even in the dATP-bound form, but the substrate does not bind. The structures implicate a complex network of interactions in activity regulation that involve the GRD more than 30 Å away from the dATP molecules, the allosteric substrate specificity site and a conserved but previously unseen flap over the active site. Taken together, the results suggest that dATP inhibition in anaerobic RNRs acts by increasing the flexibility of the flap and GRD, thereby preventing both substrate binding and radical mobilisation.