Glycyl Radical Enzymes and Sulfonate Metabolism in the Microbiome.

Sulfonates include diverse natural products and anthropogenic chemicals and are widespread in the environment. Many bacteria can degrade sulfonates and obtain sulfur, carbon, and energy for growth, playing important roles in the biogeochemical sulfur cycle. Cleavage of the inert sulfonate C-S bond involves a variety of enzymes, cofactors, and oxygen-dependent and oxygen-independent catalytic mechanisms. Sulfonate degradation by strictly anaerobic bacteria was recently found to involve C-S bond cleavage through O2-sensitive free radical chemistry, catalyzed by glycyl radical enzymes (GREs). The associated discoveries of new enzymes and metabolic pathways for sulfonate metabolism in diverse anaerobic bacteria have enriched our understanding of sulfonate chemistry in the anaerobic biosphere. An anaerobic environment of particular interest is the human gut microbiome, where sulfonate degradation by sulfate- and sulfite-reducing bacteria (SSRB) produces H2S, a process linked to certain chronic diseases and conditions.

Two radical-dependent mechanisms for anaerobic degradation of the globally abundant organosulfur compound dihydroxypropanesulfonate.

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.

Rescuing activity of oxygen-damaged pyruvate formate-lyase by a spare part protein.

Pyruvate formate-lyase (PFL) is a glycyl radical enzyme (GRE) that converts pyruvate and coenzyme A into acetyl-CoA and formate in a reaction that is crucial to the primary metabolism of many anaerobic bacteria. The glycyl radical cofactor, which is posttranslationally installed by a radical S-adenosyl-L-methionine (SAM) activase, is a simple and effective catalyst, but is also susceptible to oxidative damage in microaerobic environments. Such damage occurs at the glycyl radical cofactor, resulting in cleaved PFL (cPFL). Bacteria have evolved a spare part protein termed YfiD that can be used to repair cPFL. Previously, we obtained a structure of YfiD by NMR spectroscopy and found that the N-terminus of YfiD was disordered and that the C-terminus of YfiD duplicates the structure of the C-terminus of PFL, including a ß-strand that is not removed by the oxygen-induced cleavage. We also showed that cPFL is highly susceptible to proteolysis, suggesting that YfiD rescue of cPFL competes with protein degradation. Here, we probe the mechanism by which YfiD can bind and restore activity to cPFL through enzymatic and spectroscopic studies. Our data show that the disordered N-terminal region of YfiD is important for YfiD glycyl radical installation but not for catalysis, and that the duplicate ß-strand does not need to be cleaved from cPFL for YfiD to bind. In fact, truncation of this PFL region prevents YfiD rescue. Collectively our data suggest the molecular mechanisms by which YfiD activation is precluded both when PFL is not damaged and when it is highly damaged.

Engineering Escherichia coli for anaerobic alkane activation: Biosynthesis of (1-methylalkyl)succinates.

In anoxic environments, microbial activation of alkanes for subsequent metabolism occurs most commonly through the addition of fumarate to a subterminal carbon, producing an alkylsuccinate. Alkylsuccinate synthases are complex, multi-subunit enzymes that utilize a catalytic glycyl radical and require a partner, activating enzyme for hydrogen abstraction. While many genes encoding putative alkylsuccinate synthases have been identified, primarily from nitrate- and sulfate-reducing bacteria, few have been characterized and none have been reported to be functionally expressed in a heterologous host. Here, we describe the functional expression of the (1-methylalkyl)succinate synthase (Mas) system from Azoarcus sp. strain HxN1 in recombinant Escherichia coli. Mass spectrometry confirms anaerobic biosynthesis of the expected products of fumarate addition to hexane, butane, and propane. Maximum production of (1-methylpentyl)succinate is observed when masC, masD, masE, masB, and masG are all present on the expression plasmid; omitting masC reduces production by 66% while omitting any other gene eliminates production. Meanwhile, deleting iscR (encoding the repressor of the E. coli iron-sulfur cluster operon) improves product titer, as does performing the biotransformation at reduced temperature (18°C), both suggesting alkylsuccinate biosynthesis is largely limited by functional expression of this enzyme system.

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

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.

Insight into the Mechanism of Phenylacetate Decarboxylase (PhdB), a Toluene-Producing Glycyl Radical Enzyme.

We recently reported the discovery of phenylacetate decarboxylase (PhdB), representing one of only ten glycyl-radical-enzyme reaction types known, and a promising biotechnological tool for first-time biochemical synthesis of toluene from renewable resources. Here, we used experimental and computational data to evaluate the plausibility of three candidate PhdB mechanisms, involving either attack at the phenylacetate methylene carbon or carboxyl group [via H-atom abstraction from COOH or single-electron oxidation of COO- (Kolbe-type decarboxylation)]. In vitro experimental data included assays with F-labeled phenylacetate, kinetic studies, and tests with site-directed PhdB mutants; computational data involved estimation of reaction energetics using density functional theory (DFT). The DFT results indicated that all three mechanisms are thermodynamically challenging (beyond the range of many known enzymes in terms of endergonicity or activation energy barrier), reflecting the formidable demands on PhdB for catalysis of this reaction. Evidence that PhdB was able to bind α,α-difluorophenylacetate but was unable to catalyze its decarboxylation supported the enzyme's abstraction of a methylene H atom. Diminished activity of H327A and Y691F mutants was consistent with proposed proton donor roles for His327 and Tyr691. Collectively, these and other data most strongly support PhdB attack at the methylene carbon.

A gene cluster for taurine sulfur assimilation in an anaerobic human gut bacterium.

Aminoethylsulfonate (taurine) is widespread in the environment and highly abundant in the human body. Taurine and other aliphatic sulfonates serve as sulfur sources for diverse aerobic bacteria, which carry out cleavage of the inert sulfonate C-S bond through various O2-dependent mechanisms. Taurine also serves as a sulfur source for certain strict anaerobic fermenting bacteria. However, the mechanism of C-S cleavage by these bacteria has long been a mystery. Here we report the biochemical characterization of an anaerobic pathway for taurine sulfur assimilation in a strain of Clostridium butyricum from the human gut. In this pathway, taurine is first converted to hydroxyethylsulfonate (isethionate), followed by C-S cleavage by the O2-sensitive isethionate sulfo-lyase IseG, recently identified in sulfate- and sulfite-reducing bacteria. Homologs of the enzymes described in this study have a sporadic distribution in diverse strict and facultative anaerobic bacteria, from both the environment and the taurine-rich human gut, and may enable sulfonate sulfur acquisition in certain nutrient limiting conditions.

Solution structure and biochemical characterization of a spare part protein that restores activity to an oxygen-damaged glycyl radical enzyme.

Glycyl 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.

1,2-Propanediol Dehydration in Roseburia inulinivorans: STRUCTURAL BASIS FOR SUBSTRATE AND ENANTIOMER SELECTIVITY.

Glycyl radical enzymes (GREs) represent a diverse superfamily of enzymes that utilize a radical mechanism to catalyze difficult, but often essential, chemical reactions. In this work we present the first biochemical and structural data for a GRE-type diol dehydratase from the organism Roseburia inulinivorans (RiDD). Despite high sequence (48% identity) and structural similarity to the GRE-type glycerol dehydratase from Clostridium butyricum, we demonstrate that the RiDD is in fact a diol dehydratase. In addition, the RiDD will utilize both (S)-1,2-propanediol and (R)-1,2-propanediol as a substrate, with an observed preference for the S enantiomer. Based on the new structural information we developed and successfully tested a hypothesis that explains the functional differences we observe.

Substrate-bound structures of benzylsuccinate synthase reveal how toluene is activated in anaerobic hydrocarbon degradation.

Various bacteria perform anaerobic degradation of small hydrocarbons as a source of energy and cellular carbon. To activate non-reactive hydrocarbons such as toluene, enzymes conjugate these molecules to fumarate in a radical-catalyzed, C-C bond-forming reaction. We have determined x-ray crystal structures of the glycyl radical enzyme that catalyzes the addition of toluene to fumarate, benzylsuccinate synthase (BSS), in two oligomeric states with fumarate alone or with both substrates. We find that fumarate is secured at the bottom of a long active site cavity with toluene bound directly above it. The two substrates adopt orientations that appear ideal for radical-mediated C-C bond formation; the methyl group of toluene is positioned between fumarate and a cysteine that forms a thiyl radical during catalysis, which is in turn adjacent to the glycine that serves as a radical storage residue. Toluene is held in place by fumarate on one face and tight packing by hydrophobic residues on the other face and sides. These hydrophobic residues appear to become ordered, thus encapsulating toluene, only in the presence of BSSß, a small protein subunit that forms a tight complex with BSSα, the catalytic subunit. Enzymes related to BSS are able to metabolize a wide range of hydrocarbons through attachment to fumarate. Using our structures as a guide, we have constructed homology models of several of these "X-succinate synthases" and determined conservation patterns that will be useful in understanding the basis for catalysis and specificity in this family of enzymes.

Glycyl radical activating enzymes: structure, mechanism, and substrate interactions.

The glycyl radical enzyme activating enzymes (GRE-AEs) are a group of enzymes that belong to the radical S-adenosylmethionine (SAM) superfamily and utilize a [4Fe-4S] cluster and SAM to catalyze H-atom abstraction from their substrate proteins. GRE-AEs activate homodimeric proteins known as glycyl radical enzymes (GREs) through the production of a glycyl radical. After activation, these GREs catalyze diverse reactions through the production of their own substrate radicals. The GRE-AE pyruvate formate lyase activating enzyme (PFL-AE) is extensively characterized and has provided insights into the active site structure of radical SAM enzymes including GRE-AEs, illustrating the nature of the interactions with their corresponding substrate GREs and external electron donors. This review will highlight research on PFL-AE and will also discuss a few GREs and their respective activating enzymes.

New mechanisms for bacterial degradation of sulfoquinovose.

Sulfoquinovose (SQ, 6-deoxy-6-sulfo-D-glucose) is a sulfo-sugar with a ubiquitous distribution in the environment due to its production by plants and other photosynthetic organisms. Bacteria play an important role in degradation of SQ and recycling of its constituent sulfur and carbon. Since its discovery in 1963, SQ was noted to have a structural resemblance to glucose-6-phosphate and proposed to be degraded through a pathway analogous to glycolysis, termed sulfoglycolysis. Studies in recent years have uncovered an unexpectedly diverse array of sulfoglycolytic pathways in different bacteria, including one analogous to the Embden-Meyerhof-Parnas pathway (sulfo-EMP), one analogous to the Entner-Doudoroff pathway (sulfo-ED), and two involving sulfo-sugar cleavage by a transaldolase (sulfo-TAL) and transketolase (sulfo-TK), respectively, analogous to reactions in the pentose phosphate (PP) pathway. In addition, a non-sulfoglycolytic SQ degradation pathway was also reported, involving oxygenolytic C-S cleavage catalyzed by a homolog of alkanesulfonate monooxygenase (sulfo-ASMO). Here, we review the discovery of these new mechanisms of SQ degradation and lessons learnt in the study of new catabolic enzymes and pathways in bacteria.

The B12-independent glycerol dehydratase activating enzyme from Clostridium butyricum cleaves SAM to produce 5'-deoxyadenosine and not 5'-deoxy-5'-(methylthio)adenosine.

Glycerol dehydratase activating enzyme (GD-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential amino acid backbone radical onto glycerol dehydratase in bacteria under anaerobic conditions. Although GD-AE is closely homologous to other radical SAM activases that have been shown to cleave the S-C(5') bond of SAM to produce 5'-deoxyadenosine (5'-dAdoH) and methionine, GD-AE from Clostridium butyricum has been reported to instead cleave the S-C(γ) bond of SAM to yield 5'-deoxy-5'-(methylthio)adenosine (MTA). Here we re-investigate the SAM cleavage reaction catalyzed by GD-AE and show that it produces the widely observed 5'-dAdoH, and not the less conventional product MTA.

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

Desulfonation of isethionate by the bacterial glycyl radical enzyme (GRE) isethionate sulfite-lyase (IslA) generates sulfite, a substrate for respiration that in turn produces the disease-associated metabolite hydrogen sulfide. Here, we present a 2.7 Å resolution X-ray structure of wild-type IslA from Bilophila wadsworthia with isethionate bound. In comparison with other GREs, alternate positioning of the active site ß strands allows for distinct residue positions to contribute to substrate binding. These structural differences, combined with sequence variations, create a highly tailored active site for the binding of the negatively charged isethionate substrate. Through the kinetic analysis of 14 IslA variants and computational analyses, we probe the mechanism by which radical chemistry is used for C-S bond cleavage. This work further elucidates the structural basis of chemistry within the GRE superfamily and will inform structure-based inhibitor design of IsIA and thus of microbial hydrogen sulfide production.

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

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.

Potential Correlation between Dietary Fiber-Suppressed Microbial Conversion of Choline to Trimethylamine and Formation of Methylglyoxal.

Dietary interventions alter the formation of the disease-associated metabolite, trimethylamine (TMA), via intestinal microbial TMA lyase activity. Nevertheless, the mechanisms regulating microbial enzyme production are still unclear. Sequencing of the gut bacteria 16S rDNA demonstrated that dietary intervention changed the composition of the gut microbiota and the functional metagenome involved in the choline utilization pathway. Characterization of the functional profile of the metagenomes and metabonomics analysis revealed that a series of Kyoto Encyclopedia of Genes and Genomes orthologous groups and enzyme groups related to accumulation of methylglyoxal (MG) and glycine were enriched in red meat diet-fed animals, whereas fiber-rich diet suppressed glycine formation via the MG-dependent pathway. Our observations suggest associations between choline-TMA lyase expression and MG formation, which are indicative of a novel role of the gut microbiota in choline metabolism and highlight it as a potential target for inhibiting TMA production.

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

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 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.

Mechanistic Studies of Radical SAM Enzymes: Pyruvate Formate-Lyase Activating Enzyme and Lysine 2,3-Aminomutase Case Studies.

The radical SAM enzyme superfamily is large and diverse, with ever-increasing numbers of examples of characterized reactions. This chapter focuses on the methodology we have developed over the last 25 years for working with these enzymes, with the specific examples discussed being the pyruvate formate-lyase activating enzyme (PFL-AE) and lysine 2,3-aminomutase (LAM). Both enzymes are purified from overexpressing Escherichia coli, but differ in that PFL-AE is expressed without an affinity tag and does not require iron-sulfur cluster reconstitution, while LAM purification is carried out through use of a His6 affinity tag and the enzyme benefits from cluster reconstitution. Because of radical SAM enzymes' catalytic need for a [4Fe-4S] cluster, we present methods for characterization and incorporation of a full [4Fe-4S] cluster in addition to enzyme activity assay protocols. Synthesis of SAM (S-adenosyl-l-methionine) and its analogs have played an important role in our mechanistic studies of radical SAM enzymes, and their synthetic methods are also presented in detail.

Anaerobic 4-hydroxyproline utilization: Discovery of a new glycyl radical enzyme in the human gut microbiome uncovers a widespread microbial metabolic activity.

The discovery of enzymes responsible for previously unappreciated microbial metabolic pathways furthers our understanding of host-microbe and microbe-microbe interactions. We recently identified and characterized a new gut microbial glycyl radical enzyme (GRE) responsible for anaerobic metabolism of trans-4-hydroxy-l-proline (Hyp). Hyp dehydratase (HypD) catalyzes the removal of water from Hyp to generate Δ1-pyrroline-5-carboxylate (P5C). This enzyme is encoded in the genomes of a diverse set of gut anaerobes and is prevalent and abundant in healthy human stool metagenomes. Here, we discuss the roles HypD may play in different microbial metabolic pathways as well as the potential implications of this activity for colonization resistance and pathogenesis within the human gut. Finally, we present evidence of anaerobic Hyp metabolism in sediments through enrichment culturing of Hyp-degrading bacteria, highlighting the wide distribution of this pathway in anoxic environments beyond the human gut.

The glycyl-radical enzyme 2-ketobutyrate formate-lyase, TdcE, interacts specifically with the formate-translocating FNT-channel protein FocA.

Formate is a major product of mixed-acid fermentation in Escherichia coli. Because formate can act as an uncoupler at high concentration it must be excreted from the cell. The FNT (formate-nitrite transporter) membrane channel FocA ensures formate is translocated across the cytoplasmic membrane. Two glycyl-radical enzymes (GREs), pyruvate formate-lyase (PflB) and 2-ketobutyrate formate-lyase (TdcE), generate formate as a product of catalysis during anaerobic growth of Escherichia coli. We demonstrate in this study that TdcE, like PflB, interacts specifically with FocA. His-tagged variants of two other predicted GREs encoded in the genome of E. coli were over-produced and purified and were shown not to interact with FocA, indicating that interaction with FocA is not a general property of GREs per se. Together, these data show that only the GREs TdcE and PflB interact with the FNT channel protein and suggest that, like PflB, TdcE can control formate translocation by FocA.