Emily P. Balskus

Microbial conversion of choline to trimethylamine requires a glycyl radical enzyme

Choline and trimethylamine (TMA) are small molecules that play central roles in biological processes throughout all kingdoms of life. These ubiquitous metabolites are linked through a single biochemical transformation, the conversion of choline to TMA by anaerobic microorganisms. This metabolic activity, which contributes to methanogenesis and human disease, has been known for over a century but has eluded genetic and biochemical characterization. We have identified a gene cluster responsible for anaerobic choline degradation within the genome of a sulfate-reducing bacterium and verified its function using both a genetic knockout strategy and heterologous expression in Escherichia coli. Bioinformatics and electron paramagnetic resonance (EPR) spectroscopy revealed the involvement of a C–N bond cleaving glycyl radical enzyme in TMA production, which is unprecedented chemistry for this enzyme family. Our discovery provides the predictive capabilities needed to identify choline utilization clusters in numerous bacterial genomes, underscoring the importance and prevalence of this metabolic activity within the human microbiota and the environment.

Predicting and Manipulating Cardiac Drug Inactivation by the Human Gut Bacterium Eggerthella lenta

Digoxin Dangers A proportion of patients treated with digoxin, a cardiac glycoside used to treat heart function abnormalities, generate the inactive metabolite, dihydrodigoxin, resulting in poor efficacy. Haiser et al. (p. 295) examined a potential culprit responsible for this transformation—the actinobacterium, Eggerthella lenta—to probe the microbiota-digoxin interaction. Microbe growth was promoted by arginine, and differential expression analysis revealed a two-gene cardiac glycoside reductase (cgr) operon that was induced by digoxin in low arginine conditions. Not all strains of E. lenta could reduce digoxin and, when fecal samples from healthy people were tested, a spectrum of digoxin inactivation was detected. When the digoxin-reducing strain of E. lenta was given to germ-free mice that were fed a high-protein (that is, high-arginine) diet, digoxin levels stayed high in serum, and drug inactivation was suppressed. The heart drug digoxin can be inactivated by a strain of gut microbe bearing a cardiac glycoside reductase operon. Despite numerous examples of the effects of the human gastrointestinal microbiome on drug efficacy and toxicity, there is often an incomplete understanding of the underlying mechanisms. Here, we dissect the inactivation of the cardiac drug digoxin by the gut Actinobacterium Eggerthella lenta. Transcriptional profiling, comparative genomics, and culture-based assays revealed a cytochrome-encoding operon up-regulated by digoxin, inhibited by arginine, absent in nonmetabolizing E. lenta strains, and predictive of digoxin inactivation by the human gut microbiome. Pharmacokinetic studies using gnotobiotic mice revealed that dietary protein reduces the in vivo microbial metabolism of digoxin, with significant changes to drug concentration in the serum and urine. These results emphasize the importance of viewing pharmacology from the perspective of both our human and microbial genomes.

The genetic and molecular basis for sunscreen biosynthesis in cyanobacteria

Enzymes for Sunscreen Synthesis Many fungi, cyanobacteria, algae, and other marine organisms can biosynthesize small molecules termed mycosporine and mycosporine-like amino acids (MAAs) that, among other functions, protect them from harmful UV exposure. A formulation containing MAAs is used as a sunscreen in skin care and cosmetic products. Balskus and Walsh (p. 1653, published online 2 September) have now identified the gene cluster in a cyanobacterium that is responsible for MAA production. They expressed the cluster in Escherichia coli and characterized the four biosynthetic enzymes bio chemically. Two are adenosine triphosphate.dependent peptide bond.forming enzymes that catalyze imine bond formation through unusual mechanisms. A gene cluster encodes a four-enzyme pathway that uses an unusual mechanism to synthesize small-molecule sunscreens. Ultraviolet UV-A and UV-B radiation is harmful to living systems, causing damage to biological macromolecules. An important strategy for dealing with UV exposure is the biosynthesis of small-molecule sunscreens. Among such metabolites, the mycosporine and mycosporine-like amino acids (MAAs) are remarkable for their wide phylogenetic distribution and their unique chemical structures. Here, we report the identification of a MAA biosynthetic gene cluster in a cyanobacterium and the discovery of analogous pathways in other sequenced organisms. We have expressed the cluster in a heterologous bacterial host and characterized all four biosynthetic enzymes in vitro. In addition to clarifying the origin of the MAAs, these efforts have revealed two unprecedented enzymatic strategies for imine formation.

Investigating the Initial Steps in the Biosynthesis of Cyanobacterial Sunscreen Scytonemin

The cyanobacterial natural product scytonemin (1) functions as a sunscreen, absorbing harmful UV-A radiation. Using information from a recently identified gene cluster, we propose a biosynthetic route to this pigment. We also report the characterization of two enzymes, NpR1275 and NpR1276, which are involved in the initial stages of this pathway. A regioselective acyloin reaction between indole-3-pyruvic acid (4) and p-hydroxyphenylpyruvic acid (5) is a key step in assembling the carbon framework of a proposed monomeric scytonemin precursor (2).

Chemical transformation of xenobiotics by the human gut microbiota

One persons meat is anothers poison The human gut is packed with actively metabolizing microorganisms. These have a transformative effect on what we ingest—whether food, drugs, or pollutants. Koppel et al. review the distinguishing features of microbial xenobiotic metabolism, its interaction with somatic metabolism, and interindividual variation. Depending on the functional composition of microorganisms in the gut, the subsequent products may have nutritionally beneficial effects, modify pharmaceuticals, or be toxic. All of these consequences of our companion microbes can have important impacts on human health and well-being. Science, this issue p. eaag2770 BACKGROUND Humans ingest a multitude of small molecules that are foreign to the body (xenobiotics), including dietary components, environmental chemicals, and pharmaceuticals. The trillions of microorganisms that inhabit our gastrointestinal tract (the human gut microbiota) can directly alter the chemical structures of such compounds, thus modifying their lifetimes, bioavailabilities, and biological effects. Our knowledge of how gut microbial transformations of xenobiotics affect human health is in its infancy, which is surprising given the importance of the gut microbiota. We currently lack an understanding of the extent to which this metabolism varies between individuals, the mechanisms by which these microbial activities influence human biology, and how we might rationally manipulate these reactions. This deficiency stems largely from the difficulty of connecting this microbial chemistry to specific organisms, genes, and enzymes. ADVANCES Over the past several decades, studies of gut microbiota–mediated modification of xenobiotics have revealed that these organisms collectively have a larger metabolic repertoire than human cells. The chemical differences between human and microbial transformations of ingested compounds arise not only from the increased diversity of enzymes present in this complex and variable community but also from the distinct selection pressures that have shaped these activities. For example, whereas host metabolism evolved to facilitate excretion of many xenobiotics from the body, microbial modifications of these compounds and their human metabolites often support microbial growth through provision of nutrients or production of energy. Notably, the chemistry of microbial transformations often opposes or reverses that of host metabolism, altering the pharmacokinetic and pharmacodynamic properties of xenobiotics and associated metabolites. The range of xenobiotics subject to gut microbial metabolism is impressive and expanding. Gut microbes modify many classes of dietary compounds, including complex polysaccharides, lipids, proteins, and phytochemicals. These metabolic reactions are linked to a variety of health benefits, as well as disease susceptibilities. Gut microbes are also able to transform industrial chemicals and pollutants, altering their toxicities and lifetimes in the body. Similarly, microbial transformations of drugs can change their pharmacokinetic properties, be critical for prodrug activation, and lead to undesirable side effects or loss of efficacy. In the vast majority of cases, the individual microbes and enzymes that mediate these reactions are unknown. Fueled by findings underscoring the relevance of microbial xenobiotic metabolism to human health, scientists are increasingly seeking to discover and manipulate the enzymatic chemistry involved in these transformations. Recent work exploring how gut microbes metabolize the drugs digoxin and irinotecan, as well as the dietary nutrient choline, provides guidance for such investigations. These studies, which combine traditional methods with modern approaches, illustrate how a molecular understanding of gut microbial xenobiotic metabolism can guide hypothesis-driven research into the roles these reactions play in both microbiota and host biology. OUTLOOK We still face a myriad of challenges in understanding the gut microbiota’s contribution to xenobiotic metabolism. It is imperative that we connect the many known microbial transformations with the genes and enzymes responsible for these activities, and knowledge of enzyme mechanism and biochemical logic will facilitate this objective. There also remains a great need to uncover currently unappreciated activities associated with this community. Revealing the full scope of microbially mediated transformations in the gut may give us new insights into the many variable and contradictory studies regarding the effects of diet, pollutants, and drugs on human health. Microbial genes and enzymes will provide both specific targets for manipulation and diagnostic markers that can be incorporated into clinical studies and practice. Ultimately, a molecular understanding of gut microbial xenobiotic metabolism will inform personalized nutrition, toxicology risk assessment, precision medicine, and drug development. Human gut microbes metabolize xenobiotics. The microorganisms that inhabit the human gut alter the chemical structures of ingested compounds, including dietary components, industrial chemicals, and drugs. These changes affect xenobiotic toxicity, biological activity, and bioavailability. The gut microbial enzymes responsible for many of these transformations are poorly understood. Me, methyl. The human gut microbiota makes key contributions to the metabolism of ingested compounds (xenobiotics), transforming hundreds of dietary components, industrial chemicals, and pharmaceuticals into metabolites with altered activities, toxicities, and lifetimes within the body. The chemistry of gut microbial xenobiotic metabolism is often distinct from that of host enzymes. Despite their important consequences for human biology, the gut microbes, genes, and enzymes involved in xenobiotic metabolism are poorly understood. Linking these microbial transformations to enzymes and elucidating their biological effects is undoubtedly challenging. However, recent studies demonstrate that integrating traditional and emerging technologies can enable progress toward this goal. Ultimately, a molecular understanding of gut microbial xenobiotic metabolism will guide personalized medicine and nutrition, inform toxicology risk assessment, and improve drug discovery and development.

A prodrug resistance mechanism is involved in colibactin biosynthesis and cytotoxicity.

Commensal Escherichia coli residing in the human gut produce colibactin, a small-molecule genotoxin of unknown structure that has been implicated in the development of colon cancer. Colibactin biosynthesis is hypothesized to involve a prodrug resistance strategy that entails initiation of biosynthesis via construction of an N-terminal prodrug scaffold and late-stage cleavage of this structural motif during product export. Here we describe the biochemical characterization of the prodrug synthesis, elongation, and cleavage enzymes from the colibactin biosynthetic pathway. We show that nonribosomal peptide synthetases ClbN and ClbB assemble and process an N-acyl-D-asparagine prodrug scaffold that serves as a substrate for the periplasmic D-amino peptidase ClbP. In addition to affording information about structural features of colibactin, this work reveals the biosynthetic logic underlying the prodrug resistance strategy and suggests that cytotoxicity requires amide bond cleavage.

An Enzymatic Cyclopentyl[b]indole Formation Involved in Scytonemin Biosynthesis

Previous studies of the biosynthetic enzymes involved in the assembly of scytonemin (1), a cyanobacterial sunscreen, have identified beta-ketoacid 2 as an important intermediate that is produced by ThDP-dependent enzyme ScyA. We now report that ScyC, previously annotated as a hypothetical protein, catalyzes cyclization and decarboxylation of 2 to generate ketone 5. Assembly of the cyclopentyl[b]indole structure in this manner has little precedent in the chemical literature. Additional mechanistic experiments have revealed that cyclization likely precedes decarboxylation and that the latter event may provide a driving force for cyclopentane formation.

Asymmetric catalysis of the transannular diels-alder reaction

Transannular chemical reactions are unparalleled in their ability to generate high degrees of stereochemical and architectural complexity in a single transformation. However, the successful application of this approach in synthesis depends on the ability to predict and control the outcome of the transannular reaction. Use of a chiral catalyst in this context represents an attractive, yet unused, strategy. This report describes a catalytic, asymmetric transannnular Diels-Alder (TADA) reaction that affords polycyclic products in high enantiomeric excess. This catalyst system can also alter the inherent diastereoselectivity of cyclizations with substrates containing chiral centers. Additionally, the catalytic enantioselective TADA has been used as the key step in a total synthesis of the sesquiterpene 11,12-diacetoxydrimane; this route may provide a general approach to the polycyclic carbon framework shared by many terpene natural products.

Mechanistic insight into digoxin inactivation by Eggerthella lenta augments our understanding of its pharmacokinetics.

The human gut microbiota plays a key role in pharmacology, yet the mechanisms responsible remain unclear, impeding efforts toward personalized medicine. We recently identified a cytochrome-encoding operon in the common gut Actinobacterium Eggerthella lenta that is transcriptionally activated by the cardiac drug digoxin. These genes represent a predictive microbial biomarker for the inactivation of digoxin. Gnotobiotic mouse experiments revealed that increased protein intake can limit microbial drug inactivation. Here, we present a biochemical rationale for how the proteins encoded by this operon might inactivate digoxin through substrate promiscuity. We discuss digoxin signaling in eukaryotic systems, and consider the possibility that endogenous digoxin-like molecules may have selected for microbial digoxin inactivation. Finally, we highlight the diverse contributions of gut microbes to drug metabolism, present a generalized approach to studying microbe-drug interactions, and argue that mechanistic studies will pave the way for the clinical application of this work.

Characterization and Detection of a Widely Distributed Gene Cluster That Predicts Anaerobic Choline Utilization by Human Gut Bacteria

ABSTRACT Elucidation of the molecular mechanisms underlying the human gut microbiotas effects on health and disease has been complicated by difficulties in linking metabolic functions associated with the gut community as a whole to individual microorganisms and activities. Anaerobic microbial choline metabolism, a disease-associated metabolic pathway, exemplifies this challenge, as the specific human gut microorganisms responsible for this transformation have not yet been clearly identified. In this study, we established the link between a bacterial gene cluster, the choline utilization (cut) cluster, and anaerobic choline metabolism in human gut isolates by combining transcriptional, biochemical, bioinformatic, and cultivation-based approaches. Quantitative reverse transcription-PCR analysis and in vitro biochemical characterization of two cut gene products linked the entire cluster to growth on choline and supported a model for this pathway. Analyses of sequenced bacterial genomes revealed that the cut cluster is present in many human gut bacteria, is predictive of choline utilization in sequenced isolates, and is widely but discontinuously distributed across multiple bacterial phyla. Given that bacterial phylogeny is a poor marker for choline utilization, we were prompted to develop a degenerate PCR-based method for detecting the key functional gene choline TMA-lyase (cutC) in genomic and metagenomic DNA. Using this tool, we found that new choline-metabolizing gut isolates universally possessed cutC. We also demonstrated that this gene is widespread in stool metagenomic data sets. Overall, this work represents a crucial step toward understanding anaerobic choline metabolism in the human gut microbiota and underscores the importance of examining this microbial community from a function-oriented perspective. IMPORTANCE Anaerobic choline utilization is a bacterial metabolic activity that occurs in the human gut and is linked to multiple diseases. While bacterial genes responsible for choline fermentation (the cut gene cluster) have been recently identified, there has been no characterization of these genes in human gut isolates and microbial communities. In this work, we use multiple approaches to demonstrate that the pathway encoded by the cut genes is present and functional in a diverse range of human gut bacteria and is also widespread in stool metagenomes. We also developed a PCR-based strategy to detect a key functional gene (cutC) involved in this pathway and applied it to characterize newly isolated choline-utilizing strains. Both our analyses of the cut gene cluster and this molecular tool will aid efforts to further understand the role of choline metabolism in the human gut microbiota and its link to disease. Anaerobic choline utilization is a bacterial metabolic activity that occurs in the human gut and is linked to multiple diseases. While bacterial genes responsible for choline fermentation (the cut gene cluster) have been recently identified, there has been no characterization of these genes in human gut isolates and microbial communities. In this work, we use multiple approaches to demonstrate that the pathway encoded by the cut genes is present and functional in a diverse range of human gut bacteria and is also widespread in stool metagenomes. We also developed a PCR-based strategy to detect a key functional gene (cutC) involved in this pathway and applied it to characterize newly isolated choline-utilizing strains. Both our analyses of the cut gene cluster and this molecular tool will aid efforts to further understand the role of choline metabolism in the human gut microbiota and its link to disease.