Biology subjects

Balskus, E. P.

Publications and source records attributed to Balskus, E. P..

At least 19 recordsLinked to original sources

bioRxiv2026

The Structural Basis of Malodorant Skatole Formation by the Glycyl Radical Enzyme Indoleacetate Decarboxylase

Glycyl radical enzymes (GREs) catalyze challenging chemical reactions using a post-translationally installed glycyl radical cofactor. One such enzyme, indoleacetate decarboxylase (IAD), performs the radical-based decarboxylation of indole-3-acetate (I3A) to form the malodorant molecule skatole. In addition to being an odor nuisance, skatole is a human and livestock lung toxin, a suspected carcinogen, and a mosquito attractant, all of which impact human health, agriculture, food production, and wastewater treatment. Here, we use cryogenic electron microscopy to solve a 2.45-[A] resolution structure of indoleacetate decarboxylase from the gut bacterium Olsenella uli. We observe IAD in a homotetrameric form with the substrate I3A bound in all four protomers. The positioning of I3A in the active site is unexpected and is more consistent with a Kolbe-type decarboxylation mechanism, i.e. a decarboxylation initiated by a 1-electron oxidation of the carboxylate moiety rather than being initiated by hydrogen atom transfer (HAT). Previously, a high deuterium content in skatole from IAD assays in D2O was used to support a HAT mechanism over a Kolbe-type mechanism. However, we show here that deuterium content does not necessarily inform on mechanism as IAD can catalyze the exchange of skatole's 3'-methyl hydrogens post- turnover. Structural comparisons show that both IAD and HPAD display structural features that are not found in other characterized GREs, suggesting that they represent a distinct GRE-subclass. Collectively, these insights will inform IAD inhibitor design aimed at decreasing skatole production.

biochemistry↗

bioRxiv2026

Inhibitors of gut bacterial L-dopa decarboxylation with reduced susceptibility to host metabolism

Microorganisms in the human gut influence the efficacy and metabolism of host-targeted small molecule therapeutics, including the frontline Parkinson's disease drug levodopa (L-dopa). Previous work has identified a mechanism-based inhibitor of gut bacterial decarboxylases that degrade L-dopa, -fluoromethyltyrosine (AFMT). However, early experiments with AFMT in rodent models suggested undesirable in vivo metabolism by host tyrosine hydroxylase, producing -fluoromethyldopa, a metabolite likely to worsen Parkinson's phenotypes and prevent application as an L-dopa co-treatment. Here, we demonstrate oxidation of AFMT to -fluoromethyldopa in vitro by recombinant human tyrosine hydroxylase. We then develop AFMT analogs that retain activity against bacterial decarboxylases but have reduced susceptibility to host hydroxylation. Suitable arenes for inhibitor design were identified using assays with commercially available noncanonical amino acids, which revealed aryl difluorination as a promising modification. Difluoroaryl AFMT derivatives are less prone to degradation by tyrosine hydroxylase in vitro yet still inhibit L-dopa metabolism by bacterial decarboxylases. This work exemplifies how substrate reactivity can streamline design of mechanism-based enzyme inhibitors, as well as how constraints posed by the host can be incorporated during development of microbiome-targeted therapeutics. The compounds reported here are promising starting points for future studies in animal models and further exploration of gut bacterial effects on L-dopa treatment efficacy.

microbiology↗

bioRxiv2026

A previously unappreciated class of metal-dependent bile salt hydrolases from the human gut microbiome

Bile salt hydrolases (BSHs) are gut microbial enzymes that catalyze the deconjugation of glycine-or taurine-conjugated bile acids (BAs), a key step in shaping the BA pool in the human gastrointestinal tract and modulating host-gut microbiome interactions.1-3 All known BSHs are members of the N-terminal nucleophile (Ntn) hydrolase superfamily and share a conserved architecture and mechanism involving a nucleophilic active site cysteine.4,5 This knowledge has guided predictions and study of BSH activity in the gut microbiome6,7 as well as the development of BSH inhibitors8. Here, we report the discovery and characterization of a previously unknown BSH from the human gut bacterium Bilophila wadsworthia that belongs to the metal-dependent amidohydrolase superfamily and exhibits robust and specific activity toward taurine-conjugated bile salts. We show this secreted enzyme, metalloBSH, utilizes a metallocofactor for BA deconjugation, a mechanism distinct from that of canonical Ntn-type BSHs. MetalloBSHs are conserved in B. wadsworthia and present in many other Desulfovibrionaceae found in vertebrate gut microbiomes. Analysis of multi-omic datasets indicates metalloBSHs are expressed in vivo and correlate with BA metabolism. Overall, our findings reshape our understanding of BSH activity in the gut microbiome and highlight the promise of activity guided discovery in revealing previously overlooked gut microbial enzymes.

biochemistry↗

bioRxiv2026

Chemiluminescent probes allow for the rapid identification of colibactin-producing bacteria

The pks (or clb) gene cluster, which produces the genotoxic natural product colibactin, is encoded by human gut Enterobacteriaceae, including many commensal strains of E. coli. Colibactin crosslinks DNA and is implicated in colorectal cancer development, highlighting the importance of identifying colibactin-producing gut bacteria within biological samples. In this study, we develop phenoxy-dioxetane chemiluminescent probes that selectively react with a critical colibactin biosynthetic enzyme, the serine peptidase ClbP. We show that these chemiluminescent probes have superior sensitivity, speed, and detection capabilities compared to previously reported fluorescent ClbP probes. Furthermore, we employ these chemiluminescent probes to detect pks+ E. coli directly in complex stool suspensions. These probes will enable multiple applications requiring detection of colibactin-producing bacteria, including the identification of ClbP inhibitors and the screening of clinical samples.

biochemistry↗

bioRxiv2025

Discovery of the metalloenzyme IsmB revises a pathway for coprostanol formation by the human gut microbiome

High levels of circulating cholesterol are associated with human cardiovascular diseases and an altered gut microbiome. Still, major gaps exist in our understanding of the interactions of cholesterol with gut microbes. The reductive transformation of cholesterol to the poorly absorbed sterol coprostanol by human gut bacteria has long been known, but the genetic and biochemical basis for this activity is only partially elucidated. Here, we discover and characterize a gut bacterial enzyme that catalyzes the reduction of cholestenone to coprostanone, the second step in the intestinal sterol metabolism (ism) pathway for coprostanol production. We identify a gene encoding a previously unknown 5{beta}-reductase, IsmB, a new member of the Fe-S cluster flavoenzyme superfamily, in the coprostanol producing organism Eubacterium coprostanoligenes. Biochemical characterization of IsmB confirms it is an anaerobic Fe-S cluster flavoenzyme and reveals specificity for reduction of an unanticipated intermediate, 5-cholesten-3-one, to coprostanone, revising the ism pathway. We also identify and characterize homologs of IsmB encoded in uncultured human gut bacteria that also encode the previously identified ism pathway enzyme IsmA, further supporting the role of IsmB in coprostanol formation. Finally, analysis of human stool metagenomics and metabolomics datasets further confirms the relevance of IsmB in the human gut microbiome, and analyses of human serum metabolomics from Framingham Heart Study participants reveal negative correlation between serum cholesterol levels and the presence of IsmA/IsmB encoders in the gut. Together, these results show the utility of combining biochemistry and stool metagenomic analysis for gut microbial enzyme discovery, and suggests IsmA/IsmB-encoding gut bacteria carry potential benefits for cholesterol homeostasis and cardiovascular health.

biochemistry↗

bioRxiv2025

Colibactin produced by a honeybee symbiont defends against pathogens and shapes the gut community

Colibactin is a bacterial genotoxin that is linked to colorectal cancer and implicated in interbacterial competition; however, its role in natural communities remains unknown. Frischella perrara, a symbiont living only in honeybee guts, produces colibactin and causes DNA damage. Here, we found that F. perrara mono-colonization reduces bee lifespan but increases survivorship following challenge with an opportunistic pathogen. F. perrara reduces pathogen loads by causing colibactin-dependent DNA damage and prophage induction. clbS, a gene conferring protection from colibactin, is ubiquitous among bacteria restricted to bee guts but is absent from opportunistic colonizers including bee pathogens. Our findings provide evidence that colibactin functions in interbacterial competition, exerts selective pressure on community members, and confers protection against pathogens, while permitting co-evolved gut symbionts to persist.

microbiology↗

bioRxiv2025

Systematic identification and characterization of regulators of aryl hydrocarbon receptor signaling

The human aryl hydrocarbon receptor (AHR) integrates chemical signals derived from the environment, gut microbes, and endogenous sources to regulate processes ranging from intestinal barrier integrity to xenobiotic detoxification. Despite strong evidence that dysregulation of AHR signaling is a causal factor in metabolic and autoimmune disorders, we currently lack a comprehensive understanding of the factors that regulate AHR activity in human cells. Here, we use genome-scale CRISPR screening to systematically identify regulators of AHR signaling in hepatocytes. The resulting datasets recapitulate the core AHR signaling pathway and identify a large network of regulators. Many of these factors have roles beyond AHR signaling, reflecting that AHR signaling is deeply integrated into human cell biology. We further dissect this network to reveal novel modes of regulation of AHR expression, protein levels, and signaling. For example, we find that the E3 ubiquitin ligase UBR5 sustains AHR signaling by counteracting degradation of ligand-bound AHR. Finally, we identify components of the AHR regulatory network that are specific to cell types and ligands as potential nodes to manipulate AHR signaling in a targeted manner for therapeutic benefit. Overall, our results define the regulatory network that underpins AHR activation, with implications for our understanding of host-microbe interactions and integrative chemosensation and the etiology of metabolic and inflammatory disorders.

genomics↗

bioRxiv2025

A genotoxin associated with colorectal cancer linked to gut dysbiosis in children with cystic fibrosis

Cystic fibrosis (CF) substantially alters the gastrointestinal microbiome from an early age, leading to significant changes in microbial composition and functionality. This study explores the physiological and microbiological factors contributing to dysbiosis in children with cystic fibrosis (cwCF), characterized by an increase in potentially pathogenic Escherichia coli and a decrease in beneficial anaerobes such as Bacteroides. In this study, we employed an in vitro medium representative of the nutritional environment of the CF colon to test the role of factors including mucin, fat, bile, pH, antibiotics and features associated with inflammation (e.g., nitrate, sulfate, formate, reactive oxygen species) on growth of clinical isolates of E. coli and Bacteroides spp. We further examined interactions between these two microbes under CF-like conditions to understand modulators of microbial competition, and identified glycerol, a surrogate of increased fat, as a significant driver of altered microbial competition. Finally, we investigated genetic determinants influencing these microbial interactions, with the focus on glycerol metabolism, by performing a transposon mutagenesis screen in E. coli. Results of this screen pointed to the role of colibactin production in mediating this microbial competition; colibactin is a DNA-damaging genotoxin associated with the increased risk of colorectal cancer (CRC) in CF populations. This work enhances our understanding of mechanisms of microbial competition in the CF gut, while potentially enhancing our understanding of colorectal cancer risk in persons with CF through the identification of early-life microbial biomarkers. Significance StatementThe risk of CRC development in CF populations is significantly increased. This study examines the interplay of altered intestinal physiology in the microbial dysbiosis common in the CF gut, implicating the high fat environment in a competition-mediated depletion of immune-modulating Bacteroides. This work identifies candidate features of the young CF intestine and gut microbiome that may contribute to advanced development of CRC in these populations, informing potential therapeutic approaches.

microbiology↗

bioRxiv2025

Bacterial peptide deformylase inhibitors induce prophages in competitors

While antibiotics mediate chemical warfare among microbes, their roles in the wild extend beyond direct growth inhibition(1). Some antibiotics have the potential to mediate interference competition by triggering a bacterial stress response that subsequently activates endogenous viruses integrated in bacterial genomes (prophages). Canonically, this activation is regulated by the SOS response upon DNA damage. Here we show that a metabolite produced by natural isolates of Vibrio ordalii circumvents the SOS response by directly triggering prophage induction in other Vibrio species, co-occurring in the same environment. While the metabolite was previously classified as a broad-spectrum antibiotic, we observe how it acts as a peptide deformylase inhibitor that specifically induces certain prophages, even when target bacterial cells carry multiple other prophages. Its biosynthetic gene cluster, or ord cluster, also encodes its own peptide deformylase (OrdE) which provides self-immunity to producer strains. Likewise, among natural Vibrio isolates that carry similar prophages, resistance against the ord metabolite was found in those that had acquired a divergent second peptide deformylase. Finally, we show that prophage induction by the ord cluster prevents slower-growing producer strains from being outcompeted by their otherwise fast-growing competitors if they carry an inducible prophage. Thus, we demonstrate how natural products play additional impactful roles in communities beyond antibiotic activity and that prophage induction serves as an interference competition strategy, sustaining community diversity.

microbiology↗

bioRxiv2025

Chemical capture of diazo metabolites reveals biosynthetic hydrazone oxidation

Chemically reactive microbial natural products have enabled therapeutic development1,2 via their well-established bioactivities including anticancer,3 antibiotic,4,5 and antioxidant6 activities. However, discovery of reactive metabolites is particularly challenging because they may not tolerate traditional bioactivity-guided isolation workflows.7 Diazo-containing natural products are a subset of highly reactive microbial metabolites that display potent bioactivity8-11 and enable powerful (bio)synthetic transformations;12,13 however, instability of the diazo group to light,14,15 heat,16,17 mild acid,18 and mechanical shock19 has precluded their efficient discovery and application. Here, we develop a reactivity-based screening approach to capture diazo-containing metabolites and facilitate their discovery by mass spectrometry. This workflow revealed two novel diazo-containing natural products, 4-diazo-3-oxo-butanoic acid and diazoacetone, from the human lung pathogen Nocardia ninae. Biosynthetic investigations revealed a distinct enzymatic logic for diazo formation involving hydrazone oxidation catalyzed by the metalloenzyme Dob3, and biochemical characterization of Dob3 suggests promising future applications in biocatalysis. Overall, our work highlights the power of reactivity-guided strategies for identifying reactive metabolites and facilitating the discovery of unique enzymatic transformations.

biochemistry↗

bioRxiv2025

The specificity and structure of DNA crosslinking by the gut bacterial genotoxin colibactin

Accumulating evidence has connected the chemically unstable, DNA-damaging gut bacterial natural product colibactin to colorectal cancer, including the identification of mutational signatures that are thought to arise from colibactin-DNA interstrand crosslinks (ICLs). However, we currently lack direct information regarding the structure of this lesion. Here, we combine mass spectrometry and nuclear magnetic resonance spectroscopy to elucidate the specificity and structure of the colibactin-DNA ICL. We find that colibactin alkylates within the minor groove of AT-rich DNA, explaining the origins of mutational signatures. Unexpectedly, we discover that the chemically unstable central motif of colibactin mediates the sequence specificity of crosslinking. By directly elucidating colibactins interactions with DNA, this work enhances our understanding of the structure and genotoxic mechanisms of this cancer-linked gut bacterial natural product.

biochemistry↗

bioRxiv2025

A new heme enzyme family forms hydrazine groups in diverse biosynthetic pathways

Nitrogen-nitrogen (N-N) bond formation is an inherently challenging chemical process that plays a key role in the global nitrogen cycle. An array of microbial metalloenzyme complexes has evolved to shuttle nitrogen between biologically accessible reduced or oxidized states and its inert form as dinitrogen (N2) gas. More recently, N-N bond formation has been observed in a more specialized context, natural product biosynthesis. Here, we report the discovery of a unique metalloenzyme complex that forms hydrazine functional groups in the biosynthetic pathways of structurally diverse natural products. This heterodimeric system consists of a heme enzyme from a previously unidentified family and a partner ferredoxin. Together, these enzymes effect the unprecedented four-electron reduction of nitrite (NO2-) to form a hydrazine functional group on a substrate amino acid in an oxygen-independent reaction that resembles primary microbial nitrogen metabolism. These enzymes are unexpectedly widespread among bacteria and are present in diverse genomic contexts, including cryptic biosynthetic gene clusters, highlighting the importance of this previously uncharacterized protein family. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/656397v1_ufig1.gif" ALT="Figure 1000"> View larger version (30K): org.highwire.dtl.DTLVardef@232c7forg.highwire.dtl.DTLVardef@13e46f1org.highwire.dtl.DTLVardef@1a3a2e9org.highwire.dtl.DTLVardef@1c7e32_HPS_FORMAT_FIGEXP M_FIG TOC C_FIG

biochemistry↗

bioRxiv2025

Flavoaffinins, elusive cellulose-binding natural products from an anaerobic bacterium

Cellulose is the most abundant polymer on earth. Many anaerobic cellulose-degrading bacteria produce uncharacterized yellow-orange, cellulose-binding pigments known as yellow affinity substances (here referred to as flavoaffinins) that are associated with cellulose degradation. Here, we isolate and structurally characterize the flavoaffinins from Clostridium (Hungateiclostridium) thermocellum, a key workhorse for the industrial conversion of cellulosic feedstocks to ethanol. Flavoaffinins represent an unprecedented structural juxtaposition of an aryl polyene chain with a hydroxy-diene {gamma}-lactone. We also shed light on their biosynthesis using stable-isotope feeding experiments. This effort lays the groundwork for understanding the biological function(s) of the flavoaffinins and expands the limited number of natural products isolated from obligately anaerobic microbes.

biochemistry↗

bioRxiv2025

Distinct Classes of Gut Bacterial Molybdenum-Dependent Enzymes Produce Urolithins

Urolithin A is an anti-aging and anti-inflammatory gut bacterial metabolite derived from ellagic acid (EA), a polyphenol abundant in berries and nuts. The conversion of EA to urolithin A involves multiple chemically challenging phenol dehydroxylation steps that produce urolithins with varying bioactivities. Despite their biological and chemical significance, the bacterial enzymes responsible for urolithin production remain unidentified. Here, we use differential gene expression analysis, anaerobic protein expression, and enzyme assays to identify members of two distinct molybdenum enzyme families (the DMSO reductase family and the xanthine oxidase family) capable of regioselective dehydroxylation and urolithin generation. These two enzyme families have distinct substrate requirements, suggesting they employ different catalytic mechanisms for phenol dehydroxylation. Multi-omics analysis of a human cohort uncovers decreased levels of urolithin A and genes encoding urolithin A-producing enzymes in patients with inflammatory bowel disease (IBD), implying reduced health effects of EA consumption in this setting. Together, this study elucidates the molecular basis of urolithin production, expands the known enzymatic repertoire of the human gut microbiome, and suggests a potential link between gut bacterial urolithin production and host inflammation. Significance statementThe human gut microbiome modulates the health effects of dietary compounds by modifying their chemical structures. For example, gut microbes extensively metabolize polyphenols, a group of diverse plant-derived compounds associated with positive health outcomes. Urolithin A, a gut bacterial metabolite derived from a polyphenol abundant in berries and nuts, exhibits potent anti-inflammatory activity. However, the gut bacterial enzymes involved in its production remain largely unknown. Here, we report urolithin-producing gut bacterial enzymes, including four phenol dehydroxylases from two distinct molybdenum-dependent enzyme families. Analyzing human gut microbiomes suggests a potential link between genes encoding these enzymes and host inflammation. Together, our findings fully map urolithin A production to enzymes, increasing our understanding of how the gut microbiome can alter the impacts of diet on human health.

biochemistry↗

bioRxiv2024

Phenotypic high-throughput screening identifies modulators of gut microbial choline metabolism

Anaerobic metabolism of dietary choline to trimethylamine (TMA) by the human gut microbiome is a disease-associated pathway. The hosts impaired ability to oxidize TMA to trimethylamine-N-oxide (TMAO) results in trimethylaminuria (TMAU), while elevated serum TMAO levels have been positively correlated with cardiometabolic disease. Small molecule inhibition of gut bacterial choline metabolism attenuates the development of disease in mice, highlighting the therapeutic potential of modulating this metabolism. Inhibitors previously developed to target this pathway are often designed to mimic choline, the substrate of the key TMA-generating enzyme choline trimethylamine-lyase (CutC). Here, we use a growth-based phenotypic high-throughput screen and medicinal chemistry to identify distinct chemical scaffolds that can modulate anaerobic microbial choline metabolism and lower TMAO levels in vivo. These results illustrate the potential of using phenotypic screening to rapidly discover new inhibitors of gut microbial metabolic activities.

microbiology↗

bioRxiv2024

A cell-free workflow for detecting and characterizing RiPP recognition element-precursor peptide interactions

Post-translational modifications (PTMs) are important for the stability and function of many therapeutic proteins and peptides. Current methods for studying and engineering PTM installing proteins often suffer from low-throughput experimental techniques. Here we describe a generalizable, in vitro workflow coupling cell-free protein synthesis (CFPS) with AlphaLISA for the rapid expression and testing of PTM installing proteins. We apply our workflow to two representative classes of peptide and protein therapeutics: ribosomally synthesized and post-translationally modified peptides (RiPPs) and conjugate vaccines. First, we demonstrate how our workflow can be used to characterize the binding activity of RiPP recognition elements, an important first step in RiPP biosynthesis, and be integrated into a biodiscovery pipeline for computationally predicted RiPP products. Then, we adapt our workflow to study and engineer oligosaccharyltransferases (OSTs) involved in conjugate vaccine production, enabling the identification of mutant OSTs and sites within a carrier protein that enable high efficiency production of conjugate vaccines. In total, we expect that our workflow will accelerate design-build-test cycles for engineering PTMs.

synthetic biology↗

bioRxiv2024

The Fanconi anemia pathway repairs colibactin-induced DNA interstrand cross-links

Colibactin is a secondary metabolite produced by bacteria present in the human gut and is implicated in the progression of colorectal cancer and inflammatory bowel disease. This genotoxin alkylates deoxyadenosines on opposite strands of host cell DNA to produce DNA interstrand cross-links (ICLs) that block DNA replication. While cells have evolved multiple mechanisms to resolve ("unhook") ICLs encountered by the replication machinery, little is known about which of these pathways promote resistance to colibactin-induced ICLs. Here, we use Xenopus egg extracts to investigate replication-coupled repair of plasmids engineered to contain site-specific colibactin-ICLs. We show that replication fork stalling at a colibactin-ICL leads to replisome disassembly and activation of the Fanconi anemia ICL repair pathway, which unhooks the colibactin-ICL through nucleolytic incisions. These incisions generate a DNA double-strand break intermediate in one sister chromatid, which can be repaired by homologous recombination, and a monoadduct ("ICL remnant") in the other. Our data indicate that translesion synthesis past the colibactin-ICL remnant depends on Pol{eta} and a Pol{kappa}-REV1-Pol{zeta} polymerase complex. Although translesion synthesis past colibactin-induced DNA damage is frequently error-free, it can introduce T>N point mutations that partially recapitulate the mutation signature associated with colibactin exposure in vivo. Taken together, our work provides a biochemical framework for understanding how cells tolerate a naturally-occurring and clinically-relevant ICL.

molecular biology↗

bioRxiv2024

Elucidation of chalkophomycin biosynthesis reveals N-hydroxypyrrole-forming enzymes

Reactive functional groups, such as N-nitrosamines, impart unique bioactivities to the natural products in which they are found. Recent work has illuminated enzymatic N-nitrosation reactions in microbial natural product biosynthesis, motivating an interest in discovering additional metabolites constructed using such reactivity. Here, we use a genome mining approach to identify over 400 cryptic biosynthetic gene clusters (BGCs) encoding homologs of the N-nitrosating biosynthetic enzyme SznF, including the BGC for chalkophomycin, a CuII-binding metabolite that contains a C-type diazeniumdiolate and N-hydroxypyrrole. Characterizing chalkophomycin biosynthetic enzymes reveals previously unknown enzymes responsible for N-hydroxypyrrole biosynthesis, including the first prolyl-N-hydroxylase, and a key step in assembly of the diazeniumdiolate-containing amino acid graminine. Discovery of this pathway enriches our understanding of the biosynthetic logic employed in constructing unusual heteroatom-heteroatom bondcontaining functional groups, enabling future efforts in natural product discovery and biocatalysis.

biochemistry↗