Person: Balskus, Emily
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Balskus
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Emily
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Balskus, Emily
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Publication An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin(Springer Science and Business Media LLC, 2019-02) Ng, Tai L.; Rohac, Roman; Mitchell, Andrew J.; Boal, Amie K.; Balskus, Emily P.; Balskus, EmilyN-nitroso-containing small molecules, such as the bacterial natural product streptozotocin, are prominent carcinogens as well as important cancer chemotherapeutics. Despite this functional group’s significant impact on human health, dedicated enzymes involved in N-nitroso assembly have not been identified. Here, we describe a metalloenzyme from streptozotocin biosynthesis (SznF) that catalyzes an unprecedented oxidative rearrangement of the guanidine group of Nw-methyl-L-arginine to generate an N-nitrosourea product. Structural characterization and mutagenesis of SznF uncovered two separate active sites that promote distinct steps in this transformation using different iron-containing metallocofactors. The discovery of this biosynthetic reaction, which has little precedent in enzymology or organic synthesis, expands the catalytic capabilities of non-heme iron-dependent enzymes to include N–N bond formation. We find biosynthetic gene clusters encoding SznF homologs are widely distributed among bacteria, including environmental organisms, plant symbionts, and human pathogens, suggesting an unexpectedly diverse and uncharacterized microbial reservoir of bioactive N-nitroso metabolites.Publication Complete integration of carbene-transfer chemistry into biosynthesis(Springer Science and Business Media LLC, 2023-05-03) Huang, Jing; Quest, Andrew; Cruz-Morales, Pablo; Deng, Kai; Pereira, Jose Henrique; Van Cura, Devon; Kakumanu, Ramu; Baidoo, Edward E. K.; Dan, Qingyun; Chen, Yan; Petzold, Christopher J.; Northen, Trent R.; Adams, Paul D.; Clark, Douglas S.; Balskus, Emily; Hartwig, John F.; Mukhopadhyay, Aindrila; Keasling, Jay D.Publication Structural Basis of Colibactin Activation by the ClbP Peptidase(Springer Science and Business Media LLC, 2022-10-17) Velilla, Jose; Volpe, Matthew; Kenney, Grace; Walsh, Richard; Balskus, Emily; Gaudet, RachelleColibactin, a DNA crosslinking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability, and interactions that explain the selectivity of ClbP for the N-acyl-D-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin, with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.Publication Designer Micelles Accelerate Flux Through Engineered Metabolism in E. coli and Support Biocompatible Chemistry(Wiley, 2016-04-08) Wallace, Stephen; Balskus, EmilySynthetic biology has enabled the production of many value-added chemicals via microbial fermentation. However, the problem of low product titers from recombinant pathways has limited the utility of this approach. Methods to increase metabolic flux are therefore critical to the success of metabolic engineering. Here we demonstrate that vitaminE-derived designer micelles, originally developed for use in synthetic chemistry, are biocompatible and accelerate flux through a styrene production pathway in Escherichia coli. We show that these micelles associate non-covalently with the bacterial outer-membrane and that this interaction increases membrane permeability. In addition, these micelles also accommodate both heterogeneous and organic-soluble transition metal catalysts and accelerate biocompatible cyclopropanation in vivo. Overall, this work demonstrates that these surfactants hold great promise for further application in the field of synthetic biotechnology, and for expanding the types of molecules that can be readily accessed from renewable resources via the combination of microbial fermentation and biocompatible chemistry.Publication Mechanistic insight into digoxin inactivation by Eggerthella lenta augments our understanding of its pharmacokinetics(Landes Bioscience, 2014) Haiser, Henry J; Seim, Kristen L; Balskus, Emily; Turnbaugh, Peter JThe 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.Publication Characterization and Detection of a Widely Distributed Gene Cluster That Predicts Anaerobic Choline Utilization by Human Gut Bacteria(American Society of Microbiology, 2015) Martínez-del Campo, Ana; Bodea, Smaranda; Hamer, Hilary A.; Marks, Jonathan A.; Haiser, Henry J.; Turnbaugh, Peter J.; Balskus, EmilyABSTRACT Elucidation of the molecular mechanisms underlying the human gut microbiota’s 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.Publication Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring(2017) Zha, Li; Jiang, Yindi; Henke, Matthew T.; Wilson, Matthew; Wang, Jennifer X.; Kelleher, Neil L.; Balskus, EmilyDespite containing an α-amino acid, the versatile cofactor S-adenosylmethionine (SAM) is not a known building block for non-ribosomal peptide synthetase (NRPS) assembly lines. Here we report an unusual NRPS module from colibactin biosynthesis that uses SAM for amide bond formation and subsequent cyclopropanation. Our findings showcase a new use for SAM and reveal a novel biosynthetic route to a functional group that likely mediates colibactin’s genotoxicity.Publication The biosynthesis of cyanobacterial sunscreen scytonemin in intertidal microbial mat communities(Wiley-Blackwell, 2011) Balskus, Emily; Case, Rebecca J.; Walsh, ChristopherWe have examined the biosynthesis and accumulation of cyanobacterial sunscreening pigment scytonemin within intertidal microbial mat communities using a combination of chemical, molecular, and phylogenetic approaches. Both laminated (layered) and nonlaminated mats contained scytonemin, with morphologically distinct mats having different cyanobacterial community compositions. Within laminated microbial mats, regions with and without scytonemin had different dominant oxygenic phototrophs, with scytonemin-producing areas consisting primarily of Lyngbya aestuarii and scytonemin-deficient areas dominated by a eukaryotic alga. The nonlaminated mat was populated by a diverse group of cyanobacteria and did not contain algae. The amplification and phylogenetic assignment of scytonemin biosynthetic gene scyC from laminated mat samples confirmed that the dominant cyanobacterium in these areas, L. aestuarii, is likely responsible for sunscreen production. This study is the first to utilize an understanding of the molecular basis of scytonemin assembly to explore its synthesis and function within natural microbial communities.Publication An Enzymatic Cyclopentyl[b]indole Formation Involved in Scytonemin Biosynthesis(American Chemical Society (ACS), 2009) Balskus, Emily; Walsh, ChristopherPrevious studies of the biosynthetic enzymes involved in the assembly of scytonemin (1), a cyanobacterial sunscreen, have identified β-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.Publication Structural Analysis of Spiro β-Lactone Proteasome Inhibitors(American Chemical Society (ACS), 2008) Groll, Michael; Balskus, Emily; Jacobsen, EricSpiro β-lactone-based proteasome inhibitors were discovered in the context of an asymmetric catalytic total synthesis of the natural product (+)-lactacystin (1). Lactone 4 was found to be a potent inhibitor of the 26S proteasome, while its C-6 epimer (5) displayed weak activity. Crystallographic studies of the two analogues covalently bound to the 20S proteasome permitted characterization of the important stabilizing interactions between each inhibitor and the proteasome’s key catalytic N-terminal threonine residue. This structural data support the hypothesis that the discrepancy in potency between 4 and 5 may be due to differences in the hydrolytic stabilities of the resulting acyl enzyme complexes.
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