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    Snapshots of the catalytic cycle of an O2, pyridoxal phosphate-dependent hydroxylase.
    ACS Chem Biol 2018 Feb 21. Epub 2018 Feb 21.
    Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme- and non-heme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)-dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts L-arginine to (S)-4-hydroxy-2-ketoarginine. Read More

    Chemiluminescent Biosensors for Detection of Second Messenger Cyclic di-GMP.
    ACS Chem Biol 2018 Feb 21. Epub 2018 Feb 21.
    Bacteria colonize highly diverse and complex environments, from gastrointestinal tracts, to soil and plant surfaces. This colonization process is controlled in part by the intracellular signal cyclic di-GMP, which regulates bacterial motility and biofilm formation. To interrogate cyclic di-GMP signaling networks, a variety of fluorescent biosensors for live cell imaging of cyclic di-GMP have been developed. Read More

    A Switchable Site-Specific Antibody Conjugate.
    ACS Chem Biol 2018 Feb 20. Epub 2018 Feb 20.
    Genetic incorporation of unnatural amino acids (UAAs) provides a unique approach to the synthesis of site-specific antibody conjugates that are homogeneous and better defined constructs than random conjugates. Yet, the yield varies for every antibody, and the process is costly and time-consuming. We have developed a switchable αGCN4-Fab conjugate that incorporates UAA p-acetylphenylalanine. Read More

    Identification of protein targets of 12/15-lipoxygenase-derived lipid electrophiles in mouse peritoneal macrophages using omega-alkynyl fatty acid.
    ACS Chem Biol 2018 Feb 20. Epub 2018 Feb 20.
    The 12/15-lipoxygenase (12/15-LOX) enzyme introduces peroxyl groups, in a position-specific manner, into polyunsaturated fatty acids to form various kinds of bioactive lipid metabolites, including lipid-derived electrophiles (LDE). The resident peritoneal macrophage is the site of greatest 12/15-LOX expression in the mouse. However, the role of the enzyme in the regulation of resident macrophages is not fully understood. Read More

    More Than a Light Switch: Engineering Unconventional Fluorescent Configurations for Biological Sensing.
    ACS Chem Biol 2018 Feb 20. Epub 2018 Feb 20.
    Department of Chemistry, University of British Columbia , 2036 Main Mall, Vancouver, British Columbia V6T 1Z1, Canada.
    Fluorescence is a powerful and sensitive tool in biological detection, used widely for cellular imaging and in vitro molecular diagnostics. Over time, three prominent conventions have emerged in the design of fluorescent biosensors: a sensor is ideally specific for its target, only one fluorescence signal turns on or off in response to the target, and each target requires its own sensor and signal combination. These are conventions but not requirements, and sensors that break with one or more of these conventions can offer new capabilities and advantages. Read More

    Inhibition of HIV fusion by small molecule agonists through efficacy-engineering of CXCR4.
    ACS Chem Biol 2018 Feb 20. Epub 2018 Feb 20.
    CXC chemokine receptor 4 (CXCR4) is involved in multiple physiological and pathological processes, notably as a co-receptor for human immunodeficiency virus (HIV) cell entry. Its broad expression pattern and vital biological importance make CXCR4 a troublesome drug target, as disruption of the interaction with its endogenous ligand, CXC chemokine ligand 12 (CXCL12), has severe consequences. In fact, only one CXCR4 drug, the bicyclam antagonist and HIV entry inhibitor AMD3100 (Plerixafor/Mozobil), has been approved for clinical use, however only for stem cell mobilization-a consequence of CXCR4 antagonism. Read More

    Thienyl-Substituted α-Ketoamide: A Less Hydrophobic Reactive Group for Photo-Affinity Labeling.
    ACS Chem Biol 2018 Feb 19. Epub 2018 Feb 19.
    Synthetic Organic Chemistry Laboratory, RIKEN , Wako, Saitama 351-0198, Japan.
    Photoaffinity labeling (PAL) is an important tool in chemical biology research, but application of α-ketoamides for PAL has been hampered by their photoinstability. Here, we show that 2-thienyl-substituted α-ketoamide is a superior photoreactive group for PAL. Studies with a series of synthetic mannose-conjugated α-ketoamides revealed that 2-thienyl substitution of α-ketoamide decreased the electrophilicity of the keto group and reduced the rate of photodegradation. Read More

    Frontiers in CRISPR.
    ACS Chem Biol 2018 Feb;13(2):296-304
    CRISPR-based approaches to genetic engineering are progressing at a rapid pace and present exciting new avenues for science, medicine, and technology. Many of the most cutting-edge advances in genome engineering are encompassed in the Research Articles, Reviews, and Perspectives in this special issue, often with an eye toward future directions for the field. Yet, many questions remain at this new frontier. Read More

    Spotlight: A Conversation with Laura Kiessling and Jennifer Doudna.
    ACS Chem Biol 2018 Feb;13(2):290-295
    ACS Chemical Biology recorded a special podcast, in which Editor-in-Chief Laura Kiessling (Massachusetts Institute of Technology) interviews CRISPR investigator and former Associate Editor Jennifer Doudna (University of California, Berkeley). Listen to the podcast here . A transcript of the interview, which has been lightly edited, is published here as part of our Special Issue on the Chemical Biology of CRISPR. Read More

    Lessons Learned from Luminous Luciferins and Latent Luciferases.
    ACS Chem Biol 2018 Feb 19. Epub 2018 Feb 19.
    Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School , Worcester, Massachusetts 01605, United States.
    Compared to the broad palette of fluorescent molecules, there are relatively few structures that are competent to support bioluminescence. Here, we focus on recent advances in the development of luminogenic substrates for firefly luciferase. The scope of this light-emitting chemistry has been found to extend well beyond the natural substrate and to include enzymes incapable of luciferase activity with d-luciferin. Read More

    Incorporation of Nonproteinogenic Amino Acids in Class I and II Lantibiotics.
    ACS Chem Biol 2018 Feb 21. Epub 2018 Feb 21.
    Howard Hughes Medical Institute and Roger Adams Laboratory, Department of Chemistry, University of Illinois at Urbana-Champaign , 600 South Mathews Avenue, Urbana, Illinois 61801, United States.
    Lantibiotics are ribosomally synthesized and post-translationally modified peptide natural products that contain thioether cross-links formed by lanthionine and methyllanthionine residues. They exert potent antimicrobial activity against Gram-positive bacteria. We herein report production of analogues of two lantibiotics, lacticin 481 and nisin, that contain nonproteinogenic amino acids using two different strategies involving amber stop codon suppression technology. Read More

    Ligand-directed chemistry of AMPA receptors confers live-cell fluorescent biosensors.
    ACS Chem Biol 2018 Feb 13. Epub 2018 Feb 13.
    AMPA-type glutamate receptors (AMPARs) mediate fast excitatory synaptic transmission in the central nervous system. Disregulation of AMPAR function is associated with many kinds of neurological, neurodegenerative and psychiatric disorders. As a result, molecules capable of controlling AMPAR functions are potential therapeutic agents. Read More

    Stereospecificity of Enoylreductase Domains from Modular Polyketide Synthases.
    ACS Chem Biol 2018 Feb 15. Epub 2018 Feb 15.
    State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University , Shanghai 200240, China.
    An enoylreductase (ER) domain of a polyketide synthase module recruiting a methylmalonate extender unit sets the C2 methyl branch to either the S or R configuration during processing of a polyketide intermediate carried by an acyl carrier protein (ACP) domain. In the present study, pantetheine- and ACP-bound trans-2-methylcrotonyl substrate surrogates were used to scrutinize the stereospecificity of the ER domains. The pantetheine-bound thioester was reduced to mixtures of both 2R and 2S products, whereas the expected 2S epimer was almost exclusively generated when the cognate ACP-bound substrate surrogate was utilized. Read More

    In Situ Target Engagement Studies in Adherent Cells.
    ACS Chem Biol 2018 Feb 21. Epub 2018 Feb 21.
    Chemical Biology Consortium Sweden, Science for Life Laboratory, Karolinska Institutet , SE-171 65 Solna, Sweden.
    A prerequisite for successful drugs is effective binding of the desired target protein in the complex environment of a living system. Drug-target engagement has typically been difficult to monitor in physiologically relevant models, and with current methods, especially, while maintaining spatial information. One recent technique for quantifying drug-target engagement is the cellular thermal shift assay (CETSA), in which ligand-induced protein stabilization is measured after a heat challenge. Read More

    Light-Activated Proteomic Labeling via Photocaged Bioorthogonal Non-Canonical Amino Acids.
    ACS Chem Biol 2018 Feb 8. Epub 2018 Feb 8.
    Department of Chemical Engineering, University of Washington , 4000 15th Ave NE, Seattle, Washington 98195, United States.
    This work introduces light-activated bioorthogonal noncanonical amino acid tagging (laBONCAT) as a method to selectively label, isolate, and identify proteins newly synthesized at user-defined regions in tissue culture. By photocaging l-azidohomoalanine (Aha), metabolic incorporation into proteins is prevented. The caged compound remains stable for many hours in culture, but can be photochemically liberated rapidly and on demand with spatial control. Read More

    Discovery of Selective RNA-Binding Small Molecules by Affinity-Selection Mass Spectrometry.
    ACS Chem Biol 2018 Feb 14. Epub 2018 Feb 14.
    Merck & Co., Inc. , Boston, Massachusetts 02115, United States.
    Recent advances in understanding the relevance of noncoding RNA (ncRNA) to disease have increased interest in drugging ncRNA with small molecules. The recent discovery of ribocil, a structurally distinct synthetic mimic of the natural ligand of the flavin mononucleotide (FMN) riboswitch, has revealed the potential chemical diversity of small molecules that target ncRNA. Affinity-selection mass spectrometry (AS-MS) is theoretically applicable to high-throughput screening (HTS) of small molecules binding to ncRNA. Read More

    Trifunctional High-Throughput Screen Identifies Promising Scaffold To Inhibit Grp94 and Treat Myocilin-Associated Glaucoma.
    ACS Chem Biol 2018 Feb 20. Epub 2018 Feb 20.
    School of Chemistry & Biochemistry, Georgia Institute of Technology , Atlanta, Georgia 30332, United States.
    Gain-of-function mutations within the olfactomedin (OLF) domain of myocilin result in its toxic intracellular accumulation and hasten the onset of open-angle glaucoma. The absence of myocilin does not cause disease; therefore, strategies aimed at eliminating myocilin could lead to a successful glaucoma treatment. The endoplasmic reticulum Hsp90 paralog Grp94 accelerates OLF aggregation. Read More

    The Effects of Ultraviolet Radiation on Nucleoside Modifications in RNA.
    ACS Chem Biol 2018 Feb 5. Epub 2018 Feb 5.
    Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati , Cincinnati, Ohio 45221-0172, United States.
    Ultraviolet radiation (UVR) is a known genotoxic agent. Although its effects on DNA have been well-documented, its impact on RNA and RNA modifications is less studied. By using Escherichia coli tRNA (tRNA) as a model system, we identify the UVA (370 nm) susceptible chemical groups and bonds in a large variety of modified nucleosides. Read More

    Gene Drive: Evolved and Synthetic.
    ACS Chem Biol 2018 Feb 5;13(2):343-346. Epub 2018 Feb 5.
    Life Sciences, Imperial College , South Kensington, London, SW7 2AZ, United Kingdom.
    Drive is a process of accelerated inheritance from one generation to the next that allows some genes to spread rapidly through populations even if they do not contribute to-or indeed even if they detract from-organismal survival and reproduction. Genetic elements that can spread by drive include gametic and zygotic killers, meiotic drivers, homing endonuclease genes, B chromosomes, and transposable elements. The fact that gene drive can lead to the spread of fitness-reducing traits (including lethality and sterility) makes it an attractive process to consider exploiting to control disease vectors and other pests. Read More

    Cardiovascular Small Heat Shock Protein HSPB7 Is a Kinetically Privileged Reactive Electrophilic Species (RES) Sensor.
    ACS Chem Biol 2018 Feb 8. Epub 2018 Feb 8.
    Department of Chemistry and Chemical Biology, Cornell University , Ithaca, New York 14853, United States.
    Small heat shock protein (sHSP)-B7 (HSPB7) is a muscle-specific member of the non-ATP-dependent sHSPs. The precise role of HSPB7 is enigmatic. Here, we disclose that zebrafish Hspb7 is a kinetically privileged sensor that is able to react rapidly with native reactive electrophilic species (RES), when only substoichiometric amounts of RES are available in proximity to Hspb7 expressed in living cells. Read More

    Understanding the Molecular Mechanisms of the CRISPR Toolbox Using Single Molecule Approaches.
    ACS Chem Biol 2018 Feb 9. Epub 2018 Feb 9.
    Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.
    Adaptive immunity against foreign genetic elements conferred by the CRISPR systems in microbial species has been repurposed as a revolutionary technology for wide-ranging biological applications-chiefly genome engineering. Biochemical, structural, genetic, and genomics studies have revealed important insights into their function and mechanisms, but most ensemble studies cannot observe structural changes of these molecules during their function and are often blind to key reaction intermediates. Here, we review the use of single molecule approaches such as fluorescent particle tracking, FRET, magnetic tweezers, and atomic force microscopy imaging in improving our understanding of the CRISPR toolbox. Read More

    Antimicrobial Metallopeptides.
    ACS Chem Biol 2018 Feb 13. Epub 2018 Feb 13.
    Evans Laboratory of Chemistry, The Ohio State University , 100 West 18th Avenue, Columbus, Ohio 43210, United States.
    Antimicrobial peptides are short amphipathic peptides that are produced by the innate immune system in order to protect a host from pathogens. They have been shown to have broad-spectrum antimicrobial activity toward Gram-positive and Gram-negative bacteria, as well as antifungal, antiprotozoan, and antiviral activity. These peptides are able to exert their activity through a variety of mechanisms that include inhibiting DNA and RNA replication, inhibiting protein synthesis, permeabilizing the cell membrane, disrupting proton and ion transmembrane gradients, and inhibiting cell wall biosynthesis. Read More

    Biosynthesis of the Klebsiella oxytoca Pathogenicity Factor Tilivalline: Heterologous Expression, in Vitro Biosynthesis, and Inhibitor Development.
    ACS Chem Biol 2018 Feb 13. Epub 2018 Feb 13.
    Department of Microbial Natural Products (MINS), Helmholtz Institute for Pharmaceutical Research Saarland (HIPS) - Helmholtz Centre for Infection Research (HZI) and Department of Pharmacy, Saarland University , 66123 Saarbrücken, Germany.
    Tilvalline is a pyrrolo[4,2]benzodiazepine derivative produced by the pathobiont Klebsiella oxytoca and is the causative toxin in antibiotic associated hemorrhagic colitis (AAHC). Heterologous expression of the tilivalline biosynthetic gene cluster along with in vitro reconstitution of the respective NRPS (NpsA, ThdA, NpsB) was employed to reveal a nonenzymatic indole incorporation via a spontaneous Friedel-Crafts-like alkylation reaction. Furthermore, the heterologous system was used to generate novel tilivalline derivatives by supplementation of respective anthranilate and indole precursors. Read More

    Development of Activity-Based Chemical Probes for Human Sirtuins.
    ACS Chem Biol 2018 Feb 8. Epub 2018 Feb 8.
    Department of Pharmaceutical Sciences, Albany College of Pharmacy and Health Sciences , 261 Mountain View Drive, Colchester, Vermont 05446, United States.
    Sirtuins consume stoichiometric amounts of nicotinamide adenine dinucleotide (NAD) to remove an acetyl group from lysine residues. These enzymes have been implicated in regulating various cellular events and have also been suggested to mediate the beneficial effects of calorie restriction (CR). However, controversies on sirtuin biology also peaked during the past few years because of conflicting results from different research groups. Read More

    Biosynthesis and Structure-Activity Relationship Studies of Okaramines That Target Insect Glutamate-Gated Chloride Channels.
    ACS Chem Biol 2018 Feb 2. Epub 2018 Feb 2.
    Chemical Biology Research Group, RIKEN Center for Sustainable Resource Science , Wako, Saitama 351-0198, Japan.
    Prenylated indole alkaloid okaramines selectively target insect glutamate-gated chloride channels (GluCls). Because of their highly complex structures, including azocine and azetidine rings, total synthesis of okaramine A or B has not been achieved, preventing evaluation of the biological activities of okaramines. Biosynthetic approaches provide alternatives to accessing structurally diverse derivatives and enabling the elucidation of structure-activity relationships. Read More

    Characterization of a Prenyltransferase for Iso-A82775C Biosynthesis and Generation of New Congeners of Chloropestolides.
    ACS Chem Biol 2018 Jan 31. Epub 2018 Jan 31.
    State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences , Beijing 100101, China.
    Chloropupukeananin and chloropestolides are novel metabolites of the plant endophyte Pestalotiopsis fici, showing antimicrobial, antitumor, and anti-HIV activities. Their highly complex and unique skeletons were generated from the coisolated pestheic acid (1) and iso-A82775C (10) based on our previous studies. Here, we identified the biosynthetic gene cluster iac of 10 and characterized an iacE encoded prenyltransferase. Read More

    Biochemical Studies and Molecular Dynamic Simulations Reveal the Molecular Basis of Conformational Changes in DNA Methyltransferase-1.
    ACS Chem Biol 2018 Feb 8. Epub 2018 Feb 8.
    Drug Discovery and Design Center, CAS Key Laboratory of Receptor Research, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences , Shanghai 201203, China.
    DNA methyltransferase-1 (DNMT1) plays a crucial role in the maintenance of genomic methylation patterns. The crystal structure of DNMT1 was determined in two different states in which the helix that follows the catalytic loop was either kinked (designated helix-kinked) or well folded (designated helix-straight state). Here, we show that the proper structural transition between these two states is required for DNMT1 activity. Read More

    Smart Cell Culture Systems: Integration of Sensors and Actuators into Microphysiological Systems.
    ACS Chem Biol 2018 Feb 15. Epub 2018 Feb 15.
    Department of Biosystems Science and Engineering, Bio Engineering Laboratory, ETH Zürich , Basel, Switzerland.
    Technological advances in microfabrication techniques in combination with organotypic cell and tissue models have enabled the realization of microphysiological systems capable of recapitulating aspects of human physiology in vitro with great fidelity. Concurrently, a number of analysis techniques has been developed to probe and characterize these model systems. However, many assays are still performed off-line, which severely compromises the possibility of obtaining real-time information from the samples under examination, and which also limits the use of these platforms in high-throughput analysis. Read More

    A Cleavage-Responsive Stem-Loop Hairpin for Assaying Guide RNA Activity.
    ACS Chem Biol 2018 Feb 2;13(2):461-466. Epub 2018 Feb 2.
    Berkeley Department of Bioengineering, University of California , Berkeley, California 94720, United States.
    The scope of the CRISPR-Cas9 technology now reaches far beyond genomic engineering. While significant efforts are driving the evolution of this revolutionary biomedical tool, the in vitro cleavage assay remains the standard method implemented to validate the guide RNA that directs endonuclease Cas9 to a desired genomic target. Here, we report the development of an alternative guide RNA validation system called GUIDER. Read More

    Quantitative mRNA Imaging with Dual Channel qFIT Probes to Monitor Distribution and Degree of Hybridization.
    ACS Chem Biol 2018 Feb 7. Epub 2018 Feb 7.
    Institut für Chemie der Humboldt-Universität zu Berlin , 12489 Berlin, Germany.
    Fluorogenic oligonucleotide probes facilitate the detection and localization of RNA targets within cells. However, quantitative measurements of mRNA abundance are difficult when fluorescence signaling is based on intensity changes because a high concentration of unbound probes cannot be distinguished from a low concentration of target-bound probes. Here, we introduce qFIT (quantitative forced intercalation) probes that allow the detection both of probe-target complexes and of unbound probes on separate, independent channels. Read More

    Characterization of the Fast and Promiscuous Macrocyclase from Plant PCY1 Enables the Use of Simple Substrates.
    ACS Chem Biol 2018 Feb 12. Epub 2018 Feb 12.
    Biomedical Sciences Research Complex, University of St. Andrews , North Haugh, St. Andrews, KY16 9ST, United Kingdom.
    Cyclic ribosomally derived peptides possess diverse bioactivities and are currently of major interest in drug development. However, it can be chemically challenging to synthesize these molecules, hindering the diversification and testing of cyclic peptide leads. Enzymes used in vitro offer a solution to this; however peptide macrocyclization remains the bottleneck. Read More

    Design and Application of Sensors for Chemical Cytometry.
    ACS Chem Biol 2018 Feb 8. Epub 2018 Feb 8.
    Department of Chemistry, The University of North Carolina at Chapel Hill , Chapel Hill, North Carolina 27599, United States.
    The bulk cell population response to a stimulus, be it a growth factor or a cytotoxic agent, neglects the cell-to-cell variability that can serve as a friend or as a foe in human biology. Biochemical variations among closely related cells furnish the basis for the adaptability of the immune system but also act as the root cause of resistance to chemotherapy by tumors. Consequently, the ability to probe for the presence of key biochemical variables at the single-cell level is now recognized to be of significant biological and biomedical impact. Read More

    Turning the Spotlight on Lipids in Non-Apoptotic Cell Death.
    ACS Chem Biol 2018 Feb 6. Epub 2018 Feb 6.
    Department of Chemistry, University at Buffalo, The State University of New York , Buffalo, New York 14260, United States.
    Although apoptosis has long dominated the spotlight, studies in the past two decades have expanded the repertoire of programmed cell death (PCD). Several forms of non-apoptotic regulated cell death have been identified, with important links to organismal homeostasis and different disease pathologies. Necroptosis, ferroptosis, pyroptosis, and NETosis are the major forms of PCD that have attracted attention. Read More

    Can CRISPR-Based Gene Drive Be Confined in the Wild? A Question for Molecular and Population Biology.
    ACS Chem Biol 2018 Feb 7;13(2):424-430. Epub 2018 Feb 7.
    Section of Cell and Developmental Biology, University of California, San Diego , La Jolla, California 92093, United States of America.
    The recent discovery of CRISPR and its application as a gene editing tool has enabled a range of gene drive systems to be engineered with greater ease. In order for the benefits of this technology to be realized, in some circumstances drive systems should be developed that are capable of both spreading into populations to achieve their desired impact and being recalled in the event of unwanted consequences or public disfavor. We review the performance of three broad categories of drive systems at achieving these goals: threshold-dependent drives, homing-based drive and remediation systems, and temporally self-limiting systems such as daisy-chain drives. Read More

    Toward a Microparticle-Based System for Pooled Assays of Small Molecules in Cellular Contexts.
    ACS Chem Biol 2018 Feb 8. Epub 2018 Feb 8.
    Department of Chemistry, Columbia University , New York, New York 10027, United States.
    Experimental approaches to the discovery of small molecule probes and drug candidates often use biochemical or cell-based screening of large libraries (>10) of small molecules. Small molecules of interest are tested one at a time in individual wells of a microtiter plate, at a significant cost in time and resources. Furthermore, evaluation of large numbers of compounds in such assays requires robust cellular or biochemical screening formats that may not be relevant to the contexts found in human patients. Read More

    Multiple mechanisms of zinc-mediated inhibition for the apoptotic caspases -3, -6, -7, & -8.
    ACS Chem Biol 2018 Jan 24. Epub 2018 Jan 24.
    Zinc is emerging as a widely used and important biological regulatory signal. Cellular zinc levels are tightly regulated by a complex array of zinc importer and exporters to control processes such as apoptotic cell death. While caspase inhibition by zinc has been reported previously, the reported inhibition constants were too weak to suggest a critical biological role for zinc-mediated inhibition. Read More

    Heterologous Production and Purification of a Functional Chloroform Reductive Dehalogenase.
    ACS Chem Biol 2018 Jan 25. Epub 2018 Jan 25.
    School of Biotechnology and Biomolecular Sciences, University of New South Wales , Sydney, New South Wales 2052, Australia.
    Reductive dehalogenases (RDases) are key enzymes involved in the respiratory process of anaerobic organohalide respiring bacteria (ORB). Heterologous expression of respiratory RDases is desirable for structural and functional studies; however, there are few reports of successful expression of these enzymes. Dehalobacter sp. Read More

    Heme Binding to Porphobilinogen Deaminase from Vibrio cholerae Decelerates the Formation of 1-Hydroxymethylbilane.
    ACS Chem Biol 2018 Feb 7. Epub 2018 Feb 7.
    Department of Chemistry, Faculty of Science, Hokkaido University , Sapporo 060-0810, Japan.
    Porphobilinogen deaminase (PBGD) is an enzyme that catalyzes the formation of hydroxymethylbilane, a tetrapyrrole intermediate, during heme biosynthesis through the stepwise polymerization of four molecules of porphobilinogen. PBGD from Vibrio cholerae was expressed in Escherichia coli and characterized in this study. Unexpectedly, spectroscopic measurements revealed that PBGD bound one equivalent of heme with a dissociation constant of 0. Read More

    Site-Specific Incorporation of a Thioester Containing Amino Acid into Proteins.
    ACS Chem Biol 2018 Feb 9. Epub 2018 Feb 9.
    Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute , 10550 N Torrey Pines Road, La Jolla, California 92037, United States.
    Here, we report the site-specific incorporation of a thioester containing noncanonical amino acid (ncAA) into recombinantly expressed proteins. Specifically, we genetically encoded a thioester-activated aspartic acid (ThioD) in bacteria in good yield and with high fidelity using an orthogonal nonsense suppressor tRNA/aminoacyl-tRNA synthetase (aaRS) pair. To demonstrate the utility of ThioD, we used native chemical ligation to label green fluorescent protein with a fluorophore in good yield. Read More

    A Peptidomimetic Antibiotic Interacts with the Periplasmic Domain of LptD from Pseudomonas aeruginosa.
    ACS Chem Biol 2018 Jan 23. Epub 2018 Jan 23.
    Chemistry Department, University of Zurich , Winterthurerstrasse 190, 8057 Zurich, Switzerland.
    The outer membrane (OM) in Gram-negative bacteria is an asymmetric bilayer with mostly lipopolysaccharide (LPS) molecules in the outer leaflet. During OM biogenesis, new LPS molecules are transported from their site of assembly on the inner membrane to the OM by seven LPS transport proteins (LptA-G). The complex formed between the integral β-barrel OM protein LptD and the lipoprotein LptE is responsible for transporting LPS from the periplasmic side of the OM to its final location on the cell surface. Read More

    Potent Inhibitors of Mycobacterium tuberculosis Growth Identified by Using in-Cell NMR-based Screening.
    ACS Chem Biol 2018 Feb 2. Epub 2018 Feb 2.
    Department of Chemistry, University at Albany, State University of New York , Albany, New York 12222, United States.
    In-cell NMR spectroscopy was used to screen for drugs that disrupt the interaction between prokaryotic ubiquitin like protein, Pup, and mycobacterial proteasome ATPase, Mpa. This interaction is critical for Mycobacterium tuberculosis resistance against nitric oxide (NO) stress; interruption of this process was proposed as a mechanism to control latent infection. Three compounds isolated from the NCI Diversity set III library rescued the physiological proteasome substrate from degradation suggesting that the proteasome degradation pathway was selectively targeted. Read More

    A Predictive Approach for the Optical Control of Carbonic Anhydrase II Activity.
    ACS Chem Biol 2018 Feb 9. Epub 2018 Feb 9.
    Department of Chemistry, Ludwig-Maximilian-University Munich and Munich Center for Integrated Protein Science (CIPSM) , Butenandtstrasse 5-13, 83177 Munich, Germany.
    Optogenetics and photopharmacology are powerful approaches to investigating biochemical systems. While the former is based on genetically encoded photoreceptors that utilize abundant chromophores, the latter relies on synthetic photoswitches that are either freely diffusible or covalently attached to specific bioconjugation sites, which are often native or engineered cysteines. The identification of suitable cysteine sites and appropriate linkers for attachment is generally a lengthy and cumbersome process. Read More

    Allostery, Recognition of Nascent Peptidoglycan, and Cross-linking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of Streptococcus pneumoniae.
    ACS Chem Biol 2018 Jan 30. Epub 2018 Jan 30.
    Department of Crystallography and Structural Biology, Institute of Physical Chemistry "Rocasolano," CSIC , 28006 Madrid, Spain.
    Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze cross-linking of the bacterial cell wall. This transformation is critical for the survival of bacteria, and it is the target of inhibition by β-lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures, and molecular-dynamics simulations. Read More

    Translation Termination Factor GSPT1 Is a Phenotypically Relevant Off-Target of Heterobifunctional Phthalimide Degraders.
    ACS Chem Biol 2018 Jan 29. Epub 2018 Jan 29.
    CeMM Research Center for Molecular Medicine of the Austrian Academy of Science , Lazarettgasse 14, AKH Bt. 25.3, 1090 Vienna, Austria.
    Protein degradation is an emerging therapeutic strategy with a unique molecular pharmacology that enables the disruption of all functions associated with a target. This is particularly relevant for proteins depending on molecular scaffolding, such as transcription factors or receptor tyrosine kinases (RTKs). To address tractability of multiple RTKs for chemical degradation by the E3 ligase CUL4-RBX1-DDB1-CRBN (CRL4), we synthesized a series of phthalimide degraders based on the promiscuous kinase inhibitors sunitinib and PHA665752. Read More

    Playing with the molecules of life.
    ACS Chem Biol 2018 Jan 18. Epub 2018 Jan 18.
    Our understanding of the complex molecular processes of living organisms is growing exponentially. This knowledge, togeth-er with a powerful arsenal of tools for manipulating the structures of biological macromolecules, is allowing chemists to begin to harness and reprogram the remarkable machinery of life. Here we review one such example in which the genetic code itself has been expanded with new building blocks that allow us to probe and manipulate the structures and functions of proteins in ways previously unimaginable. Read More

    The Development of Benzimidazole-Based Clickable Probes for the Efficient Labeling of Cellular Protein Arginine Deiminases (PADs).
    ACS Chem Biol 2018 Feb 1. Epub 2018 Feb 1.
    Department of Biochemistry and Molecular Pharmacology, UMass Medical School , 364 Plantation Street, Worcester, Massachusetts 01605, United States.
    Citrullination is the post-translational hydrolysis of peptidyl-arginines to form peptidyl-citrulline, a reaction that is catalyzed by the protein arginine deiminases (PADs), a family of calcium-regulated enzymes. Aberrantly increased protein citrullination is associated with a slew of autoimmune diseases (e.g. Read More

    Human Neuraminidase Isoenzymes Show Variable Activities for 9-O-Acetyl-sialoside Substrates.
    ACS Chem Biol 2018 Feb 13. Epub 2018 Feb 13.
    Alberta Glycomics Centre, Department of Chemistry, University of Alberta , Edmonton Alberta T6G 2G2, Canada.
    Recognition of terminal sialic acids is central to many cellular processes, and structural modification of sialic acid can disrupt these interactions. A prominent, naturally occurring, modification of sialic acid is 9-O-acetylation (9-O-Ac). Study of this modification through generation and analysis of 9-O-Ac sialosides is challenging because of the lability of the acetate group. Read More

    Histone Deacetylase 11 Is a Fatty-Acid Deacylase.
    ACS Chem Biol 2018 Jan 30. Epub 2018 Jan 30.
    Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV , Prumyslova 595, 252 50 Vestec, Czech Republic.
    Histone deacetylase 11 (HDAC11) is a sole member of the class IV HDAC subfamily with negligible intrinsic deacetylation activity. Here, we report in vitro profiling of HDAC11 deacylase activities, and our data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8-18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate. Additionally, N-linked lipoic acid and biotin are removed by the enzyme, although with lower efficacy. Read More

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