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Angewandte Chemie (International Ed. in... Feb 2020The diverse secondary structures of nucleic acids are emerging as attractive chiral scaffolds to construct artificial metalloenzymes (ArMs) for enantioselective...
The diverse secondary structures of nucleic acids are emerging as attractive chiral scaffolds to construct artificial metalloenzymes (ArMs) for enantioselective catalysis. DNA-based ArMs containing duplex and G-quadruplex scaffolds have been widely investigated, yet RNA-based ArMs are scarce. Here we report that a cyclic dinucleotide of c-di-AMP and Cu ions assemble into an artificial metalloribozyme (c-di-AMP⋅Cu ) that enables catalysis of enantioselective Friedel-Crafts reactions in aqueous media with high reactivity and excellent enantioselectivity of up to 97 % ee. The assembly of c-di-AMP⋅Cu gives rise to a 20-fold rate acceleration compared to Cu ions. Based on various biophysical techniques and density function theory (DFT) calculations, a fine coordination structure of c-di-AMP⋅Cu metalloribozyme is suggested in which two c-di-AMP form a dimer scaffold and the Cu ion is located in the center of an adenine-adenine plane through binding to two N7 nitrogen atoms and one phosphate oxygen atom.
Topics: Catalysis; Copper; Cycloaddition Reaction; Density Functional Theory; Dimerization; Dinucleoside Phosphates; G-Quadruplexes; Kinetics; Metalloproteins; Stereoisomerism; Substrate Specificity; Water
PubMed: 31825550
DOI: 10.1002/anie.201912962 -
Journal of Bacteriology Sep 20213'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and...
3'3'-Cyclic di-AMP (c-di-AMP) is an important nucleotide second messenger found throughout the bacterial domain of life. c-di-AMP is essential in many bacteria and regulates a diverse array of effector proteins controlling pathogenesis, cell wall homeostasis, osmoregulation, and central metabolism. Despite the ubiquity and importance of c-di-AMP, methods to detect this signaling molecule are limited, particularly at single-cell resolution. In this work, crystallization of the Listeria monocytogenes c-di-AMP effector protein Lmo0553 enabled structure-guided design of a Förster resonance energy transfer (FRET)-based biosensor, which we have named CDA5. CDA5 is a fully genetically encodable, specific, and reversible biosensor which allows the detection of c-di-AMP dynamics both and within live cells in a nondestructive manner. Our initial studies identified a distribution of c-di-AMP in Bacillus subtilis populations first grown in Luria broth and then resuspended in diluted Luria broth compatible with fluorescence analysis. Furthermore, we found that B. subtilis mutants lacking either a c-di-AMP phosphodiesterase and cyclase have higher and lower FRET responses, respectively. These findings provide novel insight into the c-di-AMP distribution within bacterial populations and establish CDA5 as a powerful platform for characterizing new aspects of c-di-AMP regulation. c-di-AMP is an important nucleotide second messenger for which detection methods are severely limited. In this work we engineered and implemented a c-di-AMP-specific FRET biosensor to remedy this dearth. We present this biosensor, CDA5, as a versatile tool to investigate previously intractable facets of c-di-AMP biology.
Topics: Bacillus subtilis; Bacterial Proteins; Biosensing Techniques; Dinucleoside Phosphates; Fluorescence Resonance Energy Transfer; Gene Expression Regulation, Bacterial; Listeria monocytogenes; Models, Molecular; Mutation; Nucleotides; Protein Conformation
PubMed: 34309402
DOI: 10.1128/JB.00080-21 -
Proteins Nov 2016Understanding the origin of discrimination between rNTP and dNTP by DNA/RNA polymerases is important both for gaining fundamental knowledge on the corresponding systems...
Understanding the origin of discrimination between rNTP and dNTP by DNA/RNA polymerases is important both for gaining fundamental knowledge on the corresponding systems and for advancing the design of specific drugs. This work explores the nature of this discrimination by systematic calculations of the transition state (TS) binding energy in RB69 DNA polymerase (gp43) and T7 RNA polymerase. The calculations reproduce the observed trend, in particular when they included the water contribution obtained by the water flooding approach. Our detailed study confirms the idea that the discrimination is due to the steric interaction between the 2'OH and Tyr416 in DNA polymerase, while the electrostatic interaction is the source of the discrimination in RNA polymerase. Proteins 2016; 84:1616-1624. © 2016 Wiley Periodicals, Inc.
Topics: Binding Sites; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerases; Dinucleoside Phosphates; Kinetics; Models, Molecular; Protein Binding; Protein Domains; Protein Structure, Secondary; Ribonucleotides; Static Electricity; Substrate Specificity; Thermodynamics; Viral Proteins; Water
PubMed: 27480935
DOI: 10.1002/prot.25104 -
Bioorganic & Medicinal Chemistry Letters Jan 2001A series of dinucleoside 5-polyphosphates UpnU (n = 2-7) was synthesized. Their relative potencies as agonists at the G-protein-coupled receptors P2Y1, P2Y2, P2Y4, and...
A series of dinucleoside 5-polyphosphates UpnU (n = 2-7) was synthesized. Their relative potencies as agonists at the G-protein-coupled receptors P2Y1, P2Y2, P2Y4, and P2Y6 were determined by intracellular calcium measurements using fluorescent imaging techniques. The correlation of phosphate chain length to activities at these receptors is discussed.
Topics: Calcium; Dinucleoside Phosphates; Humans; Protein Binding; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2; Spectrometry, Fluorescence; Structure-Activity Relationship; Transduction, Genetic; Tumor Cells, Cultured; Uracil Nucleotides
PubMed: 11206448
DOI: 10.1016/s0960-894x(00)00612-0 -
Nucleic Acids Research Mar 1974A dinucleoside monophosphate, 6,2'-anhydro-6-oxy-1-beta-D-arabinofuranosyluracil-phosphoryl- (3'-5')-6,2'-anhydro-6-oxy-1-beta-D-arabinofuranosyluracil (I) was...
A dinucleoside monophosphate, 6,2'-anhydro-6-oxy-1-beta-D-arabinofuranosyluracil-phosphoryl- (3'-5')-6,2'-anhydro-6-oxy-1-beta-D-arabinofuranosyluracil (I) was synthesized by the condensation reaction using DCC from 5'-monomethoxytrityl derivative(VII) and 3'-acetyl-5'-phosphate(X) of the monomer units. Yield was ca. 70%. UV of compound I showed no hypochromicity and its CD spectrum showed only a trough at 252 nm. No temperature dependency of epsilon and [Q] values was observed. These results suggested an unstacked random conformation for I. Complex formation of I with cycloadenosine oligomer could not be detected under the experimental condition.
Topics: Adenine Nucleotides; Circular Dichroism; Dinucleoside Phosphates; Hydrolysis; Nucleic Acid Conformation; Oligoribonucleotides; Poly A; Spectrophotometry, Ultraviolet; Uridine
PubMed: 10793680
DOI: 10.1093/nar/1.3.479 -
Organic Letters Jun 2013A procedure for the synthesis of dinucleoside 5'-pentaphosphates (Np5N) and nucleoside 5'-tetraphosphates (Np4) is described. The procedure relies on the activation of...
A procedure for the synthesis of dinucleoside 5'-pentaphosphates (Np5N) and nucleoside 5'-tetraphosphates (Np4) is described. The procedure relies on the activation of cyclic trimetaphosphate followed by a reaction with a nucleoside 5'-monophosphate (NMP) to give intermediates of type 3. Reaction of 3 with water or an NMP gives the desired products in yields ranging from 77 to 86%.
Topics: Dinucleoside Phosphates; Magnetic Resonance Spectroscopy; Molecular Structure; Nucleosides; Nucleotides; Polyphosphates
PubMed: 23668391
DOI: 10.1021/ol4007822 -
Methods in Enzymology 2019So far, four cyclic dinucleotides (CDNs) have been discovered as important second messengers in nature, where three canonical CDNs of c-di-GMP, c-di-AMP and c-AMP-GMP...
So far, four cyclic dinucleotides (CDNs) have been discovered as important second messengers in nature, where three canonical CDNs of c-di-GMP, c-di-AMP and c-AMP-GMP were found in bacteria containing two 3'-5' phosphodiester linkages and one non-canonical CDN 2'3'-c-GMP-AMP was identified in mammals containing mixed 2'-5' and 3'-5' phosphodiester linkages. The CDNs are produced by specific cyclases and degraded by phosphodiesterases (PDEs). All of the known CDNs could bind to the stimulator of interferon genes (STING) to induce type I interferon (IFN) responses and the three bacterial CDNs are sensed by specific riboswitches to regulate gene expression. The emerging physiological functions of bacterial CDNs lead the motivation to investigate other possible canonical CDNs. In recent years, many endeavors have been devoted to develop fast, convenient and cheap strategies for chemically synthesizing CDNs and their analogues. The phosphoramidite approach using commercial starting materials has attracted much attention. Herein, we describe an adapted one-pot strategy that enables fast synthesis of crude 3'-5'-linked canonical CDNs followed by purification of the obtained CDNs using reversed phase high-performance of liquid chromatography (HPLC). Furthermore, we report the full characterization of CDNs by mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques.
Topics: Animals; Cyclic AMP; Dinucleoside Phosphates; Humans; Membrane Proteins; Nucleotides, Cyclic
PubMed: 31455536
DOI: 10.1016/bs.mie.2019.04.022 -
The Journal of Laboratory and Clinical... Aug 1996
Review
Topics: Animals; Autoimmunity; B-Lymphocytes; CpG Islands; DNA, Bacterial; Dinucleoside Phosphates; Humans; Immunity, Innate; Inflammation
PubMed: 8765208
DOI: 10.1016/s0022-2143(96)90004-9 -
The Journal of Biological Chemistry Jul 2022The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and...
The bacterial second messenger c-di-AMP controls essential cellular processes, including potassium and osmolyte homeostasis. This makes synthesizing enzymes and components involved in c-di-AMP signal transduction intriguing as potential targets for drug development. The c-di-AMP receptor protein DarB of Bacillus subtilis binds the Rel protein and triggers the Rel-dependent stringent response to stress conditions; however, the structural basis for this trigger is unclear. Here, we report crystal structures of DarB in the ligand-free state and of DarB complexed with c-di-AMP, 3'3'-cGAMP, and AMP. We show that DarB forms a homodimer with a parallel, head-to-head assembly of the monomers. We also confirm the DarB dimer binds two cyclic dinucleotide molecules or two AMP molecules; only one adenine of bound c-di-AMP is specifically recognized by DarB, while the second protrudes out of the donut-shaped protein. This enables DarB to bind also 3'3'-cGAMP, as only the adenine fits in the active site. In absence of c-di-AMP, DarB binds to Rel and stimulates (p)ppGpp synthesis, whereas the presence of c-di-AMP abolishes this interaction. Furthermore, the DarB crystal structures reveal no conformational changes upon c-di-AMP binding, leading us to conclude the regulatory function of DarB on Rel must be controlled directly by the bound c-di-AMP. We thus derived a structural model of the DarB-Rel complex via in silico docking, which was validated with mass spectrometric analysis of the chemically crosslinked DarB-Rel complex and mutagenesis studies. We suggest, based on the predicted complex structure, a mechanism of stringent response regulation by c-di-AMP.
Topics: Adenine; Adenosine Monophosphate; Bacillus subtilis; Bacterial Proteins; Dinucleoside Phosphates
PubMed: 35714772
DOI: 10.1016/j.jbc.2022.102144 -
Stresses that Raise NpA Levels Induce Protective Nucleoside Tetraphosphate Capping of Bacterial RNA.Molecular Cell Sep 2019Present in all realms of life, dinucleoside tetraphosphates (NpNs) are generally considered signaling molecules. However, only a single pathway for NpN signaling has...
Present in all realms of life, dinucleoside tetraphosphates (NpNs) are generally considered signaling molecules. However, only a single pathway for NpN signaling has been delineated in eukaryotes, and no receptor that mediates the influence of NpNs has ever been identified in bacteria. Here we show that, under disulfide stress conditions that elevate cellular NpN concentrations, diverse Escherichia coli mRNAs and sRNAs acquire a cognate Np cap. Purified E. coli RNA polymerase and lysyl-tRNA synthetase are both capable of adding such 5' caps. Cap removal by either of two pyrophosphatases, ApaH or RppH, triggers rapid RNA degradation in E. coli. ApaH, the predominant decapping enzyme, functions as both a sensor and an effector of disulfide stress, which inactivates it. These findings suggest that the physiological changes attributed to elevated NpN concentrations in bacteria may result from widespread Np capping, leading to altered RNA stability and consequent changes in gene expression.
Topics: Acid Anhydride Hydrolases; Dinucleoside Phosphates; Escherichia coli K12; Escherichia coli Proteins; RNA Stability; RNA, Bacterial
PubMed: 31178354
DOI: 10.1016/j.molcel.2019.05.031