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Journal of Applied Microbiology Jun 2021Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. This study determines whether the E. amylovora guanine-hypoxanthine...
AIM
Erwinia amylovora is the causal agent of fire blight, a devastating disease of apples and pears. This study determines whether the E. amylovora guanine-hypoxanthine transporter (EaGhxP) is required for virulence and if it can import the E. amylovora produced toxic analogue 6-thioguanine (6TG) into cells.
METHODS AND RESULTS
Characterization of EaGhxP in guanine transport deficient Escherichia coli reveals that it can transport guanine, hypoxanthine and the toxic analogues 8-azaguanine (8AG) and 6TG. Similarly, EaGhxP transports 8AG and 6TG into E. amylovora cells. EaGhxP has a high affinity for 6TG with a K of 3·7 µmol l . An E. amylovora ⊿ghxP::Cam strain shows resistance to growth on 8AG and 6TG. Although EaGhxP is expressed during active disease propagation, it is not necessary for virulence as determined on immature apple and pear assays.
CONCLUSIONS
EaGhxP is not required for virulence, but it does import 6TG into E. amylovora cells.
SIGNIFICANCE AND IMPACT OF THE STUDY
As part of the disease establishment process, E. amylovora synthesizes and exports a toxic guanine derivative 6TG. Our results are counter intuitive and show that EaGhxP, an influx transporter, can move 6TG into cells raising questions regarding the role of 6TG in disease establishment.
Topics: Azaguanine; Erwinia amylovora; Escherichia coli; Guanine; Hypoxanthine; Malus; Nucleobase Transport Proteins; Plant Diseases; Recombinant Proteins; Thioguanine
PubMed: 33152175
DOI: 10.1111/jam.14925 -
High-Throughput Screening Assay to Identify Small Molecule Inhibitors of Marburg Virus VP40 Protein.ACS Infectious Diseases Oct 2020Marburg virus (MARV) causes sporadic outbreaks of severe disease with high case fatality rates in humans. To date, neither therapeutics nor prophylactic approaches have...
Marburg virus (MARV) causes sporadic outbreaks of severe disease with high case fatality rates in humans. To date, neither therapeutics nor prophylactic approaches have been approved for MARV disease. The MARV matrix protein VP40 (mVP40) plays central roles in virus assembly and budding. mVP40 also inhibits interferon signaling by inhibiting the function of Janus kinase 1. This suppression of host antiviral defenses likely contributes to MARV virulence and therefore is a potential therapeutic target. We developed and optimized a cell-based high-throughput screening (HTS) assay in 384-well format to measure mVP40 interferon (IFN) antagonist function such that inhibitors could be identified. We performed a pilot screen of 1280 bioactive compounds and identified 3 hits, azaguanine-8, tosufloxacin hydrochloride, and linezolid, with scores > 3 and no significant cytotoxicity. Of these, azaguanine-8 inhibited MARV growth at noncytotoxic concentrations. These data demonstrate the suitability of the HTS mVP40 assay for drug discovery and suggest potential directions for anti-MARV therapeutic development.
Topics: Animals; High-Throughput Screening Assays; Humans; Interferons; Marburg Virus Disease; Marburgvirus; Virus Assembly
PubMed: 32870648
DOI: 10.1021/acsinfecdis.0c00512 -
Microbiology Resource Announcements Jul 2020Here, we describe the genome of DSM 40503, an 8-azaguanine-producing strain. The genome is the basis for future study and presents an underexplored taxonomy and...
Here, we describe the genome of DSM 40503, an 8-azaguanine-producing strain. The genome is the basis for future study and presents an underexplored taxonomy and biosynthetic potential, which expands our understanding of the diversity of microorganisms that produce nitrogen heterocyclic compounds.
PubMed: 32732231
DOI: 10.1128/MRA.00518-20 -
Pest Management Science Dec 2020Rice stripe tenuivirus (RSV) is one of the most destructive pathogens of rice and other cereal crops. The virus is transmitted by the small brown planthopper (SBPH,...
BACKGROUND
Rice stripe tenuivirus (RSV) is one of the most destructive pathogens of rice and other cereal crops. The virus is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a circulative-propagative manner. Thus, blocking transmission by the insect vector would provide an effective strategy to prevent epidemic outbreaks of the disease.
RESULTS
In this study, we explored the effect of ribavirin on acquisition and transmission of the virus by specifically inhibiting the expression of sugar transporter 6 (LsSt-6), which was recently reported as a key vector component for RSV transmission. Ribavirin at the highest concentration tested (250 μmol L ) significantly reduced RSV acquisition and transmission efficiency by SBPHs through inhibiting LsSt-6 messenger RNA (mRNA) level. Survival of the model insect Spodoptera frugiperda cell line (Sf9) was 95.0 ± 2.2 and 85.6 ± 2.1% after exposure to 250 μmol L ribavirin or 8-azaguanine, respectively. Further study confirmed that 250 μmol L ribavirin also significantly reduced LsSt-6 mRNA and protein levels in Sf9 cells. However, 8-azaguanine did not significantly inhibit viral infectivity and LsSt-6 mRNA levels in SBPH or the Sf9 cell line.
CONCLUSION
This result provides evidence that ribavirin has the potential to disrupt LsSt-6 expression but not others like viral RNAs to prevent acquiring RSV, which leads to less viral accumulation in SBPH tissues and thereby lower transmission efficiency. © 2020 Society of Chemical Industry.
Topics: Animals; Hemiptera; Insect Vectors; Oryza; Ribavirin; Sugars; Tenuivirus
PubMed: 32542993
DOI: 10.1002/ps.5963 -
Nature Communications Mar 2020The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich...
The heterocycle 1,2,3-triazole is among the most versatile chemical scaffolds and has been widely used in diverse fields. However, how nature creates this nitrogen-rich ring system remains unknown. Here, we report the biosynthetic route to the triazole-bearing antimetabolite 8-azaguanine. We reveal that its triazole moiety can be assembled through an enzymatic and non-enzymatic cascade, in which nitric oxide is used as a building block. These results expand our knowledge of the physiological role of nitric oxide synthase in building natural products with a nitrogen-nitrogen bond, and should also inspire the development of synthetic biology approaches for triazole production.
Topics: Azaguanine; Bacteria; Bacterial Proteins; Biological Products; Biosynthetic Pathways; Genes, Bacterial; Nitric Oxide; Nitric Oxide Synthase; Nitrogen; Streptomyces; Synthetic Biology; Triazoles
PubMed: 32235841
DOI: 10.1038/s41467-020-15420-8 -
Journal of Chemical Information and... Mar 2020Protein nucleoside phosphorylase (PNP) is an enzyme that catalyzes a reversible conversion process (ribosylation and phosphorolysis) between nucleobases (purines) and...
Protein nucleoside phosphorylase (PNP) is an enzyme that catalyzes a reversible conversion process (ribosylation and phosphorolysis) between nucleobases (purines) and their nucleosides. Experimental studies showed that calf PNP ribosylates purine analogues in specific positions: 2,6-diamino-8-azapurine in position 7 or 8 and 8-azaguanine in position 9 of the triazole ring. The reason for this phenomenon can be a result of different expositions of purine substrates to the channel leading to the binding site. This hypothesis was verified by the application of molecular modeling techniques to two complexes of purine analogues 2,6-diamino-azapurine, calf PNP (pdb-code: 1LVU), and 8-azaguanine, calf PNP (pdb-code: 2AI1). The results obtained with a combination of quantum chemistry, docking, and molecular dynamics methods showed qualitative validity of our hypothesis. Binding free energies of protein-ligand systems showed that most probable binding poses expose N8 nitrogen for 2,6-diamino-8-azapurine and N9 nitrogen for 8-azaguanine into the binding channel and ruled out the exposition of N9 for 2,6-diamino-8-azapurine and N7 for 8-azaguanine, partially in agreement with the experimental data. The other important result obtained in this study is a significantly higher population of the protonated form of crucial residue Glu-201 present in the binding pocket, compared to the standard protonation of free glutamic acid in solution. This result combined with populations of tautomeric forms of both investigated systems strongly suggests that 2,6-diamino-8-azapurine and 8-azaguanine are recognized by proteins with deprotonated and protonated Glu-201 residues, respectively. A comparison of computed binding poses of the investigated ligands to the inhibitors present in crystal structures suggests that the modification of the ()-PMPDAP inhibitor, in which a 2-(phosphonomethoxy)propyl chain is attached at position 8 instead of position 9, might increase its binding affinity.
Topics: Binding Sites; Ligands; Models, Molecular; Purine-Nucleoside Phosphorylase; Purines
PubMed: 31944095
DOI: 10.1021/acs.jcim.9b00985 -
Biochemistry Jul 2019Guanine deaminase is a metabolic enzyme, found in all forms of life, which catalyzes the conversion of guanine to xanthine. Despite the availability of several crystal...
Guanine deaminase is a metabolic enzyme, found in all forms of life, which catalyzes the conversion of guanine to xanthine. Despite the availability of several crystal structures, the molecular determinants of substrate orientation and mechanism remain to be elucidated for the amidohydrolase family of guanine deaminase enzymes. Here, we report the crystal structures of and guanine deaminase enzymes (EcGuaD and Gud1, respectively), both members of the amidohydrolase superfamily. EcGuaD and Gud1 retain the overall TIM barrel tertiary structure conserved among amidohydrolase enzymes. Both proteins also possess a single zinc cation with trigonal bipyrimidal coordination geometry within their active sites. We also determined a liganded structure of Gud1 bound to the product, xanthine. Analysis of this structure, along with kinetic data of native and site-directed mutants of EcGuaD, identifies several key residues that are responsible for substrate recognition and catalysis. In addition, after a small library of compounds had been screened, two guanine derivatives, 8-azaguanine and 1-methylguanine, were identified as EcGuaD substrates. Interestingly, both EcGuaD and Gud1 also exhibit secondary ammeline deaminase activity. Overall, this work details key structural features of substrate recognition and catalysis of the amidohydrolase family of guanine deaminase enzymes in support of our long-term goal to engineer these enzymes with altered activity and substrate specificity.
Topics: Amidohydrolases; Catalytic Domain; Escherichia coli Proteins; Guanine Deaminase; Protein Binding; Protein Structure, Secondary; Saccharomyces cerevisiae Proteins; Substrate Specificity
PubMed: 31283204
DOI: 10.1021/acs.biochem.9b00341 -
Chembiochem : a European Journal of... Sep 2019The impact of 7-deaza-8-azaguanine (DAG) and 7-deaza-8-azaisoguanine (DAiG) modifications on the geometry and stability of the G:C Watson-Crick (cWW) base pair and the...
Structural and Energetic Impact of Non-natural 7-Deaza-8-azaguanine, 7-Deaza-8-azaisoguanine, and Their 7-Substituted Derivatives on Hydrogen-Bond Pairing with Cytosine and Isocytosine.
The impact of 7-deaza-8-azaguanine (DAG) and 7-deaza-8-azaisoguanine (DAiG) modifications on the geometry and stability of the G:C Watson-Crick (cWW) base pair and the G:iC and iG:C reverse Watson-Crick (tWW) base pairs has been characterized theoretically. In addition, the effect on the same base pairs of seven C7-substituted DAG and DAiG derivatives, some of which have been previously experimentally characterized, has been investigated. Calculations indicate that all of these modifications have a negligible impact on the geometry of the above base pairs, and that modification of the heterocycle skeleton has a small impact on the base-pair interaction energies. Instead, base-pair interaction energies are dependent on the nature of the C7 substituent. For the 7-substituted DAG-C cWW systems, a linear correlation between the base-pair interaction energy and the Hammett constant of the 7-substituent is found, with higher interaction energies corresponding to more electron-withdrawing substituents. Therefore, the explored modifications are expected to be accommodated in both parallel and antiparallel nucleic acid duplexes without perturbing their geometry, while the strength of a base pair (and duplex) featuring a DAG modification can, in principle, be tuned by incorporating different substituents at the C7 position.
Topics: Azaguanine; Base Pairing; Cytosine; Hydrogen Bonding; Molecular Structure; Thermodynamics
PubMed: 30983115
DOI: 10.1002/cbic.201900245 -
The Journal of Physical Chemistry. A Apr 2019The photochemical reaction path approach, the MS-CASPT2 quantum-chemical method, and double-ζ basis sets (cc-pVDZ) were used to study 9 H-8-azaguanine and 8...
The photochemical reaction path approach, the MS-CASPT2 quantum-chemical method, and double-ζ basis sets (cc-pVDZ) were used to study 9 H-8-azaguanine and 8 H-8-azaguanine relaxation pathways. Several potential energy hypersurfaces were characterized by means of equilibrium geometries, surface crossings (conical intersections and singlet-triplet intersystem crossings), minimum energy paths, and linear interpolation in internal coordinates. The 9 H-8-azaguanine main photochemical event begins with the direct population of the (ππ* L) state, which evolves toward a conical intersection with the ground state after surmounting a small energy barrier, explaining why it is nonfluorescent. For 8 H-8-azaguanine, two relaxation mechanisms are possible, depending on the excitation energy. If the S (ππ*) state is initially populated (lower-energy region), the system evolves barrierless to the S (ππ*) region, from where the excess energy is released. If the (ππ* L) state is populated (higher-energy radiation), the system will encounter conical intersections with the S (nπ*) and S (ππ*) states before evolving to the (ππ* L) region, from where a conical intersection with the ground state is accessible, favoring radiationless deactivation to the ground state. However, because a fraction of the population can be transferred from (ππ* L) to the S (ππ*) state, emission from the S (ππ*) region is also expected, although weaker than it would be if the S (ππ*) state were populated directly. Irrespective of the excitation energy, the emissive state is the same and a single fluorescence band is observed, with the strongest emission occurring upon excitation in the lower-energy region, as observed experimentally. Therefore, our computational study corroborates experimental results, attributing the emission of the neutral form of 8-azaguanine in solution to the presence of the minor 8 H-8-azaguanine tautomer, while the 9 H-8-azaguanine major tautomer is nonfluorescent.
PubMed: 30901221
DOI: 10.1021/acs.jpca.9b01397 -
Scientific Reports Feb 2019Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have...
Myelodysplastic syndromes (MDS) are haematopoietic malignancies that are characterised by a heterogeneous clinical course. In recent years, sequencing efforts have uncovered recurrent somatic mutations within RNA splicing factors, including SF3B1, SRSF2, U2AF1 and ZRSR2. The most frequently mutated gene is SF3B1, mutated in 17% of MDS patients. While SF3B1 mutations and their effects on splicing have been well characterised, much remains to be explored about their more far-reaching effects on cellular homeostasis. Given that mRNA splicing and nuclear export are coordinated processes, we hypothesised that SF3B1 mutation might also affect export of certain mRNAs and that this may represent a targetable pathway for the treatment of SF3B1-mutant MDS. We used CRISPR/Cas9-genome editing to create isogenic cellular models. Comprehensive transcriptome and proteome profiling of these cells identified alterations in the splicing and export of components of the translational machinery, primarily tRNA synthetases, in response to the SF3B1 K700E mutation. While steady-state protein synthesis was unaffected, SF3B1 mutant cells were more sensitive to the clinically-relevant purine analogue, 8-azaguanine. In this study, we also demonstrated that 8-azaguanine affects splicing. Our results suggest that the simultaneous targeting of RNA metabolism and splicing by 8-azaguanine represents a therapeutic opportunity for SF3B1-mutant myelodysplastic syndromes.
Topics: Amino Acyl-tRNA Synthetases; Cell Line, Tumor; Cytoplasm; Gene Editing; Gene Expression Profiling; HEK293 Cells; Humans; K562 Cells; Mutation; Myelodysplastic Syndromes; Phosphoproteins; Protein Biosynthesis; Proteome; Proteomics; RNA Splicing; RNA Splicing Factors
PubMed: 30804405
DOI: 10.1038/s41598-019-39591-7