scholarly journals "Structural characterization of aminoglycoside modifying enzyme ANT(2"")-Ia"

2014 ◽  
Vol 70 (a1) ◽  
pp. C702-C702
Author(s):  
Angelia Bassenden ◽  
Dmitry Rodionov ◽  
Nilu Sabet-Kassouf ◽  
Tahereh Haji ◽  
Kun Shi ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Specific Activity ◽  
Ribosomal Subunit ◽  
Bactericidal Effect ◽  
Nmr Studies ◽  
Structure Based Drug Design ◽  
X Ray Crystallography ◽  
16S Rna

Aminoglycosides are a class of broad-spectrum antibiotics used in the treatment of serious Gram-negative bacterial infections, they target the 16S RNA subunit and upon binding cause errors in translation, eventually inducing a bactericidal effect [1]. Aminoglycoside nucleotidyltransferase (2")-Ia (ANT(2")-Ia) is an aminoglycoside modifying enzyme that prevents aminoglycosides from binding to the ribosomal subunit, making this enzyme a principle candidate structure-based drug design [2]. Characterization of ANT(2")-Ia has been proven to be difficult due to the low stability and solubility of overexpressed protein, where 95% of the protein being expressed is in the form of inclusion bodies [3]. We describe a protocol that has lead to successful expression and purification of ANT(2")-Ia. A successful enzymatic assay has also been adapted and the protein is active and stable under these conditions with a specific activity of 0.14 U/mg. Furthermore, nuclear magnetic resonance (NMR) studies have allowed for the assignment of 144 of the 176 non-proline backbone residues. Substrate binding NMR experiments have shown unique global chemical shift perturbations upon binding ATP and tobramycin, suggesting unique binding sites for each substrate. Structural determination of ANT(2")-Ia using NMR in conjunction with x-ray crystallography can be utilized in order to develop small molecules that will act as more effective aminoglycosides in order to inhibit ANT(2")-Ia from binding and modifying these antibiotics.

Synthesis and characterization of 19F NMR chelators for measurement of cytosolic free Ca

1987 ◽  
Vol 252 (4) ◽  
pp. C441-C449 ◽  
Author(s):  
L. A. Levy ◽  
E. Murphy ◽  
R. E. London
Keyword(s):  
Chemical Shifts ◽  
Cell Types ◽  
Free Calcium ◽  
Tetraacetic Acid ◽  
Nmr Studies ◽  
Cytosolic Free Calcium ◽  
Fluorine Nmr ◽  
Dissociation Constant Kd

Fluorine 19 nuclear magnetic resonance (NMR) studies of intracellular fluorinated calcium chelators provide a useful strategy for the determination of cytosolic free calcium levels in cells and perfused organs. However, the fluorinated chelator with the highest affinity for calcium ions which has been described to date. 1,2-bis-(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), exhibits a dissociation constant (Kd) value 5- to 10-fold greater than the intracellular calcium concentration levels in most cell types, thus limiting the ability of fluorine NMR to report these concentrations reliably. We have consequently designed and synthesized several fluorinated calcium chelators with higher affinity for calcium. The best of these, 2-(2-amino-4-methyl-5-fluorophenoxy)-methyl-8 aminoquinidine-N,N,N',N'-tetraacetic acid (quinMF), has a Kd value approximately 10 times lower than that of 5FBAPTA. Several of the newly synthesized indicators have different chemical shifts for the calcium complexed and uncomplexed chelators to allow the simultaneous use of two indicators. In addition to providing information about the level of cytosolic free calcium, chelators containing a quinoline ring exhibit considerable sensitivity to magnesium levels and hence have potential application for the determination of cytosolic-magnesium concentrations. Application of these chelators is illustrated by determination of the cytosolic-free calcium level in erythrocytes. Use of quinMF, the chelator with the lowest Kd value, gives a calcium value of 25-30 nM.

scholarly journals Characterization of reactive organometallic species via MicroED

2019 ◽  
Author(s):  
Christopher Jones ◽  
Matthew Asay ◽  
Lee Joon Kim ◽  
Jack Kleinsasser ◽  
Ambarneil Saha ◽  
...  
Keyword(s):  
Transition Metal ◽  
Metal Complexes ◽  
Transition Metal Complexes ◽  
Cryogenic Cooling ◽  
Structural Determination ◽  
X Ray ◽  
X Ray Crystallography ◽  
Diamagnetic Transition

Here we apply microcrystal electron diffraction (MicroED) to the structural determination of transition metal complexes. We find that the simultaneous use of 300 keV electrons, very low electron doses, and an ultra-sensitive camera allows for the collection of data without cryogenic cooling of the stage. This technique reveals the first crystal structures of the classic zirconocene hydride, colloquially known as “Schwartz’s reagent”, a novel Pd(II) complex not amenable to solution-state NMR or X-ray crystallography, and five other paramagnetic or diamagnetic transition metal complexes.

Exoglycosidase markers of diseases

2011 ◽  
Vol 39 (1) ◽  
pp. 406-409 ◽  
Author(s):  
Sylwia Chojnowska ◽  
Alina Kępka ◽  
Sławomir Dariusz Szajda ◽  
Napoleon Waszkiewicz ◽  
Marcin Bierć ◽  
...  
Keyword(s):  
Bacterial Infections ◽  
Specific Activity ◽  
Body Fluids ◽  
Biliary Cirrhosis ◽  
Sensitive Test ◽  
Lysosomal Degradation ◽  
Pathological Evaluation ◽  
Inflammatory Processes ◽  
Abuse Risk

Exoglycosidases are hydrolases involved in lysosomal degradation of oligosaccharide chains of glycoconjugates (glycoproteins, glycolipids and proteoglycans). In tissues and body fluids, a higher exoglycosidase specific activity is found in N-acetyl-β-hexosaminidase, than β-glucuronidase, α-L-fucosidase, β-galactosidase, α-mannosidase and α-glucosidase. Determination of exoglycosidases (especially N-acetyl-β-hexosaminidase and β-glucuronidase) in body fluids could be an inexpensive, easy to perform and sensitive test for pathological evaluation, as well as in screening and monitoring many diseases, including alcohol abuse, risk of arteriosclerosis, bacterial infections (e.g. Lyme borreliosis), chronic inflammatory processes, such as rheumatoid arthritis and juvenile idiopathic arthritis, asthma, autoimmune hepatitis and primary biliary cirrhosis, as well as cancers.

scholarly journals Characterization of BRPMBL, the Bleomycin Resistance Protein Associated with the Carbapenemase NDM

2017 ◽  
Vol 61 (3) ◽  
Author(s):  
Laurent Dortet ◽  
Delphine Girlich ◽  
Anne-Laure Virlouvet ◽  
Laurent Poirel ◽  
Patrice Nordmann ◽  
...  
Keyword(s):  
Specific Activity ◽  
Cloning And Expression ◽  
Cross Resistance ◽  
Dna Protection ◽  
Mobility Shift ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Resistance Protein

ABSTRACT The metallo-β-lactamase NDM-1 is among the most worrisome resistance determinants and is spreading worldwide among Gram-negative bacilli. A bleomycin resistance gene, ble MBL, downstream of the bla NDM-1 gene has been associated with resistance almost systematically. Here, we characterized the corresponding protein, BRPMBL, conferring resistance to bleomycin, an antitumoral glycopeptide molecule. We have determined whether the expression of the bla NDM-1-ble MBL operon is inducible in the presence of carbapenems and/or bleomycin-like molecules using quantitative reverse transcription-PCR (qRT-PCR), determination of imipenem and zeocin MICs, and carbapenemase-specific activity assays. We showed that the bla NDM-1-ble MBL operon is constitutively expressed. Using electrophoretic mobility shift and DNA protection assays performed with purified glutathione S-transferase (GST)-BRPMBL, we demonstrated that BRPMBL is able to bind and sequester bleomycin-like molecules, thus preventing bleomycin-dependent DNA degradation. In silico modeling confirmed that the mechanism of action required the dimerization of the BRPMBL protein in order to sequester bleomycin and prevent DNA damage. BRPMBL acts specifically on bleomycin-like molecules since cloning and expression of ble MBL in Staphyloccoccus aureus did not confer cross-resistance to any other antimicrobial glycopeptides such as vancomycin and teicoplanin.

scholarly journals Applications of Cryo-EM in small molecule and biologics drug design

2021 ◽  
Author(s):  
Joshua A. Lees ◽  
Joao M. Dias ◽  
Seungil Han
Keyword(s):  
Drug Design ◽  
Structural Characterization ◽  
Viral Vectors ◽  
Biological Macromolecules ◽  
Structure Based Drug Design ◽  
X Ray Crystallography ◽  
Technological Advances ◽  
High Resolution Analysis ◽  
Resolution Analysis

Electron cryo-microscopy (cryo-EM) is a powerful technique for the structural characterization of biological macromolecules, enabling high-resolution analysis of targets once inaccessible to structural interrogation. In recent years, pharmaceutical companies have begun to utilize cryo-EM for structure-based drug design. Structural analysis of integral membrane proteins, which comprise a large proportion of druggable targets and pose particular challenges for X-ray crystallography, by cryo-EM has enabled insights into important drug target families such as G protein-coupled receptors (GPCRs), ion channels, and solute carrier (SLCs) proteins. Structural characterization of biologics, such as vaccines, viral vectors, and gene therapy agents, has also become significantly more tractable. As a result, cryo-EM has begun to make major impacts in bringing critical therapeutics to market. In this review, we discuss recent instructive examples of impacts from cryo-EM in therapeutics design, focusing largely on its implementation at Pfizer. We also discuss the opportunities afforded by emerging technological advances in cryo-EM, and the prospects for future development of the technique.

scholarly journals Structure determination of GPCRs: cryo-EM compared with X-ray crystallography

2021 ◽  
Author(s):  
Javier García-Nafría ◽  
Christopher G. Tate
Keyword(s):  
Structural Biology ◽  
Structure Determination ◽  
Drug Targets ◽  
Cellular Systems ◽  
Single Family ◽  
Structure Based Drug Design ◽  
X Ray ◽  
Surface Receptors ◽  
X Ray Crystallography

G protein-coupled receptors (GPCRs) are the largest single family of cell surface receptors encoded by the human genome and they play pivotal roles in co-ordinating cellular systems throughout the human body, making them ideal drug targets. Structural biology has played a key role in defining how receptors are activated and signal through G proteins and β-arrestins. The application of structure-based drug design (SBDD) is now yielding novel compounds targeting GPCRs. There is thus significant interest from both academia and the pharmaceutical industry in the structural biology of GPCRs as currently only about one quarter of human non-odorant receptors have had their structure determined. Initially, all the structures were determined by X-ray crystallography, but recent advances in electron cryo-microscopy (cryo-EM) now make GPCRs tractable targets for single-particle cryo-EM with comparable resolution to X-ray crystallography. So far this year, 78% of the 99 GPCR structures deposited in the PDB (Jan–Jul 2021) were determined by cryo-EM. Cryo-EM has also opened up new possibilities in GPCR structural biology, such as determining structures of GPCRs embedded in a lipid nanodisc and multiple GPCR conformations from a single preparation. However, X-ray crystallography still has a number of advantages, particularly in the speed of determining many structures of the same receptor bound to different ligands, an essential prerequisite for effective SBDD. We will discuss the relative merits of cryo-EM and X-ray crystallography for the structure determination of GPCRs and the future potential of both techniques.

scholarly journals Structural Characterization of an Alternative Mode of Tigecycline Binding to the Bacterial Ribosome

2015 ◽  
Vol 59 (5) ◽  
pp. 2849-2854 ◽  
Author(s):  
Andreas Schedlbauer ◽  
Tatsuya Kaminishi ◽  
Borja Ochoa-Lizarralde ◽  
Neha Dhimole ◽  
Shu Zhou ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Resistance Mechanisms ◽  
Side Chain ◽  
Alternative Mode ◽  
X Ray Crystallography ◽  
Bacterial Ribosome ◽  
A Site

ABSTRACTAlthough both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in bothin vitroandin vivoactivity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to thetert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins.

What can structure tell us about in vivo function? The case of aminoglycoside-resistance genes

2003 ◽  
Vol 31 (3) ◽  
pp. 520-522 ◽  
Author(s):  
M. Vetting ◽  
S.L. Roderick ◽  
S. Hegde ◽  
S. Magnet ◽  
J.S. Blanchard
Keyword(s):  
Bacterial Infections ◽  
Ribosomal Subunit ◽  
Clinical Problem ◽  
Bactericidal Effect ◽  
Common Mechanism ◽  
Acceptor Site ◽  
Aminoglycoside Resistance ◽  
Clinical Resistance ◽  
Aminoglycoside Resistance Genes

Resistance to antibiotics used in the treatment of bacterial infections is an expanding clinical problem. Aminoglycosides, one of the oldest classes of natural product antibiotics, exert their bactericidal effect as the result of inhibiting bacterial protein synthesis by binding to the acceptor site of the 30 S ribosomal subunit. The most common mechanism of clinical resistance to aminoglycosides results from the expression of enzymes that covalently modify the aminoglycoside. We will discuss the enzymology and structure of two representative chromosomally encoded aminoglycoside N-acetyltransferases, Mycobacterium tuberculosis AAC(2´)-Ic and Salmonella enterica AAC(6´)-Iy, and speculate about their possible physiological function and substrates.

DNA and RNA: NMR studies of conformations and dynamics in solution

1987 ◽  
Vol 20 (1-2) ◽  
pp. 35-112 ◽  
Author(s):  
Dinshaw J. Patel ◽  
Lawrence Shapiro ◽  
Dennis Hare
Keyword(s):  
Aqueous Solution ◽  
Data Analysis ◽  
Nucleic Acids ◽  
Nmr Studies ◽  
Magnet Design ◽  
Dna And Rna ◽  
Qualitative Nature ◽  
Abundant Supply

The early NMR research on nucleic acids was of a qualitative nature and was restricted to partial characterization of short oligonucleotides in aqueous solution. Major advances in magnet design, spectrometer electronics, pulse techniques, data analysis and computational capabilities coupled with the availability of pure and abundant supply of long oligonucleotides have extended these studies towards the determination of the 3-D structure of nucleic acids in solution.

scholarly journals Structural characterization of the virulence factor nuclease A fromStreptococcus agalactiae

2014 ◽  
Vol 70 (11) ◽  
pp. 2937-2949 ◽  
Author(s):  
Andrea F. Moon ◽  
Philippe Gaudu ◽  
Lars C. Pedersen
Keyword(s):  
Virulence Factor ◽  
Substrate Binding ◽  
Nuclease Activity ◽  
Structure Based Drug Design ◽  
X Ray Crystallography ◽  
High Resolution Structure ◽  
Group B ◽  
Increased Activity ◽  
Dna Substrate

The group B pathogenStreptococcus agalactiaecommonly populates the human gut and urogenital tract, and is a major cause of infection-based mortality in neonatal infants and in elderly or immunocompromised adults. Nuclease A (GBS_NucA), a secreted DNA/RNA nuclease, serves as a virulence factor forS. agalactiae, facilitating bacterial evasion of the human innate immune response. GBS_NucA efficiently degrades the DNA matrix component of neutrophil extracellular traps (NETs), which attempt to kill and clear invading bacteria during the early stages of infection. In order to better understand the mechanisms of DNA substrate binding and catalysis of GBS_NucA, the high-resolution structure of a catalytically inactive mutant (H148G) was solved by X-ray crystallography. Several mutants on the surface of GBS_NucA which might influence DNA substrate binding and catalysis were generated and evaluated using an imidazole chemical rescue technique. While several of these mutants severely inhibited nuclease activity, two mutants (K146R and Q183A) exhibited significantly increased activity. These structural and biochemical studies have greatly increased our understanding of the mechanism of action of GBS_NucA in bacterial virulence and may serve as a foundation for the structure-based drug design of antibacterial compounds targeted toS. agalactiae.

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