Hana McFeeters

University of Alabama in Huntsville, Huntsville, Alabama, United States

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Publications (8)8.04 Total impact

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    ABSTRACT: Peptidyl-tRNA hydrolase (Pth; EC 3.1.1.29) from the pathogenic bacterium Salmonella typhimurium has been cloned, expressed in Escherichia coli and crystallized for X-ray analysis. Crystals were grown using hanging-drop vapor diffusion against a reservoir solution consisting of 0.03 M citric acid, 0.05 M bis-tris propane, 1% glycerol, 3% sucrose, 25% PEG 6000 pH 7.6. Crystals were used to obtain the three-dimensional structure of the native protein at 1.6 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P212121 with unit-cell parameters a = 62.1, b = 64.9, c = 110.5 Å, α = β = γ = 90°. The asymmetric unit of the crystallographic lattice was composed of two copies of the enzyme molecule with a 51% solvent fraction, corresponding to a Matthews coefficient of 2.02 Å(3) Da(-1). The structural coordinates reported serve as a foundation for computational and structure-guided efforts towards novel small-molecule Pth1 inhibitors and potential antibacterial development.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 07/2014; 70(Pt 7):872-7. · 0.55 Impact Factor
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    ABSTRACT: Peptidyl-tRNA hydrolase 1 cleaves the ester bond of peptidyl-tRNA thereby recycling peptidyl-tRNAs generated from premature termination of translation and expression of minigenes and short ORFs. Bacterial Pth1 is essential, highly conserved, and has no essential eukaryotic homolog making it a good target for antibacterial development. Herein we describe the cloning of pth1 gene from Bacillus cereus as an N-terminal hexahistidine fusion protein. Solubility was optimized for overexpression in E. coli. Purity greater than 95% was achieved in one chromatography step. Yields greater than 12 mg of purified Pth1 per liter of minimal media were achieved and buffer conditions for long-term solubility were determined. Enzymatic activity of Pth1 from B. cereus was confirmed and quantification of Michaelis-Menten parameters reported.
    Protein Expression and Purification 01/2014; · 1.43 Impact Factor
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    ABSTRACT: Bacterial Pth1 is essential for viability. Pth1 cleaves the ester bond between the peptide and nucleotide of peptidyl-tRNA generated from aborted translation, expression of mini-genes, and short ORFs. We have determined the shape of the Pth1:peptidyl-tRNA complex using small angle neutron scattering. Binding of piperonylpiperazine, a small molecule constituent of a combinatorial synthetic library common to most compounds with inhibitory activity, was mapped to Pth1 via NMR spectroscopy. We also report computational docking results, modeling piperonylpiperazine binding based on chemical shift perturbation mapping. Overall these studies promote Pth1 as a novel antibiotic target, contribute to understanding how Pth1 interacts with its substrate, advance the current model for cleavage, and demonstrate feasibility of small molecule inhibition.
    International Journal of Molecular Sciences 01/2013; 14(11):22741-22752. · 2.46 Impact Factor
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    ABSTRACT: The peptidyl-tRNA hydrolase enzyme from the pathogenic bacterium Pseudomonas aeruginosa (Pth; EC 3.1.1.29) has been cloned, expressed in Escherichia coli and crystallized for X-ray structural analysis. Suitable crystals were grown using the sitting-drop vapour-diffusion method after one week of incubation against a reservoir solution consisting of 20% polyethylene glycol 4000, 100 mM Tris pH 7.5, 10%(v/v) isopropyl alcohol. The crystals were used to obtain the three-dimensional structure of the native protein at 1.77 Å resolution. The structure was determined by molecular replacement of the crystallographic data processed in space group P6(1)22 with unit-cell parameters a = b = 63.62, c = 155.20 Å, α = β = 90, γ = 120°. The asymmetric unit of the crystallographic lattice was composed of a single copy of the enzyme molecule with a 43% solvent fraction, corresponding to a Matthews coefficient of 2.43 Å(3) Da(-1). The crystallographic structure reported here will serve as the foundation for future structure-guided efforts towards the development of novel small-molecule inhibitors specific to bacterial Pths.
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 12/2012; 68(Pt 12):1472-6. · 0.55 Impact Factor
  • Agriculture. 12/2012; 2(3):154-164.
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    ABSTRACT: Peptidyl-tRNA Hydrolase (Pth) is a highly conserved, essential enzyme in bacteria. It removes the peptide portion from peptidyl-tRNA, returning free tRNAs to participate in translation. Build-up of peptidyl-tRNAs is toxic and defects in Pth function result in cell death. Herein we use in vitro activity of recombinant E. coli Pth to screen tropical plant extracts for inhibition. Multiple extracts were found to have inhibitory activity with some exhibiting different inhibitory effects depending on extraction conditions. IC50 values ranged from 0.02 to > 53.8 microg of extract per 1 unit of Pth, holding promise for in vivo screening. The inhibitory components in these extracts may serve as lead compounds for development of novel antibacterials.
    Natural product communications 08/2012; 7(8):1107-10. · 0.96 Impact Factor
  • H. McFeeters, R. L. McFeeters
    International Journal of Modern Physics A 01/2012; 2(5):127-144. · 1.13 Impact Factor
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    ABSTRACT: With the rapid rise of antibiotic resistance in pathogenic bacteria, the need for new antibacterial agents is overwhelming. Herein we report the limited screening of tropical plant extracts for inhibitory activity against the essential enzyme peptidyl-tRNA hydrolase (Pth). Initial screening was conducted through an electrophoretic mobility assay and Northern blot detection. The ability of Pth to cleave the peptide-tRNA ester bond was assessed. The ethanol bark extract of Syzygium johnsonii showed strong inhibitory potential. Molecular docking studies point to Syzygium polyphenolics as the potential source of inhibition. This work is the forerunner of activity-directed isolation, purification, and structure elucidation of the inhibitory components from Syzygium johnsonii extracts and studies of compound interaction with Pth.
    Natural product communications 10/2011; 6(10):1421-4. · 0.96 Impact Factor