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Targeting the ESKAPE pathogens: Kinetic characterization and inhibition of Acinetobacter baumannii and Klebsiella pneumoniae 1‐Deoxy‐D‐Xylulose 5‐Phosphate Reductoisomerase
Author(s) -
Ball Haley,
Girma Misgina,
Zainab Mosufa,
Riley Nora,
Soojhawon Iswarduth,
Dowd Cynthia,
Noble Schroeder,
Couch Robin
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.470.2
Subject(s) - acinetobacter baumannii , klebsiella pneumoniae , microbiology and biotechnology , antibiotics , bacteria , enzyme , biology , chemistry , biochemistry , escherichia coli , gene , pseudomonas aeruginosa , genetics
The ESKAPE pathogens comprise a group of multidrug resistant bacteria that are the leading cause of nosocomial infections worldwide. The prevalence of antibiotic resistant strains and the relative ease by which bacteria acquire resistant genes highlights the continual need for the development of novel antibiotics against new targets. The methylerythritol phosphate (MEP) pathway is an attractive target for the development of new antimicrobial drugs. The MEP pathway governs the synthesis of isoprenoids, which are key lipid precursors for vital cell components such ubiquinone and bacteriohopanoids. Additionally, the MEP pathway is entirely distinct from the corresponding mammalian pathway, the mevalonic acid (MVA) pathway. Therefore, the first committed enzyme of the MEP pathway, 1‐Deoxy‐D‐Xylulose 5‐Phosphate Reductoisomerase (IspC), is an attractive target for antibiotic development. To facilitate drug development against two of the ESKAPE pathogens, Acinetobacter baumannii and Klebsiella pneumoniae , we cloned, expressed, purified, and characterized IspC from A. baumannii and K. pneumoniae . We have determined the apparent kinetic constants for A. baumannii and K. pneumoniae IspC, as well as the IC 50 values for two natural IspC inhibitors, fosmidomycin and FR900098, along with a suite of rationally designed analogs of fosmidomycin and FR900098. Additionally, minimum inhibitory concentrations (MICs) for fosmidomycin and FR900098 suggest that IspC is an excellent target for the development of novel inhibitors of A. baumannii and K. pneumoniae . Support or Funding Information Financial support was provided the U.S. Army Medical Research and Materiel Command (W81XWH‐17‐C‐0066 to RDC).Coomassie stained SDS‐PAGE shows two lanes of purified His‐tagged AbIspC and KpIspC alongside a molecular weight marker (MW). A. baumannii IspC is 398 aa in length; the His‐tagged enzyme has a predicted molecular weight of 44.7 kDa. K. pneumoniae IspC is 400 aa in length; the His‐tagged enzyme has a predicted molecular weight of 45.4 kDa.Protein crystallization conditions: 8.4 mg/mL AbIspC + 10% DMSO, 1:1 drop ratio, 16°C, 0.1 M sodium citrate: HCl pH 5.60, 0.15 M ammonium sulfate, 18% PEG 4000.Cation specificity of A. baumannii and K. pneumoniae IspC. Enzyme assays were performed with fixed NADPH (150 μM), DXP (400 μM), and divalent cation (25 mM) concentration. IspC from both organisms preferentially utilizes Mg 2+ . Assays were performed in duplicate.The substrate dependent catalytic activity of A. baumannii and K. pneumoniae IspC. Shown are Michaelis‐Menten plots of reaction velocity as a function of A) DXP concentration and B) NADPH concentration. Least‐squares best fit of the data to the Michaelis‐Menten equation produces the kinetic parameters displayed on the figures. The R2 value for each plot is indicated. Assays were performed in duplicate.Dose‐dependent inhibition of A. baumannii IspC. IC50 values were determined using A) fosmidomycin or B) FR900098. The R2 value for each plot is indicated. Assays were performed in duplicate.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .