title: Francisella tularensis 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase: kinetic characterization and phosphoregulation.
abstract: Deliberate and natural outbreaks of infectious disease, the prevalence of antibiotic resistant strains, and the ease by which antibiotic resistant bacteria can be intentionally engineered all underscore the necessity of effective vaccines and continued development of novel antimicrobial/antiviral therapeutics. Isoprenes, a group of molecules fundamentally involved in a variety of crucial biological functions, are derived from either the mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathway. While mammals utilize the MVA pathway, many bacteria utilize the MEP pathway, highlighting the latter as an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP cytidylyltransferase, a MEP pathway enzyme and potential target for antibiotic development. Size exclusion chromatography indicates the protein exists as a dimer in solution. Enzyme assays produced an apparentK(MEP)(M) = 178 μM, K(CTP)(M) = 73 μM , k(MEP)(cat) = 1(s-1), k(CTP)(cat) = 0.8( s-1), and a k(MEP)(cat)/ K(MEP)(M) = 3.4 x 10(5) M(-1) min(-1). The enzyme exhibits a strict preference for Mg(+2) as a divalent cation and CTP as the nucleotide. Titanium dioxide chromatography-tandem mass spectrometry identified Thr141 as a site of phosphorylation. T141D and T141E site-directed mutants are catalytically inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP cytidylyltransferase is an excellent target for the development of novel antibiotics against F. tularensis.
authors.affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.
authors.forename: Arthur
authors.initials: A
authors.lastname: Tsang
authors.forename: Heather
authors.initials: H
authors.lastname: Seidle
authors.forename: Safdar
authors.initials: S
authors.lastname: Jawaid
authors.forename: Weidong
authors.initials: W
authors.lastname: Zhou
authors.forename: Clint
authors.initials: C
authors.lastname: Smith
authors.forename: Robin D
authors.initials: RD
authors.lastname: Couch
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001665:Binding Sites; D003001:Cloning, Molecular; D055808:Drug Discovery; D005604:Francisella tularensis / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D015203:Reproducibility of Results; D013379:Substrate Specificity
chemical_list: D000900:Anti-Bacterial Agents; D009711:Nucleotides; C435808:2-C-methyl erythritol 4-phosphate cytidylyltransferase; D009713:Nucleotidyltransferases