Francisella tularensis 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase: kinetic characterization and phosphoregulation

Autor: Clint B. Smith, Heather Seidle, Arthur Tsang, Robin D. Couch, Safdar Jawaid, Weidong Zhou
Rok vydání: 2011
Předmět:
Models
Molecular
Cytidylyltransferase
Mutant
Enzyme Metabolism
lcsh:Medicine
Biochemistry
Substrate Specificity
chemistry.chemical_compound
Drug Discovery
Nucleotide
Cloning
Molecular
Phosphorylation
lcsh:Science
Francisella tularensis
chemistry.chemical_classification
Chromatography
Multidisciplinary
biology
Nucleotides
Vaccination
Chemical Reactions
Nucleotidyltransferases
Anti-Bacterial Agents
Enzymes
Chemistry
Medicine
Metabolic Pathways
Research Article
Biotechnology
Drugs and Devices
Drug Research and Development
Mevalonic acid
Biosynthesis
Catalysis
Microbiology
Enzyme Regulation
Vaccine Development
Binding site
Protein Structure
Quaternary
Biology
Enzyme Kinetics
Size Exclusion Chromatography
Binding Sites
lcsh:R
Immunity
Reproducibility of Results
biology.organism_classification
Kinetics
Enzyme
Metabolism
chemistry
Biocatalysis
lcsh:Q
Clinical Immunology
Bacteria
Zdroj: PLoS ONE
PLoS ONE, Vol 6, Iss 6, p e20884 (2011)
ISSN: 1932-6203
Popis: 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.
Databáze: OpenAIRE
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