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

Tsang A, Seidle H, Jawaid S, Zhou W, Smith C, Couch RD - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.

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.

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Purification of recombinant F. tularensis MEP cytidylyltransferase.A) Coomassie stained SDS-PAGE showing a molecular weight marker (MW) and purified His-tagged MEP cytidylyltransferase. His-tagged MEP cytidylyltransferase has a predicted molecular weight of 29.5 kDa. B) Western blot hybridization analysis of purified MEP synthase using an anti-His antibody results in an intense band of the expected size.
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pone-0020884-g002: Purification of recombinant F. tularensis MEP cytidylyltransferase.A) Coomassie stained SDS-PAGE showing a molecular weight marker (MW) and purified His-tagged MEP cytidylyltransferase. His-tagged MEP cytidylyltransferase has a predicted molecular weight of 29.5 kDa. B) Western blot hybridization analysis of purified MEP synthase using an anti-His antibody results in an intense band of the expected size.

Mentions: The 690 bp ispD gene (FTL_1525) from F. tularensis subsp. holarctica LVS encodes a polypeptide of 229 amino acids with a calculated molecular mass of 25.9 kDa. The subsp. holarctica amino acid sequence shares 99.1, 98.3, and 99.6% identity with the MEP cytidylyltransferase sequence from subsp. tularensis (FTT_0711), subsp. novicida (FTN_0623), and subsp. mediasiatica (FTM_1371), respectively. PCR was used to amplify subsp. holarctica ispD and the resulting product was cloned into an expression plasmid designed to express a C-terminal His-tagged protein in E. coli. The recombinant protein was purified by immobilized metal affinity chromatography and the protein was visualized by SDS-PAGE and Western blotting using an anti-His antibody (Fig. 2). Size-exclusion chromatography using a calibrated column indicates that the F. tularensis MEP cytidylyltransferase exists in solution as a dimer (Fig. S1), similar to the MEP cytidylyltransferase from Mycobacterium tuberculosis [13] and E. coli [14], [15].


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

Tsang A, Seidle H, Jawaid S, Zhou W, Smith C, Couch RD - PLoS ONE (2011)

Purification of recombinant F. tularensis MEP cytidylyltransferase.A) Coomassie stained SDS-PAGE showing a molecular weight marker (MW) and purified His-tagged MEP cytidylyltransferase. His-tagged MEP cytidylyltransferase has a predicted molecular weight of 29.5 kDa. B) Western blot hybridization analysis of purified MEP synthase using an anti-His antibody results in an intense band of the expected size.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3111433&req=5

pone-0020884-g002: Purification of recombinant F. tularensis MEP cytidylyltransferase.A) Coomassie stained SDS-PAGE showing a molecular weight marker (MW) and purified His-tagged MEP cytidylyltransferase. His-tagged MEP cytidylyltransferase has a predicted molecular weight of 29.5 kDa. B) Western blot hybridization analysis of purified MEP synthase using an anti-His antibody results in an intense band of the expected size.
Mentions: The 690 bp ispD gene (FTL_1525) from F. tularensis subsp. holarctica LVS encodes a polypeptide of 229 amino acids with a calculated molecular mass of 25.9 kDa. The subsp. holarctica amino acid sequence shares 99.1, 98.3, and 99.6% identity with the MEP cytidylyltransferase sequence from subsp. tularensis (FTT_0711), subsp. novicida (FTN_0623), and subsp. mediasiatica (FTM_1371), respectively. PCR was used to amplify subsp. holarctica ispD and the resulting product was cloned into an expression plasmid designed to express a C-terminal His-tagged protein in E. coli. The recombinant protein was purified by immobilized metal affinity chromatography and the protein was visualized by SDS-PAGE and Western blotting using an anti-His antibody (Fig. 2). Size-exclusion chromatography using a calibrated column indicates that the F. tularensis MEP cytidylyltransferase exists in solution as a dimer (Fig. S1), similar to the MEP cytidylyltransferase from Mycobacterium tuberculosis [13] and E. coli [14], [15].

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia, United States of America.

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.

Show MeSH
Related in: MedlinePlus