Limits...
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.

Show MeSH

Related in: MedlinePlus

Regulation of F. tularensis MEP cytidylyltransferase.A) Intrinsic fluorescence spectra of MEP cytidylyltransferase and two mutant derivatives. Wildtype and mutant (T141D and T141E) proteins were adjusted to 5 µM in 0.1 M Tris pH 7.5, 1 mM NaCl and analyzed using an excitation wavelength of 290 nm. The emission spectra was measured from 310 to 400 nm. The Em λmax of all three proteins was detected at 320 nm. Identical spectra indicate little structural change in the protein globular fold accompanies the introduction of either Asp or Glu. B) The relative activity of wildtype MEP cytidylyltransferase and the T141D and T141E mutants. Assays were performed in duplicate with 200 µM MEP and 200 µM CTP.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3111433&req=5

pone-0020884-g007: Regulation of F. tularensis MEP cytidylyltransferase.A) Intrinsic fluorescence spectra of MEP cytidylyltransferase and two mutant derivatives. Wildtype and mutant (T141D and T141E) proteins were adjusted to 5 µM in 0.1 M Tris pH 7.5, 1 mM NaCl and analyzed using an excitation wavelength of 290 nm. The emission spectra was measured from 310 to 400 nm. The Em λmax of all three proteins was detected at 320 nm. Identical spectra indicate little structural change in the protein globular fold accompanies the introduction of either Asp or Glu. B) The relative activity of wildtype MEP cytidylyltransferase and the T141D and T141E mutants. Assays were performed in duplicate with 200 µM MEP and 200 µM CTP.

Mentions: In light of the above observations, we hypothesized that phosphorylation of Thr141 would disrupt the enzyme-substrate interaction, negatively affecting enzyme activity. To test this hypothesis, two mutants of F. tularensis MEP cytidylyltransferase were created, T141D and T141E, wherein Thr141 was changed to an aspartate or glutamate, respectively, which serve to mimic a phosphothreonine. Each mutant was expressed in E. coli and purified to near homogeneity via a C-terminal His-tag. Relative to the wildtype enzyme, little difference was observed in the intrinsic fluorescence maximum (Em λmax) for either of the mutants (Fig. 7a), indicating that the introduction of the Asp or Glu has little structural influence on the protein globular fold. Size-exclusion chromatography indicates that both mutants exist as dimers in solution, as was observed with the wildtype isoform (Fig. S1). Enzyme assays using the purified proteins reveal that 75% of wildtype activity is lost with the T141D mutation, whereas the T141E mutant is completely inactive (Fig. 7b). Collectively, these results suggest that phosphorylation of Thr141 in F. tularensis MEP cytidylyltransferase inhibits the enzyme, possibly serving as a control mechanism for regulating metabolic flux through the MEP pathway (analogous to the phosphoregulation of HMG-CoA reductase [19], a kinase/phosphatase cycle may control metabolic flux by phosphoregulating the activity of MEP synthase and MEP cytidylyltransferase). Additionally, these results highlight Thr141 for the rational design of small molecule inhibitors of MEP cytidylyltransferase.


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)

Regulation of F. tularensis MEP cytidylyltransferase.A) Intrinsic fluorescence spectra of MEP cytidylyltransferase and two mutant derivatives. Wildtype and mutant (T141D and T141E) proteins were adjusted to 5 µM in 0.1 M Tris pH 7.5, 1 mM NaCl and analyzed using an excitation wavelength of 290 nm. The emission spectra was measured from 310 to 400 nm. The Em λmax of all three proteins was detected at 320 nm. Identical spectra indicate little structural change in the protein globular fold accompanies the introduction of either Asp or Glu. B) The relative activity of wildtype MEP cytidylyltransferase and the T141D and T141E mutants. Assays were performed in duplicate with 200 µM MEP and 200 µM CTP.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020884-g007: Regulation of F. tularensis MEP cytidylyltransferase.A) Intrinsic fluorescence spectra of MEP cytidylyltransferase and two mutant derivatives. Wildtype and mutant (T141D and T141E) proteins were adjusted to 5 µM in 0.1 M Tris pH 7.5, 1 mM NaCl and analyzed using an excitation wavelength of 290 nm. The emission spectra was measured from 310 to 400 nm. The Em λmax of all three proteins was detected at 320 nm. Identical spectra indicate little structural change in the protein globular fold accompanies the introduction of either Asp or Glu. B) The relative activity of wildtype MEP cytidylyltransferase and the T141D and T141E mutants. Assays were performed in duplicate with 200 µM MEP and 200 µM CTP.
Mentions: In light of the above observations, we hypothesized that phosphorylation of Thr141 would disrupt the enzyme-substrate interaction, negatively affecting enzyme activity. To test this hypothesis, two mutants of F. tularensis MEP cytidylyltransferase were created, T141D and T141E, wherein Thr141 was changed to an aspartate or glutamate, respectively, which serve to mimic a phosphothreonine. Each mutant was expressed in E. coli and purified to near homogeneity via a C-terminal His-tag. Relative to the wildtype enzyme, little difference was observed in the intrinsic fluorescence maximum (Em λmax) for either of the mutants (Fig. 7a), indicating that the introduction of the Asp or Glu has little structural influence on the protein globular fold. Size-exclusion chromatography indicates that both mutants exist as dimers in solution, as was observed with the wildtype isoform (Fig. S1). Enzyme assays using the purified proteins reveal that 75% of wildtype activity is lost with the T141D mutation, whereas the T141E mutant is completely inactive (Fig. 7b). Collectively, these results suggest that phosphorylation of Thr141 in F. tularensis MEP cytidylyltransferase inhibits the enzyme, possibly serving as a control mechanism for regulating metabolic flux through the MEP pathway (analogous to the phosphoregulation of HMG-CoA reductase [19], a kinase/phosphatase cycle may control metabolic flux by phosphoregulating the activity of MEP synthase and MEP cytidylyltransferase). Additionally, these results highlight Thr141 for the rational design of small molecule inhibitors of MEP cytidylyltransferase.

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