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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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The divalent cation specificity of F. tularensis MEP cytidylyltransferase.Enzyme assays were performed with the indicated divalent cations at a fixed MEP (200 µM) and CTP (200 µM) concentration. Relative enzyme activity reveals the strict preference of the enzyme for Mg+2. Each assay was performed in duplicate.
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pone-0020884-g004: The divalent cation specificity of F. tularensis MEP cytidylyltransferase.Enzyme assays were performed with the indicated divalent cations at a fixed MEP (200 µM) and CTP (200 µM) concentration. Relative enzyme activity reveals the strict preference of the enzyme for Mg+2. Each assay was performed in duplicate.

Mentions: The kinetic activity of purified MEP cytidylyltransferase was spectrophotometrically evaluated by monitoring the substrate dependent enzyme catalyzed production of pyrophosphate (Fig. 1B). Nonlinear regression fitting of enzyme velocity versus substrate concentration was used to determine the apparent kinetic constants (Fig. 3 and Table 1). The for MEP was obtained using assays performed with a saturating concentration of CTP (200 µM), whereas the for CTP was determined using assays with saturating levels of MEP (400 µM). Overall, the for the F. tularensis MEP cytidylyltransferase is similar to the reported values for the E. coli and M. tuberculosis orthologs, when assayed accordingly (Table 1), while the is considerably larger for the F. tularensis enzyme. Additionally, the catalytic turnover of the E. coli and F. tularensis enzymes are comparable, while each are slightly depressed relative to the M. tuberculosis ortholog (Table 1). Combined with differences in apparent values, the disparity between the turnover numbers results in considerably different specificity constants for the F. tularensis and M. tuberculosis enzymes. The biological significance of this remains unclear. Interestingly, cation specificity assays reveal a strict preference of the F. tularensis MEP cytidylyltransferase for MgCl2 (Fig. 4), unlike the E. coli and Mycobacterium orthologs which also accommodate Mn+2 or Co+2 [13], [14]. Additionally, the F. tularensis MEP cytidylyltransferase displays a clear preference for CTP over alternative nucleotides (Fig. 5).


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)

The divalent cation specificity of F. tularensis MEP cytidylyltransferase.Enzyme assays were performed with the indicated divalent cations at a fixed MEP (200 µM) and CTP (200 µM) concentration. Relative enzyme activity reveals the strict preference of the enzyme for Mg+2. Each assay was performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020884-g004: The divalent cation specificity of F. tularensis MEP cytidylyltransferase.Enzyme assays were performed with the indicated divalent cations at a fixed MEP (200 µM) and CTP (200 µM) concentration. Relative enzyme activity reveals the strict preference of the enzyme for Mg+2. Each assay was performed in duplicate.
Mentions: The kinetic activity of purified MEP cytidylyltransferase was spectrophotometrically evaluated by monitoring the substrate dependent enzyme catalyzed production of pyrophosphate (Fig. 1B). Nonlinear regression fitting of enzyme velocity versus substrate concentration was used to determine the apparent kinetic constants (Fig. 3 and Table 1). The for MEP was obtained using assays performed with a saturating concentration of CTP (200 µM), whereas the for CTP was determined using assays with saturating levels of MEP (400 µM). Overall, the for the F. tularensis MEP cytidylyltransferase is similar to the reported values for the E. coli and M. tuberculosis orthologs, when assayed accordingly (Table 1), while the is considerably larger for the F. tularensis enzyme. Additionally, the catalytic turnover of the E. coli and F. tularensis enzymes are comparable, while each are slightly depressed relative to the M. tuberculosis ortholog (Table 1). Combined with differences in apparent values, the disparity between the turnover numbers results in considerably different specificity constants for the F. tularensis and M. tuberculosis enzymes. The biological significance of this remains unclear. Interestingly, cation specificity assays reveal a strict preference of the F. tularensis MEP cytidylyltransferase for MgCl2 (Fig. 4), unlike the E. coli and Mycobacterium orthologs which also accommodate Mn+2 or Co+2 [13], [14]. Additionally, the F. tularensis MEP cytidylyltransferase displays a clear preference for CTP over alternative nucleotides (Fig. 5).

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