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Characterization of Aquifex aeolicus 4-diphosphocytidyl-2C-methyl-d-erythritol kinase - ligand recognition in a template for antimicrobial drug discovery.

Sgraja T, Alphey MS, Ghilagaber S, Marquez R, Robertson MN, Hemmings JL, Lauw S, Rohdich F, Bacher A, Eisenreich W, Illarionova V, Hunter WN - FEBS J. (2008)

Bottom Line: The enzyme displays the characteristic alpha/beta galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains.Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism.The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, University of Dundee, UK.

ABSTRACT
4-Diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP alpha-phosphate not the binding site for the methyl-D-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic alpha/beta galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.

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Mg2+ and Mn2+ dependence. The optimum concentration of both metals is 2 mm.
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fig04: Mg2+ and Mn2+ dependence. The optimum concentration of both metals is 2 mm.

Mentions: The catalytic activity of the recombinant IspE protein was measured by 13C NMR spectroscopy and photometry. The 13C NMR measurement was performed using the multiply 13C-labelled substrate [1,3,4-13C3]CDPME to enhance the sensitivity and selectivity of 13C observation. 13C NMR signals detected in a typical NMR assay are shown in Fig. 2. The chemical shifts as well as the 13C13C-coupling constants were in excellent agreement with the published data on CDPME [15] and CDPME2P [9]. Optimal activity was identified at pH 8.5 and ∼ 60 °C (Fig. 3). AaIspE was catalytically active in the presence of various metal ions (Table 1), and a highest rate of 1.5 μmol·min−1·mg−1 was achieved in the presence of 2 mm Mg2+ or Mn2+ (Fig. 4). The photometric assay was performed according to a published procedure [8] using auxiliary enzymes to observe the decrease in NADH absorption at 340 nm. The KM values for CDPME and ATP were determined as 121 and 222 μm, respectively (Table 2, Fig. 5). The data are similar to the kinetic properties reported for IspE from E. coli [16] and tomato [17], respectively.


Characterization of Aquifex aeolicus 4-diphosphocytidyl-2C-methyl-d-erythritol kinase - ligand recognition in a template for antimicrobial drug discovery.

Sgraja T, Alphey MS, Ghilagaber S, Marquez R, Robertson MN, Hemmings JL, Lauw S, Rohdich F, Bacher A, Eisenreich W, Illarionova V, Hunter WN - FEBS J. (2008)

Mg2+ and Mn2+ dependence. The optimum concentration of both metals is 2 mm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2655357&req=5

fig04: Mg2+ and Mn2+ dependence. The optimum concentration of both metals is 2 mm.
Mentions: The catalytic activity of the recombinant IspE protein was measured by 13C NMR spectroscopy and photometry. The 13C NMR measurement was performed using the multiply 13C-labelled substrate [1,3,4-13C3]CDPME to enhance the sensitivity and selectivity of 13C observation. 13C NMR signals detected in a typical NMR assay are shown in Fig. 2. The chemical shifts as well as the 13C13C-coupling constants were in excellent agreement with the published data on CDPME [15] and CDPME2P [9]. Optimal activity was identified at pH 8.5 and ∼ 60 °C (Fig. 3). AaIspE was catalytically active in the presence of various metal ions (Table 1), and a highest rate of 1.5 μmol·min−1·mg−1 was achieved in the presence of 2 mm Mg2+ or Mn2+ (Fig. 4). The photometric assay was performed according to a published procedure [8] using auxiliary enzymes to observe the decrease in NADH absorption at 340 nm. The KM values for CDPME and ATP were determined as 121 and 222 μm, respectively (Table 2, Fig. 5). The data are similar to the kinetic properties reported for IspE from E. coli [16] and tomato [17], respectively.

Bottom Line: The enzyme displays the characteristic alpha/beta galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains.Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism.The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Chemistry and Drug Discovery, University of Dundee, UK.

ABSTRACT
4-Diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2C-methyl-D-erythritol (CDPME) to 4-diphosphocytidyl-2C-methyl-d-erythritol 2-phosphate with the release of ADP. This reaction occurs in the non-mevalonate pathway of isoprenoid precursor biosynthesis and because it is essential in important microbial pathogens and absent from mammals it represents a potential target for anti-infective drugs. We set out to characterize the biochemical properties, determinants of molecular recognition and reactivity of IspE and report the cloning and purification of recombinant Aquifex aeolicus IspE (AaIspE), kinetic data, metal ion, temperature and pH dependence, crystallization and structure determination of the enzyme in complex with CDP, CDPME and ADP. In addition, 4-fluoro-3,5-dihydroxy-4-methylpent-1-enylphosphonic acid (compound 1) was designed to mimic a fragment of the substrate, a synthetic route to 1 was elucidated and the complex structure determined. Surprisingly, this ligand occupies the binding site for the ATP alpha-phosphate not the binding site for the methyl-D-erythritol moiety of CDPME. Gel filtration and analytical ultracentrifugation indicate that AaIspE is a monomer in solution. The enzyme displays the characteristic alpha/beta galacto-homoserine-mevalonate-phosphomevalonate kinase fold, with the catalytic centre positioned in a deep cleft between the ATP- and CDPME-binding domains. Comparisons indicate a high degree of sequence conservation on the IspE active site across bacterial species, similarities in structure, specificity of substrate recognition and mechanism. The biochemical characterization, attainment of well-ordered and reproducible crystals and the models resulting from the analyses provide reagents and templates to support the structure-based design of broad-spectrum antimicrobial agents.

Show MeSH
Related in: MedlinePlus