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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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Related in: MedlinePlus

Ribbon diagram of AaIspE. α and 310 helices are red and β strands are cyan. Secondary structure elements have been labelled, N- and C-termini are marked. ADP is gold, the substrate CDPME green.
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fig06: Ribbon diagram of AaIspE. α and 310 helices are red and β strands are cyan. Secondary structure elements have been labelled, N- and C-termini are marked. ADP is gold, the substrate CDPME green.

Mentions: AaIspE displays the typical GHMP kinase fold that comprises two domains (Fig. 6) [13]. The N-terminal domain (residues 1–155) consists of an elongated six-stranded β sheet (β1–β6). In addition, the concave side of the β sheet is flanked by three α helices (α1–α3) and two 310 helices (θ1 and θ2). The C-terminal domain comprises an antiparallel four-stranded β sheet (β7–β10), bordering β1 and β2 of the N-terminal domain, four α helices (α4–α7) and two 310 helices (θ3 and θ4).


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)

Ribbon diagram of AaIspE. α and 310 helices are red and β strands are cyan. Secondary structure elements have been labelled, N- and C-termini are marked. ADP is gold, the substrate CDPME green.
© Copyright Policy
Related In: Results  -  Collection

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

fig06: Ribbon diagram of AaIspE. α and 310 helices are red and β strands are cyan. Secondary structure elements have been labelled, N- and C-termini are marked. ADP is gold, the substrate CDPME green.
Mentions: AaIspE displays the typical GHMP kinase fold that comprises two domains (Fig. 6) [13]. The N-terminal domain (residues 1–155) consists of an elongated six-stranded β sheet (β1–β6). In addition, the concave side of the β sheet is flanked by three α helices (α1–α3) and two 310 helices (θ1 and θ2). The C-terminal domain comprises an antiparallel four-stranded β sheet (β7–β10), bordering β1 and β2 of the N-terminal domain, four α helices (α4–α7) and two 310 helices (θ3 and θ4).

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