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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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Stereo-view Cα overlay of AaIspE and EcIspE. AaIspE is shown in red with EcIspE in black. Also depicted are the ADP (gold) and CDPME (green) from the AaIspE complex I. The positions of α5 and α7 are marked.
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fig08: Stereo-view Cα overlay of AaIspE and EcIspE. AaIspE is shown in red with EcIspE in black. Also depicted are the ADP (gold) and CDPME (green) from the AaIspE complex I. The positions of α5 and α7 are marked.

Mentions: AaIspE shares the highest sequence identity with TtIspE (32%) and slightly less with EcIspE (30%). The alignment of EcIspE with AaIspE in shown in Fig. 7. The DALI server [20] identified EcIspE (Z-score: 29.1) and TtIspE (Z-score: 27.7) as most similar to AaIspE. (The Z-score is a measure of the statistical significance of the best subunit–subunit alignment and was determined by DALI. Typically, dissimilar proteins will have a Z-score < 2.0.) Cα superpositions of EcIspE and TtIspE with AaIspE give r.m.s.d values of 1.6 and 1.8 Å, respectively, which mainly correspond to differences in the C-terminal domain distant from the active site. An overlay of AaIspE and EcIspE structures is depicted in Fig. 8. In general, the fold and secondary structure are well conserved. With respect to AaIspE, α5 and α7 are situated at different positions in EcIspE (Fig. 8) and TtIspE structures, and an additional helix following β10 is inserted in EcIspE. The two structures are further decorated with a 310 helix following α7 or α4 respectively (not shown). The structural comparison and alignment of AaIspE with EcIspE reveals a high degree of conservation within the active site (Fig. 8). Alignment of the Mycobacterium tuberculosis IspE (MtbIspE) sequence with those of AaIspE and EcIspE indicates sequence identities of around 25% (Fig. 7). This comparison is detailed later.


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)

Stereo-view Cα overlay of AaIspE and EcIspE. AaIspE is shown in red with EcIspE in black. Also depicted are the ADP (gold) and CDPME (green) from the AaIspE complex I. The positions of α5 and α7 are marked.
© Copyright Policy
Related In: Results  -  Collection

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

fig08: Stereo-view Cα overlay of AaIspE and EcIspE. AaIspE is shown in red with EcIspE in black. Also depicted are the ADP (gold) and CDPME (green) from the AaIspE complex I. The positions of α5 and α7 are marked.
Mentions: AaIspE shares the highest sequence identity with TtIspE (32%) and slightly less with EcIspE (30%). The alignment of EcIspE with AaIspE in shown in Fig. 7. The DALI server [20] identified EcIspE (Z-score: 29.1) and TtIspE (Z-score: 27.7) as most similar to AaIspE. (The Z-score is a measure of the statistical significance of the best subunit–subunit alignment and was determined by DALI. Typically, dissimilar proteins will have a Z-score < 2.0.) Cα superpositions of EcIspE and TtIspE with AaIspE give r.m.s.d values of 1.6 and 1.8 Å, respectively, which mainly correspond to differences in the C-terminal domain distant from the active site. An overlay of AaIspE and EcIspE structures is depicted in Fig. 8. In general, the fold and secondary structure are well conserved. With respect to AaIspE, α5 and α7 are situated at different positions in EcIspE (Fig. 8) and TtIspE structures, and an additional helix following β10 is inserted in EcIspE. The two structures are further decorated with a 310 helix following α7 or α4 respectively (not shown). The structural comparison and alignment of AaIspE with EcIspE reveals a high degree of conservation within the active site (Fig. 8). Alignment of the Mycobacterium tuberculosis IspE (MtbIspE) sequence with those of AaIspE and EcIspE indicates sequence identities of around 25% (Fig. 7). This comparison is detailed later.

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