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Characterization of Aquifex aeolicus 4-diphosphocytidyl-2 C-methyl-d-erythritol kinase – ligand recognition in a template for antimicrobial drug discovery.
- Source :
- FEBS Journal; Jun2008, Vol. 275 Issue 11, p2779-2794, 16p, 8 Diagrams, 4 Charts, 4 Graphs
- Publication Year :
- 2008
-
Abstract
- 4-Diphosphocytidyl-2 C-methyl-d-erythritol kinase (IspE) catalyses the ATP-dependent conversion of 4-diphosphocytidyl-2 C-methyl-d-erythritol (CDPME) to 4-diphosphocytidyl-2 C-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 α-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 α/β 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 1742464X
- Volume :
- 275
- Issue :
- 11
- Database :
- Complementary Index
- Journal :
- FEBS Journal
- Publication Type :
- Academic Journal
- Accession number :
- 32000772
- Full Text :
- https://doi.org/10.1111/j.1742-4658.2008.06418.x