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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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Primary and secondary structure of AaIspE and sequence alignment of A. aeolicus (Aa), E. coli (Ec) and M. tuberculosis (Mtb) orthologues. Residues conserved in all three sequences are boxed in black, those conserved in two of the three sequences are boxed in red. Three GHMP kinase superfamily conserved motifs are marked. The secondary structure of AaIspE is shown with helices as red cylinders and strands as cyan arrows. Residues marked with a star interact with substrate (yellow indicates a direct interaction, blue interactions bridged by water molecules). Thr171 is marked with a red star to indicate that it interacts with both CDPME and ADP. Residues marked with a dot interact with ADP (green indicates a direct interaction, blue water mediated interactions).
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fig07: Primary and secondary structure of AaIspE and sequence alignment of A. aeolicus (Aa), E. coli (Ec) and M. tuberculosis (Mtb) orthologues. Residues conserved in all three sequences are boxed in black, those conserved in two of the three sequences are boxed in red. Three GHMP kinase superfamily conserved motifs are marked. The secondary structure of AaIspE is shown with helices as red cylinders and strands as cyan arrows. Residues marked with a star interact with substrate (yellow indicates a direct interaction, blue interactions bridged by water molecules). Thr171 is marked with a red star to indicate that it interacts with both CDPME and ADP. Residues marked with a dot interact with ADP (green indicates a direct interaction, blue water mediated interactions).

Mentions: GHMP kinases have three conserved motifs that help create the catalytic centre [12,18,19]. In AaIspE (Fig. 7) the residues of motif 1 (Lys9 to Leu14) are on β1 of the N-terminal domain and interact with CDPME, residues of motif 2 (Ile87 to Ser98) and motif 3 (Val235 to Val242), interact with the triphosphate component of ATP (see later). Motif 3 comprises the β8–β9 loop, which does not interact with the ligands but stabilizes the conformation of motifs 1 and 2 via hydrogen-bonding contacts. For example, Ser236 OG and Gly237 N form contacts with Asn11 OD1 of motif 1, Ser236 OG interacts with Gly92 O of motif 2, whereas Ser240 N and Thr241 OG1 donate hydrogen bonds to Gly90 O and Ala91 O respectively (not shown). Motif 2 is part of the β4–α2 loop, commonly described as the phosphate binding or P-loop. The main-chain amides of this glycine-rich motif and the α3 dipole surround the negatively charged phosphates of the nucleotide. The details of interactions formed between the enzyme motifs together with additional parts of the active site and the ligands are presented after comparison with structures of IspE orthologues.


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)

Primary and secondary structure of AaIspE and sequence alignment of A. aeolicus (Aa), E. coli (Ec) and M. tuberculosis (Mtb) orthologues. Residues conserved in all three sequences are boxed in black, those conserved in two of the three sequences are boxed in red. Three GHMP kinase superfamily conserved motifs are marked. The secondary structure of AaIspE is shown with helices as red cylinders and strands as cyan arrows. Residues marked with a star interact with substrate (yellow indicates a direct interaction, blue interactions bridged by water molecules). Thr171 is marked with a red star to indicate that it interacts with both CDPME and ADP. Residues marked with a dot interact with ADP (green indicates a direct interaction, blue water mediated interactions).
© Copyright Policy
Related In: Results  -  Collection

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

fig07: Primary and secondary structure of AaIspE and sequence alignment of A. aeolicus (Aa), E. coli (Ec) and M. tuberculosis (Mtb) orthologues. Residues conserved in all three sequences are boxed in black, those conserved in two of the three sequences are boxed in red. Three GHMP kinase superfamily conserved motifs are marked. The secondary structure of AaIspE is shown with helices as red cylinders and strands as cyan arrows. Residues marked with a star interact with substrate (yellow indicates a direct interaction, blue interactions bridged by water molecules). Thr171 is marked with a red star to indicate that it interacts with both CDPME and ADP. Residues marked with a dot interact with ADP (green indicates a direct interaction, blue water mediated interactions).
Mentions: GHMP kinases have three conserved motifs that help create the catalytic centre [12,18,19]. In AaIspE (Fig. 7) the residues of motif 1 (Lys9 to Leu14) are on β1 of the N-terminal domain and interact with CDPME, residues of motif 2 (Ile87 to Ser98) and motif 3 (Val235 to Val242), interact with the triphosphate component of ATP (see later). Motif 3 comprises the β8–β9 loop, which does not interact with the ligands but stabilizes the conformation of motifs 1 and 2 via hydrogen-bonding contacts. For example, Ser236 OG and Gly237 N form contacts with Asn11 OD1 of motif 1, Ser236 OG interacts with Gly92 O of motif 2, whereas Ser240 N and Thr241 OG1 donate hydrogen bonds to Gly90 O and Ala91 O respectively (not shown). Motif 2 is part of the β4–α2 loop, commonly described as the phosphate binding or P-loop. The main-chain amides of this glycine-rich motif and the α3 dipole surround the negatively charged phosphates of the nucleotide. The details of interactions formed between the enzyme motifs together with additional parts of the active site and the ligands are presented after comparison with structures of IspE orthologues.

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