Biosynthesis of isoprenoids: crystal structure of 4-diphosphocytidyl-2C-methyl-D-erythritol kinase

Abstract

4-Diphosphocytidyl-2C-methyl-d-erythritol kinase, an essential enzyme in the nonmevalonate pathway of isopentenyl diphosphate and dimethylallyl diphosphate biosynthesis, catalyzes the single ATP-dependent phosphorylation stage affording 4-diphosphocytidyl-2C-methyl-d-erythritol-2-phosphate. The 2-A resolution crystal structure of the Escherichia coli enzyme in a ternary complex with substrate and a nonhydrolyzable ATP analogue reveals the molecular determinants of specificity and catalysis. The enzyme subunit displays the alpha/beta fold characteristic of the galactose kinase/homoserine kinase/mevalonate kinase/phosphomevalonate kinase superfamily, arranged into cofactor and substrate-binding domains with the catalytic center positioned in a deep cleft between domains. Comparisons with related members of this superfamily indicate that the core regions of each domain are conserved, whereas there are significant differences in the substrate-binding pockets. The nonmevalonate pathway is essential in many microbial pathogens and distinct from the mevalonate pathway used by mammals. The high degree of sequence conservation of the enzyme across bacterial species suggests similarities in structure, specificity, and mechanism. Our model therefore provides an accurate template to facilitate the structure-based design of broad-spectrum antimicrobial agents.

Fig. 1.

The reaction catalyzed by CDP-ME kinase.

Fig. 2.

The CDP-ME kinase dimer. (Left) Ribbon diagram viewed parallel to the NCS 2-fold. The residues shown as red or blue balls and sticks (Arg-21, Ala-22, Asp-80, and Gly-87) contribute to dimer formation through electrostatic interactions. Substrate and cofactor are shown as black balls and sticks. (Right) Surface representation is colored blue for basic and red for acidic residues. Ligands are depicted as black spheres.

Fig. 3.

Sequence and structure of E. coli CDP-ME kinase. (Upper) Stereoview ribbon diagram of a monomer (β-strand numbers are black, and α-helix numbers blue). Strands and helices in the cofactor-binding domain are red and yellow, respectively. Strands associated with the two β-sheets in the substrate-binding domain are light and dark green, and the helices are orange. Substrate and AMP-PNP are depicted as black ball-and-stick models. N and C identify the amino and carboxyl termini. (Lower) Amino acid sequence of E. coli CDP-ME kinase with elements of secondary structure assigned and colored according to Upper. Residues strictly conserved or highly homologous in enzymes from S. typhi, Vibrio cholera, Haemophilus influenzae, Pseudomonas aeruginosa, Neisseria meningitidis, M. tuberculosis, Chlamydia trachomatis, and Clostridium perfringens are black on a purple background; when conserved in at least six of the sequences they are enclosed in a purple box. Sequences were retrieved from expasy (www.expasy.ch) and aligned (clustalw, ref. 63). ▵ identify residues interacting with the substrate using side chains; ▴ are for those using main chain. His-26 uses both. θ1 and θ2 identify 3₁₀-helices, and three red lines mark motifs I, II, and III.

Fig. 4.

Stereoview of the active site. Bonds in AMP-PNP are black, bonds in the protein are yellow, and bonds in CDP-ME are gray. Selected hydrogen-bonding interactions are shown as green dashed lines. Water molecules have been omitted; Gly-101 and Gly-103 are semitransparent for the purpose of clarity.

Fig. 5.

The proposed mechanism for CDP-ME kinase.