A triclinic crystal form of Escherichia coli 4-diphosphocytidyl-2C-methyl-D-erythritol kinase and reassessment of the quaternary structure

Abstract

4-Diphosphocytidyl-2C-methyl-D-erythritol kinase (IspE; EC 2.7.1.148) contributes to the 1-deoxy-D-xylulose 5-phosphate or mevalonate-independent biosynthetic pathway that produces the isomers isopentenyl diphosphate and dimethylallyl diphosphate. These five-carbon compounds are the fundamental building blocks for the biosynthesis of isoprenoids. The mevalonate-independent pathway does not occur in humans, but is present and has been shown to be essential in many dangerous pathogens, i.e. Plasmodium species, which cause malaria, and gram-negative bacteria. Thus, the enzymes involved in this pathway have attracted attention as potential drug targets. IspE produces 4-diphosphosphocytidyl-2C-methyl-D-erythritol 2-phosphate by ATP-dependent phosphorylation of 4-diphosphocytidyl-2C-methyl-D-erythritol. A triclinic crystal structure of the Escherichia coli IspE-ADP complex with two molecules in the asymmetric unit was determined at 2 A resolution and compared with a monoclinic crystal form of a ternary complex of E. coli IspE also with two molecules in the asymmetric unit. The molecular packing is different in the two forms. In the asymmetric unit of the triclinic crystal form the substrate-binding sites of IspE are occluded by structural elements of the partner, suggesting that the ;triclinic dimer' is an artefact of the crystal lattice. The surface area of interaction in the triclinic form is almost double that observed in the monoclinic form, implying that the dimeric assembly in the monoclinic form may also be an artifact of crystallization.

Figure 1

Ribbon representation of IspE. ADP is presented as van der Waals spheres coloured grey for C, red for O, orange for P and blue for N. The N- and C-termini are labelled. β-Strands are shown in yellow and α-helices are shown in red; they are numbered in black and red, respectively.

Figure 2

Comparison of the distinct asymmetric units observed for E. coli IspE. (a) The monoclinic crystal form (Miallau et al., 2003; PDB code 1oj4). (b) The triclinic form of the enzyme. For each structure, molecule A is shown in the same orientation and the position of the β2–β3 turn of molecule B is marked.

Figure 3

One EcIspE active site in the triclinic structure is occluded by the asymmetric unit partner. The substrate-binding pocket of the triclinic form molecule A is shown together with the β2–β3 turn of molecule B. CDP-ME from molecule A of the monoclinic structure, after least-squares superposition of the A molecules, is shown as semi-transparent sticks and with the same atomic colouring scheme as employed in Fig. 1 ▶.

Figure 4

ADP in the cofactor-binding pocket. The OMIT F _o − F _c difference density map is shown as black chicken wire and contoured at 1.5σ. α1 and α2 are labelled in red.