The enzyme 2C
-erythritol-2,4-cyclodiphosphate (MECP) synthase catalyzes the fifth stage in the biosynthesis of isoprenoid precursors by the 1-deoxy-d
-xylulose-5-phosphate (DOXP) pathway. This biosynthetic route, sometimes also referred to as the 2C
-erythritol-4-phosphate (MEP) pathway (Eisenreich et al.
; Lichtenthaler, 1999
), is present in many bacteria, the plastids of plants and in apicomplexan parasites. The genes encoding enzymes of the DOXP pathway have been shown to be essential in bacteria. Since mammals use a different biosynthetic route dependent on mevalonate, inhibitors of the DOXP enzymes might assist the development of novel broad-spectrum antimicrobial drugs. The biomedical implications of understanding the DOXP enzymes have in part been responsible for the interest in this area of research.
There are seven enzymes in the DOXP pathway, which catalyze the formation of the universal isoprenoid precursor isopentenyl diphosphate (IPP) and the isomer dimethylallyl diphosphate (DMAPP). The first step is the condensation of glyceraldehyde 3-phosphate and pyruvate by DOXP synthase (Lois et al.
; Sprenger et al.
), followed by conversion of DOXP to MEP by a reductoisomerase (Reuter et al.
; Steinbacher et al.
). Next, a cytidyltransferase produces 4-diphosphocytidyl-2C
-erythritol (CDP-ME; Richard et al.
). In an ATP-dependent reaction, CDP-ME kinase then phosphorylates the erythritol moiety (Miallau et al.
; Wada et al.
). MECP synthase then catalyzes the formation of the unusual cyclic diphosphate MECP with release of CMP (Fig. 1; Kemp et al.
). MECP is finally converted to IPP in two further steps catalyzed by IspG and IspH proteins (Hecht et al.
; Rohdich et al.
). Some of the IPP produced is isomerized to DMAPP by an IPP isomerase (Eisenreich et al.
), thereby providing the reagents necessary for chain elongation leading to more complex isoprenoids. The sequential additions of IPP to the allylic precursor are catalyzed by isoprenyl diphosphate synthases to give geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and longer chain products (Kellogg & Poulter, 1997
; Leyes et al.
). These compounds then serve as precursors for the biosynthesis of more complex natural products including sterols, dolichols, triterpenes, ubiquinones and plastoquinones (Sacchettini & Poulter, 1997
The reaction catalyzed by MECP synthase.
Recent studies have revealed that isoprenoids bind to MECP synthase, raising the possibility of a feedback mechanism to regulate this important biosynthetic pathway (Kemp et al.
). Electrospray mass spectrometry indicated that recombinant Escherichia coli
MECP synthase binds a mixture of IPP, GPP and FPP in an approximate ratio of 1:4:2 and it is assumed that these ligands are acquired as the protein is produced and are retained during purification. Crystal structures of MECP synthase in complex with GPP or FPP have been deposited in the PDB (Gabrielsen et al.
; Kemp et al.
; Ni et al.
) and reveal the isoprenoid diphosphate to be placed at an interface created by the three subunits. The terminal phosphate group is positioned by six hydrogen bonds with three Arg142 residues, each held in place by Glu144 on the surface of the trimeric prism. The second phosphate group participates in three hydrogen bonds with amide groups of three Phe139 residues, whilst the tail of the isoprenoid ligands extends down into a hydrophobic cavity (Figs. 2 and 3
Figure 2 Ribbon representation of the functional MECP synthase trimer with the single subunits colored yellow, magenta and blue. The CDP molecules in stick representation (C positions are white, O red, N blue) bind in the active site formed at the interface of (more ...)
Figure 3 The entrance of the isoprenoid diphosphate-binding site in (a) the wild-type and (b) the mutated enzyme. GPP molecules are shown as in Fig. 2. Selected amino-acid residues are depicted in stick mode and colored C yellow, N blue, O red. Hydrogen-bonding (more ...)
To investigate the potential contributions that isoprenoid diphosphate binding might make to MECP synthase activity, we sought to prepare a sample with a conserved structure but for which isoprenoid diphosphate binding was disrupted. For this purpose, an E. coli MECP synthase double mutant was generated where Arg142 and Glu144 are replaced with methionine and leucine, respectively, and the crystal structure determined to examine the consequences of such mutations.